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INNOVATION WITHIN REACH
InvivoGen’s primary objective is to combine cutting-edge research with our technical
expertise to create tools for the bioresearch community. Our unparalleled skill in
microbial fermentation allows us to provide ultra-pure antibiotics made entirely without
animal components, novel anti-mycoplasma treatments, and the largest collection of
PRR agonists derived from a wide range of micro-organisms.
Our collection of innovative Blue Reporter Cells is the result of the intensive study of the
innate immune system matched with our vector design capabilities and cell culture
experience. We are expanding the popular HEK-Blue family of cells to include
murine TLRs and NODs. Also new this year is an expanded collection of
pNiFty plasmids for monitoring a variety of signaling pathways.
The 2011 Catalog has a new chapter devoted to Antibodies and Vaccination. In this section
you will find our novel pFUSE plasmids for antibody generation utilizing recombinant DNA
technology. Harnessing this technology allows us to provide monoclonal isotype collections
of therapeutically relevant antibodies. We also offer a unique set of vaccine adjuvants
including our preclinical VacciGrade PRR Ligands.
InvivoGen remains the only company devoted to marketing CpG-free plasmid DNA.
We’ve added two new pCpGfree plasmid backbones to facilitate a better understanding of
how CpGs affect gene expression, which we believe is important to
the future of gene therapy.
Please browse through this catalog and visit our website to learn about all the tools
we provide. If there is something you are looking for that you cannot find in this catalog or
on our website please let us know. We are always looking for new ways to
bring innovation within reach.

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CONTENTS AT A GLANCE
1 CELL CULTURE & TRANSFECTION
2 MAMMALIAN EXPRESSION VECTORS
3 INNATE IMMUNITY
4 CYTOKINE SIGNALING
5 ANTIBODIES & VACCINATION
6 CpG-FREE DNA
7 RNA INTERFERENCE
8 INHIBITORS
9 CUSTOM SERVICES

TABLE of CONTENTS
1 CELL CULTURE & TRANSFECTION
Mycoplasma Detection & Elimination
PlasmoTest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Plasmocin - Plasmocure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Primocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Normocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Fungin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Reporter Detection Kits
LacZ Detection Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
SEAP Reporter Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
QUANTI-Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
HEK-Blue Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Selective Antibiotics
Blasticidin S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
G418 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Hygromycin B - HygroGold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Phleomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Puromycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Zeocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Transfection Reagent
LyoVec . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Induced Pluripotent Stem Cells
Reprogramming Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Reprogramming Enhancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2 MAMMALIAN EXPRESSION VECTORS
Lentiviral Vectors
LENTI-Smart (INT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
LENTI-Smart NIL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
LENTI-Smart OSKM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Cloning Vectors
Recombinant IgGs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Fc Fusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Rodent Transgenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
In vitro & in vivo Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Tagged Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Genes - Open Reading Frames
Gene A-List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Cellular Promoters
Prom A-List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
PromTest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Custom-made pDRIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
E. coli Growth & Selection
E. coli Competent Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
E. coli Selection Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3 INNATE IMMUNITY
Pattern Recognition Receptors (PRRs)
Toll-Like Receptors (TLRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
NOD-Like Receptors (NLRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
RIG-I-Like Receptors (RLRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
C-type Lectin Receptors (CLRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
TLR, NLR, RLR & Related Genes
Native Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
HA-Tagged Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
TLR-GFP Fusion Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Dominant Negative Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Reporter Cell Lines
293/TLR & 293/NOD Clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
HEK-Blue TLR & HEK-Blue NOD Cells . . . . . . . . . . . . . . . . . . 66
THP1-XBlue & THP1-XBlue -defMyD Cells . . . . . . . . . . . . . . . 68
Ramos-Blue & Ramos-Blue -defMyD Cells . . . . . . . . . . . . . . . . 69
RAW-Blue Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
C3H/TLR4mut & C3H/WT MEFs . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
C57/WT MEFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Antibodies
Antibodies for Neutralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Antibodies for Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
PRR Signaling
pNiFty Reporter Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Pathogen-Associated Molecular Patterns
TLR Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
TLR Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77, 80
Labeled TLR Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
NOD Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Labeled NOD Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
RLR & CDS Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Dectin-1 Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
TLR & NOD Agonist Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
NF-kB Activators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
PAMPs Detection
HEK-Blue LPS Detection Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
TLR/NOD Ligand Screening Service . . . . . . . . . . . . . . . . . . . . . . . . . 91
PRR Inhibition
PRR & Related shRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Signal Transduction Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Inflammasomes
Inflammasome Inducers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Inflammasome Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
IL-1b Sensor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Autophagy & TLRs
Autophagy Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Autopagy Inducers & Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

4 CYTOKINE SIGNALING
TABLE of CONTENTS
Cytokine Expression
Cytokine and Related Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Cytokine Detection
Anti-Cytokine Neutralizing IgA Antibodies . . . . . . . . . . . . . . . . . . . 104
Blue Cytokine Reporter Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
HEK-Blue Cytokine Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
5 ANTIBODIES & VACCINATION
Antibodies
Antibody Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Antibody Isotype Collections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
IgA Antibody Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
IgA Antibody Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
IgA Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
IgG Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Vaccination
Vaccine Adjuvants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
OVA Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
DNA Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
6 CpG-FREE DNA
CpG-Free Plasmids
pCpGfree Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
CpG-Free Genes
Synthetic CpG-free Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Custom-Made CpG-free Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
7 RNA INTERFERENCE
psiRNA System
psiRNA Cloning Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
psiTEST System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Interferon response: Off Target effect
IFNr qRT-Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
psiRNA Vectors
Ready-made psiRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Custom-made psiRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
8 INHIBITORS
PRR Signaling Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Cytokine Receptor Inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Ion Channel Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Nuclear Export Inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
NF-kB Activation Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
8 INHIBITORS
MAPK Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Protein Kinase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Epigenetic Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Heat Shock Protein Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
DNA Synthesis Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
9 CUSTOM SERVICES
TLR/NOD Ligand Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Custom-Made CpG-free Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Custom Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
Large Scale Plasmid DNA Production . . . . . . . . . . . . . . . . . . . . . . . . 163
PRODUCT INFORMATION. . . . . . . . . . . . . . . . . . . . . 165
INTERNATIONAL DISTRIBUTORS. . . . . . . . . . 176

1
CELL CULTURE & TRANSFECTION
Mycoplasma Detection and Elimination 7
Reporter Detection Kits 12
Selective Antibiotics 16
Transfection Reagents 19
Induced Pluripotent Stem Cells 20

Mycoplasma Detection &
Elimination
Mycoplasma contamination remains a significant problem to the culture of mammalian cells. Mycoplasmas can
cause disastrous effects on eukaryotic cells as they can alter every cellular parameter from proliferation to virus
susceptibility and production leading to unreliable experimental results and potentially unsafe biological products.
Mycoplasmas are the smallest and simplest self-replicating organisms. They lack a rigid cell wall and grow mostly
associated with the mammalian cell membranes. In most cases, there are no signs of mycoplasma contamination.
They cannot be detected by visual inspection and do not cause consistent perceptible changes in a cell culture
such as rapid pH change and medium turbidity. Thus, mycoplasmas can remain undetected in the cell cultures for
long periods.
Mycoplasma Detection
The only way to confirm mycoplasma contamination is by routine testing using special techniques. InvivoGen has developed PlasmoTest , a
breakthrough in mycoplasma detection for cell cultures. PlasmoTest is the first mycoplasma detection kit that uses engineered cells and
therefore can be easily established as a routine procedure in the lab.
Mycoplasma Elimination
Once the mycoplasma contamination has been confirmed, it is usually recommended that the infected cell culture be immediately autoclaved
to prevent the infection from spreading and to use only mycoplasma-free cultures. However, some cell lines are irreplaceable and require an
effective eradication treatment. InvivoGen offers a choice of antimicrobial solutions designed to eliminate and prevent mycoplasma
contaminations. Some are also active against bacteria and/or fungi.
• Plasmocin - Elimination and Prevention of Mycoplasma
• Plasmocure - Elimination of Mycoplasma
• Normocin - Prevention Against Mycoplasma, Bacteria and Fungi
• Primocin - Prevention Against Mycoplasma, Bacteria and Fungi for Primary Cells
• Fungin - Prevention and Elimination of Fungi
www.invivogen.com/mycoplasma
7
CELL CULTURE & TRANSFECTION 1

CELL CULTURE & TRANSFECTION 1
PlasmoTest - Mycoplasma Detection
PlasmoTest provides a simple, rapid and reliable assay for the visual detection of mycoplasma contamination in cell cultures. This assay is the
first to utilize cells to signal the presence of mycoplasma.
- Simple - Requires only basic cell culture knowledge. No need for specific lab equipment. Results are easily determined with the naked eye or
quantified with a spectrophotometer.
- Rapid - Hands-on time less than 1 hour. Gives results after overnight incubation.
- Versatile - Detects all Mycoplasma and Acholeplasma species known to infect cell cultures, as well as other cell culture contaminants such as bacteria.
- Sensitive - Detects 5.10 2 -5.10 5 cfu/ml mycoplasmas. No false positive: a positive result indicates the presence of a cell culture contaminant.
- Complete - Contains the Mycoplasma sensor cells and all the reagents needed to perform the assay, including positive and negative controls. Up
to 500 samples can be tested with the kit. To perform further assays, only the reagents need to be reordered.
Principle
PlasmoTest features two major constituents: the Mycoplasma sensor cells
and the HEK-Blue Detection medium. The Mycoplasma sensor cells detect
the presence of mycoplasmas leading to a color change of the HEK-Blue
Detection medium. The Mycoplasma sensor cells recognize mycoplasmas
through Toll-Like Receptor 2 (TLR2), a pathogen recognition receptor. In
the presence of mycoplasmas, TLR2 initiates a signaling cascade leading to
the activation of NF-kB and other transcription factors. These transcription
factors induce the secretion of SEAP (secreted embryonic alkaline
phosphatase) in the supernatant which is readily detected by the
purple/blue coloration of the HEK-Blue Detection medium.
Key Features
HEK-Blue -2 cells, the Mycoplasma sensor cells, are engineered HEK293
cells. These cells stably express TLR2 and multiple genes from the TLR2
pathway and coexpress an optimized SEAP reporter gene, placed under
the control of a promoter inducible by the transcription factors NF-kB and
AP-1.
HEK-Blue Selection is a solution that combines several selective
antibiotics. These antibiotics guarantee the persistent expression of the
various transgenes introduced in HEK-Blue -2 cells. Furthermore,
Normocin is included in the kit to protect HEK-Blue -2 cells from any
potential microbial contamination, whether caused by mycoplasmas,
bacteria or fungi.
HEK-Blue Detection is a medium specifically designed for the detection
of SEAP. It contains a color substrate that produces a purple/blue color
following its hydrolysis by SEAP (see page 15).
8
www.invivogen.com/mycoplasma
PlasmoTest Principle

PlasmoTest Contents
- HEK-Blue -2 cells (3-5 x 10 6 cells)
- HEK-Blue Selection (4 x 2 ml) - Antibiotic mix
- Normocin (200 mg - 4 x 1 ml)
- HEK-Blue Detection (2 pouches of 50 ml each)
- HEK-Blue water (2 x 60 ml)
- Positive control
- Negative control
Buy the PlasmoTest kit once then reorder only the reagents to perform
further assays. The reagents can be purchased individually or together as
the PlasmoTest Reagent Kit which includes the following:
- HEK-Blue Selection (4 x 2 ml) - Antibiotic mix
- Normocin (4 x 1 ml)
- HEK-Blue Detection (2 pouches)
- HEK-Blue water (2 x 60 ml)
- Positive control
- Negative control
www.invivogen.com/mycoplasma
PlasmoTest Procedure
1- Collect 500 μl cell culture
supernatant and transfer into a
microtube.
2- Heat samples at 100°C for 15 mins.
3- Prepare HEK-Blue Detection by
adding 50 ml HEK-Blue water to the
powder.
4- Add 50 μl of each sample in a well of
a 96-well plate.
5- Add 50 μl of each control in a well of
a 96-well plate.
6- Prepare HEK-Blue -2 cell suspension
using HEK-Blue Detection medium.
7- Add 200 μl (~50,000 cells) of cell
suspension to each well containing the
samples or controls.
8- Incubate the plate at 37°C in a CO2
incubator overnight (16-24h).
9- Detect the presence of Mycoplasma
with the naked eye or by reading the
OD at 620-655 nm.
PRODUCT QUANTITY CAT. CODE
PlasmoTest 1 kit rep-pt2
HEK-Blue Detection 2 pouches hb-det1
HEK-Blue Selection 5 x 2 ml hb-sel
Normocin 500 mg ant-nr-1
PlasmoTest Controls 200 tests pt-ctr2
PlasmoTest Reagent Kit 500 samples rep-ptrk
9
CELL CULTURE & TRANSFECTION 1

CELL CULTURE & TRANSFECTION 1
Plasmocin - The Mycoplasma Removal Agent
Description
Plasmocin is a well-established antimycoplasma reagent. It contains two
bactericidal components strongly active against mycoplasmas that allow
their elimination in only 2 weeks. The first component acts on the protein
synthesis machinery while the second acts on the DNA replication. These
two specific and separate targets are found only in mycoplasmas and
many other bacteria and are completely absent in eukaryotic cells.
In contrast to other anti-mycoplasma compounds, Plasmocin is active
on both free mycoplasmas and intracellular forms. This advantage is
conferred by one component of Plasmocin which is actively transported
into mammalian cells. It ensures that following treatment with Plasmocin
a cell culture is not reinfected by mycoplasmas released from intracellular
compartments of infected cells.
In all animal cell lines tested to date, even at five times the working
concentration, no apparent adverse effect on cellular metabolism is
observed.
No resistance in liquid cultures of mycoplasmas has ever been identified
in repeated experiments attempting to measure the mutation rate.
Therefore, development of resistant mycoplasma strains is virtually
eliminated.
Plasmocin is also active at low concentrations on a broad range of Gram
positive and Gram negative bacteria that are otherwise resistant to the
mixture of streptomycin and penicillin, and exhibits no toxicity in
eukaryotic cells.
Many cell lines infected by mycoplasmas have been successfully treated
with Plasmocin , including embryonic stem cells, hybridomas and
retrovirus packaging cells.
10
www.invivogen.com/mycoplasma
• Plasmocin Treatment
To eliminate mycoplasmas use Plasmocin (ant-mpt) at
25 μg/ml for two weeks in the infected culture.
• Plasmocin Prophylactic
To prevent mycoplasma contamination, use Plasmocin
(ant-mpp) at 2.5 - 5 μg/ml on a regular basis in cell culture.
Plasmocure - The Alternative Mycoplasma Removal Agent
Description
In very rare cases, mycoplasmas resistant to Plasmocin have been
reported. To eradicate these mycoplasmas, InvivoGen has developed a
new antimycoplasma agent called Plasmocure . Plasmocure combines
two antibiotics that act through different mechanisms of action than those
in Plasmocin . A two week treatment with Plasmocure was found
sufficient to completely eliminate the mycoplasmas. A moderate toxicity
can be observed during the course of the treatment but full recovery of
the cell line is expected once mycoplasmas are eliminated.
Plasmocure is a sterile ready-to-use solution. Simply add to mycoplasma
contaminated cell cultures for 2 weeks at the recommended
concentration of 50 μg/ml.
Contents and Storage
Plasmocin is provided as a yellow solution either at a concentration of 25
mg/ml (Plasmocin Treatment) or 2.5 mg/ml (Plasmocin Prophylactic). One
ml Plasmocin Treatment is enough to treat 1 L medium. Plasmocin is
shipped at room temperature. Store at -20°C. Plasmocin is stable 6 months
at 4°C and at least one year at -20°C.
PRODUCT QUANTITY CAT. CODE
Plasmocin Treatment 50 mg (2 x 1 ml) ant-mpt
Plasmocin Prophylactic 25 mg (5 x 2 ml) ant-mpp
Contents and Storage
Plasmocure is provided as a colorless solution at a concentration of 100
mg/ml. One ml Plasmocure is enough to treat 2 L medium. Plasmocure
is shipped at room temperature. Store at -20°C. Plasmocure is stable
one year at 4°C or -20°C.
PRODUCT QUANTITY CAT. CODE
Plasmocure 100 mg (1 x 1 ml) ant-pc

Primocin - The Antimicrobial Shield for Primary Cells
Description
Primocin is an antibiotic formulation specifically designed to protect
primary cell lines from cell culture contaminations. Primocin is active
against both Gram+ and Gram- bacteria, mycoplasmas and fungi.
Primocin is the first formulation to offer complete protection against
microbial contaminants. There is no need to add Pen/Strep.
Primocin is non-toxic to primary cells. It acts on targets found only in
micro-organisms. Bacterial targets are the DNA gyrase and the
prokaryotic ribosomal subunits, 30S and 50S. The fungal target is
ergosterol, a molecule only found in the cell membrane of fungi and
yeasts.
Primocin is a sterile water soluble solution that can be added directly
to the cell culture medium. The solution is at 50 mg/ml and the
recommended working concentration is 100 μg/ml.
Contents and Storage
Normocin - The First Line of Defense for Your Cells
Description
Normocin is an innovative formulation of three antibiotics active against
mycoplasmas, bacteria and fungi. Normocin contains two compounds
that act on mycoplasmas and both Gram+ and Gram- bacteria by
blocking DNA and protein synthesis. The third compound eradicates
yeasts and fungi by disrupting ionic exchange through the cell membrane.
The active concentration of Normocin (100 μg/ml) displays no
toxicity to the cell line being treated. Normocin is used in combination
with Pen / Strep solutions to broaden the anti-bacterial spectrum.
Fungin - The Solution for Fungal Contaminations
Description
Fungin is a new soluble form of Pimaricin, a polyene introduced in the 1950’s
as an anti-fungal agent. This antimycotic compound kills yeasts, molds and fungi
by disrupting ionic exchange through the cell membrane.
Fungin is cell culture tested, and may be added to media containing
commonly used antibacterial agents.
Fungin is a highly stable compound. It is shipped at room temperature
and is still active after 6 days at 37°C. Fungin is water soluble unlike
Amphotericin B which needs to be dissolved in toxic deoxycholate.
Fungin exhibits no cytotoxicity to cells being treated, and no deleterious
effects on cell metabolism. The active concentration of Fungin can be
stepped up from 10 μg/ml to 50 μg/ml if needed without displaying any
negative effects.
Primocin is provided as a sterile light yellow solution at a concentration
of 50 mg/ml. Primocin is available in 1 ml vials or 20 ml bottles. One ml
Primocin is enough to treat 500 ml medium. Primocin is shipped at
room temperature and upon receipt should be stored at -20°C.
Primocin is stable one month at 4°C and 18 months at -20°C.
Contents and Storage
Normocin is provided as a sterile red solution at a concentration of 50
mg/ml. Normocin is available in 1 ml vials or 20 ml bottles. One ml
Normocin is enough to treat 500 ml medium. Product is shipped at
room temperature and should be stored at -20°C. Normocin is stable
three months at 4°C and 18 months at -20°C.
Contents and Storage
www.invivogen.com/mycoplasma
PRODUCT QUANTITY CAT. CODE
Primocin 500 mg (10 x 1 ml)
1 g (1 x 20 ml)
PRODUCT QUANTITY CAT. CODE
Normocin 500 mg (10 x 1 ml)
1 g (1 x 20 ml)
Fungin is provided as a pale yellow solution at a concentration of 10
mg/ml. One 1.5 ml vial is able to treat 300 ml to 1.5 liters of medium
depending on the severity of contamination. Fungin is shipped at room
temperature, store at -20°C. Fungin is stable 18 months at 4°C or
-20°C.
PRODUCT QUANTITY CAT. CODE
Fungin 75 mg (5 x 1.5 ml)
200 mg (1 x 20 ml)
ant-pm-1
ant-pm-2
ant-nr-1
ant-nr-2
ant-fn-1
ant-fn-2
11
CELL CULTURE & TRANSFECTION 1

CELL CULTURE & TRANSFECTION 1
Reporter Detection Kits
Gene reporter systems are extensively used for the study of eukaryotic gene expression and regulation. Although
they play a significant role in numerous applications, they most frequently serve as indicators of transcriptional
activity in cells. The reporter gene of choice is combined to a promoter sequence and can be subsequently assayed
in vitro or in vivo. An ideal reporter gene is not endogenously expressed by the target cells creating background
signals and can be detected through assays that are easy, sensitive and reliable.
InvivoGen provides two reporter gene systems that can be measured by histochemical staining of cells or tissues:
• b-Galactosidase (LacZ) reporter gene system
• Secreted embryonic alkaline phosphatase (SEAP) reporter gene system
Furthermore, InvivoGen has developed two specific media that allow fast and convenient detection of secreted
AP, called QUANTI-Blue and HEK-Blue Detection (see pages 14-15).
LacZ Reporter Gene System
The E. coli lacZ gene encoding b-galactosidase is the classical histochemical reporter gene. b-Galactosidase catalyzes the hydrolysis of X-Gal
producing a blue precipitate that can be easily visualized under a microscope. The LacZ Staining Kits provide simple and convenient methods
for the visual detection of LacZ expression within cells or tissues.
LacZ Reporter Gene
InvivoGen provides the LacZDCpG gene, a humanized and CpG-free
allele of the LacZ gene. This CpG-free gene is ten times more active than
the wild-type gene in mammalian cells. It can be used for in vitro or in
vivo applications. The LacZDCpG gene is provided in the pSELECT-zeo-
LacZ plasmid under the control of the EF-1a/HTLV composite promoter.
The pSELECT-zeo-LacZ plasmid is selectable with Zeocin in mammalian
cells and in bacteria.
Contents and Storage
The LacZDCpG gene is provided as 20 μg of lyophilized DNA with 4
pouches of E. coli Fast-Media ® Zeo (2 TB and 2 Agar, see p45). Plasmid is
shipped at room temperature. Store at -20°C for up to one year.
12
LacZ Staining Kits
www.invivogen.com/reporter-detection
InvivoGen provides two LacZ staining kits:
- LacZ Cell Staining Kit allows you to determine the percentage of
transfected cells expressing the lacZ gene. All reagents are provided to
prepare the fixative, staining and rinse solutions. The assay can be
completed in 30 minutes, and the blue precipitate can take from 30
minutes to overnight to appear. The LacZ Cell Staining Kit contains
sufficient reagent to stain one hundred 35 mm plates.
- LacZ Tissue Staining Kit allows the detection of transduced cells
expressing the lacZ gene within fresh or frozen tissues. All reagents are
provided to prepare the buffer, fixative and staining solutions. The staining
of the tissues can be observed in 5 to 24 hours. The LacZ Tissue Staining
Kit contains sufficient reagent to prepare 100 ml solutions.
Contents and Storage
The LacZ Cell Staining Kit contains: 10X PBS, X-Gal, Potassium
Ferrocyanide, Potassium Ferricyanide, MgCl2 and 10X Fixative Solution.
The LacZ Tissue Staining Kit contains: 10X PBS, X-Gal, Potassium
Ferrocyanide, Potassium Ferricyanide, MgCl2, Glutaraldehyde, Igepal and
Na deoxycholate.
Products are shipped at room teperature. Store at 4°C or -20°C
according to the product label. All reagents are stable for 6 months.
PRODUCT QUANTITY CAT. CODE
pSELECT-zeo-LacZ 20 μg psetz-lacz
LacZ Cell Staining Kit 1 kit rep-lz-c
LacZ Tissue Staining Kit 1 kit rep-lz-t

SEAP Reporter Gene System
Secreted embryonic alkaline phosphatase (SEAP) is a reporter widely used to study promoter activity or gene expression. It is a truncated
form of human placental alkaline phosphatase (PLAP) by deletion of the GPI anchor. Unlike endogenous alkaline phosphatases, PLAP is extremely
heat stable and resistant to the inhibitor L-homoarginine. SEAP is secreted into cell culture supernatant and therefore offers many advantages
over intracellular reporters. It allows to determine reporter activity without disturbing the cells, does not require the preparation of cell
lysates and can be used for kinetic studies.
SEAP Reporter Gene
InvivoGen provides the secreted embryonic alkaline phosphatase (SEAP)
gene in the pSELECT-zeo-SEAP plasmid. It can be used in vivo and in vitro
to transfect mammalian cells stably or transiently. The SEAP gene
expression is driven by the EF-1a/HTLV composite promoter that
combines the elongation factor 1 alpha core promoter and the
5’untranslated region of the Human T-cell Leukemia Virus. The pSELECTzeo-SEAP
plasmid is selectable with Zeocin in both mammalian cells
and bacteria.
Contents and Storage
The SEAP gene is provided as 20 μg of lyophilized DNA. It is supplied
with 4 pouches of E. coli Fast-Media ® Zeo (2 TB and 2 Agar, see p44-45).
Plasmid is shipped at room temperature. Store at -20°C for up to one
year.
InvivoGen offers three different methods to determine SEAP activity:
• SEAP Reporter Assay Kit - Detection kit for the quantification of SEAP
• QUANTI-Blue - Detection medium for the detection and quantification of any AP
• HEK-Blue Detection - Cell culture medium for real-time, one step detection of SEAP
SEAP Reporter Assay Kit
The SEAP Reporter Assay Kit provides a rapid and convenient method
to determine the levels of SEAP in the culture medium of transfected
cells expressing the seap gene. SEAP catalyzes the hydrolysis of
p-Nitrophenyl phosphate producing a yellow end product that can be
read spectrophotometrically at 405 nm.
- Hands-on-time: less than 1 hour
- Obtention of results: 10 to 40 minutes
- Sensitivity: as little as 10 -10 g/ml of SEAP
- Concentration range: 1 ng - 1 μg/ml
Contents and Storage
www.invivogen.com/reporter-detection
PRODUCT QUANTITY CAT. CODE
pSELECT-zeo-SEAP 20 μg psetz-seap
These colorimetric assays are somewhat less sensitive than the chemiluminescent method but are much less expensive and can be performed
using a conventional ELISA plate spectrophotometer.
The SEAP Reporter Assay Kit contains all reagents necessary to measure
SEAP activity in 150 samples: 50X Dilution Buffer, 5X Assay Buffer, 100
mM L-Homoarginine, tablets of SEAP substrate. Store 50X Dilution Buffer
and 5X Assay Buffer at 4°C. Store all other components at -20°C. All
reagents are stable for 6 months when properly stored.
PRODUCT QUANTITY CAT. CODE
SEAP Reporter Assay Kit 1 kit rep-sap
13
CELL CULTURE & TRANSFECTION 1

CELL CULTURE & TRANSFECTION 1
QUANTI-Blue - Detection and Quantification of Alkaline Phosphatase
QUANTI-Blue is a detection medium developed to determine the activity of any alkaline phosphatase (AP) present in a biological sample.
QUANTI-Blue offers many advantages over the conventional SEAP Reporter Assay Kit based on the pNPP substrate:
➥ Shorter hands-on-time: no longer than 10 minutes
➥ Simpler preparation: just add water
➥ Allows high-throughput screening
➥ Higher saturation threshold
Key Features
Requires small samples of cell supernatants
Samples of 10 μl are sufficient.
Determine SEAP activity without disturbing cells
The same cell cultures can be repeatedly sampled for kinetic studies or
further experimentation.
Assay can be completed in 30 min
The enzymatic activity can be detected as early as 20 min after incubation
of the samples in QUANTI-Blue .
Highly sensitive for quantitative measurement
The assay allows to detect low and high levels of SEAP. No need to
perform multiple sample dilutions.
Wide dynamic range for accurate measurement
Higher saturation threshold than with pNPP resulting in more significant
differences between non or low SEAP expression and high SEAP
expression.
Contents and Storage
QUANTI-Blue is provided in a 5- or 10-pouch unit. Each pouch allows
the preparation of 100 ml of detection medium. Store at room
temperature. Pouches are stable 12 months at room temperature. After
preparation, product is stable 2 weeks at 4°C and 2 months at -20°C.
14
PRODUCT QUANTITY CAT. CODE
QUANTI-Blue 5 pouches (5 x 100 ml)
10 pouches (10 x 100 ml)
* Bulk quantities readily available
rep-qb1
rep-qb2
www.invivogen.com/reporter-detection
QUANTI-Blue Procedure

HEK-Blue Detection - Real-Time Detection of SEAP
HEK-Blue Detection is a cell culture medium that allows the detection of SEAP as the reporter protein is secreted by the cells. HEK-Blue Detection contains all the nutrients necessary for cell growth and a specific SEAP color substrate. The hydrolysis of the substrate by SEAP
produces a purple/blue color that can be easily detected with the naked eye or measured with a spectrophotometer.
➥ One step procedure
➥ Extremely simple to use
➥ Designed for high-throughput screening
Key Features
No need to process samples
Preparation of cell lysates or heating of samples are not required.
Determine SEAP activity without disturbing cells
The same cell cultures can be repeatedly sampled for kinetic studies.
Measure the time course of SEAP
Detection of SEAP occurs as the reporter protein is secreted by the cells
grown in HEK-Blue Detection. Being a cell culture medium,
in contrast to a simple detection medium such as QUANTI-Blue ,
HEK-Blue Detection is about 10-fold less sensitive than the former.
Virtually no hands-on time
1- Resuspend HEK-Blue Detection by adding water.
2- Grow SEAP-expressing cells in HEK-Blue Detection.
3- Detect SEAP expression with the naked eye or read at OD620-655.
Contents and Storage
HEK-Blue Detection is provided in a 2- or 5-pouch unit. Each pouch
allows the preparation of 50 ml of detection medium. Store at room
temperature. Pouches are stable 12 months at room temperature. After
preparation, product is stable 2 weeks at 4°C and 2 months at -20°C.
PRODUCT QUANTITY CAT. CODE
HEK-Blue Detection 2 pouches (2 x 50 ml)
5 pouches (5 x 50 ml)
* Bulk quantities readily available
hb-det1
hb-det2
HEK-Blue Procedure
www.invivogen.com/reporter-detection
15
CELL CULTURE & TRANSFECTION 1

CELL CULTURE & TRANSFECTION 1
Selective Antibiotics
InvivoGen is a leader in the production of selective antibiotics. We manufacture the largest choice of antibiotics
for selection. Our state-of-the-art facilities allow us to produce large quantities of high quality antibiotics with
purity levels exceeding 95%. As we manufacture our products, we are able to offer the best prices on the market.
All our products are provided as cell-culture tested, sterile solutions that are ready-to-use.
High Quality
As all the products are manufactured by InvivoGen, our antibiotics
meet rigorous standards and have passed stringent quality control,
which includes verification of potency, purity and stability using
microbiological and chromatographic methods. This leads to
consistently high quality.
Purity and Stability
All our antibiotics are ultra pure: more than 95% purity for most of
them. Their excellent stability allows for exceptional selection and
reproducible results.
An Excellent Choice of Antibiotics for Selection
in Both Mammalian Cells and E. coli
Matches up to the antibiotic resistance genes carried by InvivoGen
plasmids, built for selection in both mammalian and bacterial cells.
Ready-to-use Cell Culture Tested Solutions
No weighing needed - Our antibiotics are available in solution
filtered to sterility for customer convenience and validated for cell
culture usage.
16
www.invivogen.com/selective-antibiotics
Endotoxin-free Guaranteed
InvivoGen’s selective antibiotics contain no detectable levels of
endotoxin (

Blasticidin S
Description
Blasticidin S is a peptidyl nucleoside antibiotic isolated from Streptomyces
griseochromogenes. It specifically inhibits protein synthesis in both
prokaryotes and eukaryotes by interfering with the peptide bound
formation in the ribosomal machinery. Resistance to blasticidin is
conferred by the blasticidin resistance gene from Bacillus cereus (bsr)
which encodes a deaminase. Typically, bacteria are sensitive to blasticidin
concentrations of 25-100 μg/ml, and mammalian cells to 1-10 μg/ml.
Contents and Storage
Blasticidin is provided as a colorless solution at 10 mg/ml. Blasticidin is
shipped at room temperature and should be stored at -20°C. Blasticidin
is stable at least one year when stored at -20°C.
G418 Sulfate
Description
G418 is an aminoglycoside antibiotic similar in structure to gentamicin
B1, produced by Micromonospora rhodorangea. G418 blocks polypeptide
synthesis by inhibiting the elongation step in both prokaryotic and
eukaryotic cells. Resistance to G418 is conferred by the neo gene from
Tn5 encoding an aminoglycoside 3’-phosphotransferase, APH 3’ II.
Selection in mammalian cells is usually achieved in three to seven days
with concentrations ranging from 400 to 1000 μg/ml. Cells that are
dividing are affected sooner than those that are not.
Contents and Storage
G418 is provided as a colorless solution at 100 mg/ml. G418 is shipped
at room temperature and should be stored at -20°C. G418 is stable for
at least one year when stored at -20°C.
Puromycin
Description
Puromycin is an aminonucleoside antibiotic produced by Streptomyces
alboniger. It specifically inhibits peptidyl transfer on both prokaryotic and
eukaryotic ribosomes. This antibiotic inhibits the growth of Gram positive
bacteria and various animal and insect cells. Puromycin can also be used
in some particular conditions for the selection of E. coli transformants.
Resistance to puromycin is conferred by the Pac gene encoding a
puromycin N-acetyl-transferase (PAC) that was found in a Streptomyces
producer strain. Animal cells are generally sensitive to concentrations from
1 to 10 μg/ml.
Contents and Storage
Puromycin hydrochloride is provided as a colorless solution at 10 mg/ml.
Puromycin is shipped at room temperature and should be stored at -20°C.
Puromycin is stable at least one year when stored at -20°C.
PRODUCT QUANTITY CAT. CODE
Blasticidin (solution) 100 mg (5 x 2 ml)
500 mg (25 x 2 ml)
500 mg (1 x 50 ml)
ant-bl-1
ant-bl-5
ant-bl-5b
Blasticidin (powder) 1 g ant-bl-10p
PRODUCT QUANTITY CAT. CODE
G418 Sulfate 1 g (5 x 2 ml)
5 g (1 x 50 ml)
ant-gn-1
ant-gn-5
PRODUCT QUANTITY CAT. CODE
Puromycin 100 mg (5 x 2 ml)
www.invivogen.com/selective-antibiotics
500 mg (25 x 2 ml)
ant-pr-1
ant-pr-5
17
CELL CULTURE & TRANSFECTION 1

CELL CULTURE & TRANSFECTION 1
Hygromycin B
Description
Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces
hygroscopicus. It inhibits protein synthesis by interfering with translocation
and causing mistranslation at the 70S ribosome. Hygromycin B is effective
on most bacteria, fungi and higher eukaryotes. Resistance to hygromycin is
conferred by the hph gene from E. coli. Hygromycin B is normally used at
a concentration of 50-200 μg/ml in mammalian cells and 100 μg/ml in
bacteria.
Two grades of Hygromycin B are available:
• Hygromycin B (purity >85%)
• HygroGold (purity >98%)
Phleomycin
Description
Phleomycin is a glycopeptide antibiotic of the bleomycin family, isolated
from a mutant strain of Streptomyces verticillus. It binds and intercalates
DNA thus destroying the integrity of the double helix. Phleomycin is active
against most bacteria, filamentous fungi, yeast, plant and animal cells. Use
of phleomycin is recommended for cells poorly sensitive to Zeocin , i.e.
filamentous fungi and some yeasts. Phleomycin resistance is conferred by
the Sh ble gene from Streptoalloteichus hindustanus which encodes a protein
that binds to phleomycin, inhibiting its DNA cleavage activity. Typically,
phleomycin is used at a concentration of 10 μg/ml for yeasts and 25-150
μg/ml for filamentous fungi.
Zeocin
Description
Zeocin is a formulation of phleomycin D1, a copper-chelated
glycopeptide antibiotic produced by Streptomyces CL990. Zeocin causes
cell death by intercalating into DNA and cleaving it. This antibiotic is
effective on most aerobic cells and is therefore useful for selection in bacteria,
eukaryotic microorganisms, plant and animal cells. Resistance to Zeocin is
conferred by the Sh ble gene product which inactivates Zeocin by binding
to the antibiotic. Zeocin is used at a concentration of 50-300 μg/ml for
selection in mammalian cells and 25 μg/ml for bacterial selection.
Contents and Storage
Hygromycin B and HygroGold are provided as 100 mg/ml yellow
solutions. HygroGold is also provided as a powder. Products are shipped
at room temperature. Store at -20°C. Hygromycin B solutions are stable
for at least one year when stored at -20°C.
PRODUCT QUANTITY CAT. CODE
Hygromycin B 1 g (5 x 2 ml)
5 g (1 x 50 ml)
HygroGold 1 g (5 x 2 ml)
5 g (1 x 50 ml)
10 g (powder)
Contents and Storage
Phleomycin is provided as a blue solution at 20 mg/ml or as a powder.
Phleomycin is shipped at room temperature. Store the solution at -20°C
and the powder at 4°C. Phleomycin is stable for at least one year when
properly stored.
PRODUCT QUANTITY CAT. CODE
Phleomycin (solution) 100 mg (5 x 1 ml)
500 mg (25 x 1 ml)
Phleomycin (powder) 250 mg
500 mg
1 g
Contents and Storage
ant-ph-1
ant-ph-5
ant-ph-2p
ant-ph-5p
ant-ph-10p
Zeocin is provided as a blue solution at 100 mg/ml. Zeocin is shipped at
room temperature and upon receipt should be stored at -20°C. Zeocin
is stable for at least one year at -20°C.
PRODUCT QUANTITY CAT. CODE
Zeocin (solution) 1 g (5 x 2 ml)
5 g (25 x 2 ml)
5 g (1 x 50 ml)
Zeocin (powder) 1 g
5 g
18 www.invivogen.com/selective-antibiotics
ant-hm-1
ant-hm-5
ant-hg-1
ant-hg-5
ant-hg-10p
ant-zn-1
ant-zn-5
ant-zn-5b
ant-zn-1p
ant-zn-5p

LyoVec New formulation
LyoVec is the first lyophilized cationic lipid-based transfection reagent. The major constituent of LyoVec is the phosphonolipid DTCPTA, which
is coupled with DiPPE, a neutral lipid that helps destabilizing membrane bilayers, therefore increasing the in vitro transfection efficiency of LyoVec .
Features and Benefits
Rapid and Simple-to-use
- Hands-on time: No more than 15 min
- No preparation required the day prior transfection
Optimized Procedure
- Works similarly at various lipid/DNA ratios
- No optimization of transfection conditions
- Soluble in water up to 5X
Maximum Transfection Efficiency
- Transfects with high efficiencies a broad spectrum of mammalian cells,
including primary cells and non-adherent cells
- Effective for transient and stable transfections
Minimal Cytotoxicity
- Works in the presence of serum
- No need to wash cells after transfection
Increased Stability
- Stable over one year when lyophilized
- Stable up to six months when rehydrated
Long shelf-life of pDNA/LyoVec Complexes
In contrast with other cationic lipids, plasmid DNA (pDNA)/
LyoVec complexes remain fully active for transfection for at least two
months at 4°C. Thus, preparation of large volumes of complexes can be
made and reused repeatedly, saving you time.
Structure
DTCPTA
Di-tetradecylphosphoryl-N,N,N-trimethylmethanaminium chloride
1. Floch et al. 1997. Cationic phosphonolipids as non viral vectors for DNA transfection
in hematopoietic cell lines and CD34+ cells. Blood Cells, Molec.& Diseases 23: 69-87.
2. Guillaume-Gable et al. 1998. Cationic phosphonolipids as nonviral gene transfer
agents in the lung of mice. Hum. Gene Ther. 9: 2309-2319
Contents and Storage
LyoVec is provided as a lyophilized powder, sterile and packaged in sealed
2 ml glass vials (4 or 9 vials). After reconstitution, each vial yields 2 ml of
transfection reagent allowing 40 transfections of 1-4 x 10 5 cells/well in a 12well
plate. Each order of LyoVec comes with 1 vial of control LyoVec /GFP-
LacZ complex. LyoVec is shipped at room temperature. Upon receipt, store
at 4°C. LyoVec is stable up to 1 year when properly stored.
PRODUCT QUANTITY CAT. CODE
LyoVec 8 ml (160 reactions)
18 ml (360 reactions)
lyec-12
lyec-22
Percentage of b-Gal-expressing cells
Invivo
LyoV
LyoVec Procedure
STEP 1 - LyoVec Reconstitution
Reconstitute LyoVec with 2 ml sterile
H2O or PBS
Note: Reconstituted LyoVec can be stored
at 4°C for 6 months.
STEP 2 - Transfection of Adherent and
Suspension Cells
1-10 μl pDNA (1-3 μg) + 100 μl LyoVec
-> 20’ at room temperature
Note: Bulk quantities of pDNA/LyoVec
complexes can be prepared for multiple
wells.
1 ml cell suspension (0.25-1.10 6 cells) +
100 μl pDNA/LyoVec complexes ->
mix gently. Incubate at 37°C in 5% CO2
for 24-72 hours before testing transgene
expression or applying antibiotic
selection.
DiPPE
1,2-Diphytanoyl-sn-Glycero-3-Phosphoethanolamine
Transfection efficiencies of LyoVec : 2 to 8 x 10 5 cells per 6-well plates were
transfected with 1 μg pVITRO2-GFP/LacZ complexed with 100 μl LyoVec in 2 ml
of culture medium containing 10% FBS. LacZ expression was assessed 48h posttransfection.
Note: Reagent X is one of the most utilized transfection reagents on
the market. Transfection was performed according to the manufacturer’s protocol.
www.invivogen.com/transfection-reagents 19
CELL CULTURE & TRANSFECTION 1

CELL CULTURE & TRANSFECTION 1
Induced Pluripotent Stem Cells
Recently, pioneering work has revealed that terminally differentiated somatic
cells can be reprogrammed to generate induced pluripotent stem (iPS) cells
via overexpression of a defined set of transcription factors. These iPS cells
are morphologically and phenotypically similar to embryonic stem (ES) cells
and thus offer exciting possibilities in stem cell research and regenerative
medicine. Moreover, iPS cells are useful tools for studying the pathogenesis
of human disease, for drug discovery and toxicity screening 1, 2 .
The most widely used set of reprogramming factors, Oct4, Sox2, Klf4 and
c-Myc, was identified initially by screening 24 pre-selected factors in mouse
embryonic fibroblasts (MEFs) by Takahashi and Yamanaka 3 . This cocktail of
transcription factors, OSKM, was shown to work for different types of
somatic cells and for different species, including rhesus monkey 4 and human
cells 5 . Direct reprogramming was also achieved by another team who used
a partially overlapping combination, Oct4, Sox2, Nanog and Lin28, suggesting
that Oct4 and Sox2 are indispensable whereas Nanog, Lin28, Klf4 and
c-Myc are alternative supporting factors 6 . Subsequent studies have
demonstrated that fewer factors are required; the c-Myc gene, an oncogene,
is dispensable (although the efficiency of iPS cell formation is significantly
lower) 7 and in some cases, such as neural stem cells, expression of only one
factor (Oct4) is sufficient 8 .
The generation of iPS cells is usually achieved by genetic transduction of the
reprogramming genes using retroviral or lentiviral vectors. However, the use
of integrating viral vectors represent an obstacle to the therapeutic
translation of iPS cells as this technology can produce insertional mutagenic
lesions that are potentially tumorigenic. Two recent publications detail the
use of polycistronic lentiviral vectors delivering the OSKM quartet to
somatic cells in a single lentiviral construct reducing the number of genomic
insertions 9, 10 . Alternative approaches to deliver the reprogramming factors
with minimal or total absence of genetic modifications have been developed.
These approaches include the use of LoxP sites and Cre-induced excision
and piggyBac transposon excision of integrated reprogramming vector
sequences 11, 12 , and the use of an oriP/EBNA1-based episomal vector 13 .
Non-integrating lentiviral vectors may also represent a promising approach 14 .
One possible strategy to entirely replace gene delivery is protein
transduction. Previous studies have demonstrated that various proteins can
be delivered into cells by conjugating them with a short peptide that
mediates cell penetration, such as poly-arginine 15 . Zhou et al. have designed
and purified poly-arginine tagged Oct4, Sox2, Klf4 and c-Myc proteins that
were found to readily enter cells and translocate into the nucleus 16 . After
several cycles of protein supplementation, iPS cells were successfully
generated from MEFs. Using a similar approach, Kim et al. obtained
protein-induced pluripotent stem cells from human newborn fibroblasts
20
Induction of pluripotent
stem cells
Somatic cells are obtained from
adult organism.
The reprogramming factors are
introduced into the cultured
somatic cells.
The cells are grown under ES cells
conditions. After 2-3 weeks, iPS cells
emerge.
These induced pluripotent stem
cells may be differentiated into
various cell types for regenerative
medicine applications.
www.invivogen.com/ipsc
after several rounds of treatment with cell extracts of HEK293 cell lines
expressing poly-arginine tagged OSKM genes 17 .
Recent studies have reported an emerging role for microRNAs (miRNAs)
in the generation of iPS cells. It has been demonstrated that the
reprogramming of iPS cells and subsequent differentiation to lineage specific
cells can be observed by monitoring the differential expression of
miRNAs, small non-coding RNAs that are critical regulators of gene
expression 18 . Furthermore, the inhibition of tissue-specific miRNAs
promotes the formation of iPS cells 19 .
Direct reprogramming of somatic cells is currently a slow and inefficient
process, in particular when the c-Myc oncogene is omitted in an effort to
reduce tumorigenicity. Several chemicals have recently been reported to
either enhance reprogramming efficiencies or substitute for specific
reprogramming factors. Among the reported chemicals, some are known
to affect chromatin modifications while others influence signal transduction
pathways. Small molecules that modulate chromatin modifications include
DNA methyltransferase inhibitors (RG108, 5-azacytidine), histone
deacetylase inhibitors (valproic acid, trichostatin A) and a G9a histone
methyltransferase (Bix-01294) 20,21,22 . Small molecules reported to potentiate
reprogramming by targeting signaling pathways include the MEK inhibitor
PD035901 and the TGF-beta receptor inhibitor SB431542 23 .
Among the various reprogramming strategies, the chemical approach
combined to protein transduction may represent the most promising.
However, substantial research and development is still required before iPS
cells are ready for therapeutic applications.
1. Yokoo N. et al., 2009. The effects of cardiovactive drugs on cardiomyocytes derived from
human induced pluripotent stem cells. Biochem Biophys Res Commun. 387:482-8. 2. Lian
Q. et al., 2010. Future perspective of induced pluripotent stem cells for diagnosis, drug
screening and treatment of human diseases. Thromb Haemost. 104(1):39-44. 3. Takahashi
K. & Yamanaka S., 2006. Induction of pluripotent stem cells from mouse embryonic and
adult fibroblast cultures by defined factors. Cell. 126(4):663-76. 4. Liu H. et al., 2008.
Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell
Stem Cell. 3(6):587-90. 5. Takahashi K. et al., 2007. Induction of pluripotent stem cells from
adult human fibroblasts by defined factors. Cell. 131(5):861-72. 6. Yu J. et al., 2007. Induced
pluripotent stem cell lines derived from human somatic cells. Science. 318(5858):1917-20.
7. Nakagawa M. et al., 2008. Generation of induced pluripotent stem cells without Myc from
mouse and human fibroblasts. Nat Biotechnol. 26(1):101-6. 8. Kim JB. et al., 2009. Oct4induced
pluripotency in adult neural stem cells. Cell. 136(3):411-9. 9. Sommer CA. et al.,
2009. Induced pluripotent stem cell generation using a single lentiviral stem cell cassette.
Stem Cells. 27(3):543-9. 10. Chang CW. et al., 2009. Polycistronic lentiviral vector for "hit
and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells. Stem
Cells. 27(5):1042-9. 11. Soldner F. et al., 2009. Parkinson's disease patient-derived induced
pluripotent stem cells free of viral reprogramming factors. Cell. 136(5):964-77. 12. Kaji K.
et al., 2009. Virus-free induction of pluripotency and subsequent excision of reprogramming
factors. Nature. 458(7239):771-5. 13. Yu J. et al., 2009. Human induced pluripotent stem
cells free of vector and transgene sequences. Science. 324(5928):797-801.14. Sarkis C. et
al., 2008. Non-integrating lentiviral vectors. Curr Gene Ther. 8(6):430-7. Review. 15. Ogawa
T. et al., 2007. Novel Protein Transduction Method by Using 11R: An Effective New Drug
Delivery System for the Treatment of Cerebrovascular Diseases. Stroke, 38: 1354 - 1361.
16. Zhou H. et al., 2009. Generation of induced pluripotent stem cells using recombinant
proteins. Cell Stem Cell. 4(5):381-4. 17. Kim D. et al., 2009. Generation of human induced
pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 4(6):472-
6. 18. Kamata M. et al. 2010. Live cell monitoring of hiPSC generation and differentiation
using differential expression of endogenous microRNAs. PLoS One. 5(7):e11834.
19. Mallanna SK. & Rizzino A., 2010. Emerging roles of microRNAs in the control of
embryonic stem cells and the generation of induced pluripotent stem cells. Dev Biol. 344:16-
25. 20. Shi Y et al., 2008b. Induction of pluripotent stem cells from mouse embryonic
fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell. 2008 Nov
6;3(5):568-74. 21. Huangfu D. et al., 2008. Induction of pluripotent stem cells by defined
factors is greatly improved by small-molecule compounds. Nat Biotechnol. 2008
Jul;26(7):795-7. 22. Durcova-Hills G. et al., 2008. Reprogramming primordial germ cells into
pluripotent stem cells. PLoS One. 3(10):e3531. 23. Lin T. et al., 2009. A chemical platform
for improved induction of human iPSCs. Nat Methods. 6(11):805-8.

Poly-Arginine-HA Tagged Reprogramming Factors
Description
Poly-arginine-HA tagged reprogramming factors allow the production of
recombinant cell-penetrating reprogramming factors. They correspond to
the four transcription factors, Oct4, Sox2, Klf4, and c-Myc (OSKM), fused at
their C terminus to a poly-arginine (i.e. 11R) peptide in tandem with 3 motifs
of the hemaglutinine (HA) tag. The poly-arginine peptide enables the
recombinant proteins to readily enter the cells and have been shown to
allow their translocation into the nucleus 1, 2 . The HA tag is useful for their
detection by Western blot or their purification by affinity chromatography.
Poly-arginine-HA tagged reprogramming factors are cloned in the pUNO1
plasmid within a mammalian expression cassette comprising the EF-1a/HTLV
composite promoter and the SV40 poly adenylation sequence. pUNO1
plasmids are selectable in E. coli and mammalian cells with blasticidin.
Application
Poly-arginine-HA tagged reprogramming factors are designed for the
generation of protein-induced pluripotent cells. Following their
transfection into mammalian cell lines, such as HEK293, the cell extracts
can be used crude to treat the target cells or processed to purifiy the
poly-arginine-HA-tagged proteins on an anti-HA affinity column.
Contents and Storage
Each pUNO1 plasmid is provided as a lyophilized transformed E. coli strain
on a paper disk. Transformed strains are shipped at room temperature and
should be stored at -20°C. Each pUNO1 is provided with 4 pouches of
E. coli Fast-Media ® Blas (2 TB and 2 Agar). For more information about
Fast-Media ® , see pages 44-45.
Reprogramming Enhancers
1. Zhou H. et al., 2009. Generation of induced pluripotent stem cells using recombinant
proteins. Cell Stem Cell. 4(5):381-4. 2. Kim D. et al., 2009. Generation of human induced
pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell.
4(6):472-6.
PRODUCT
CAT. CODE
(HUMAN)
CAT. CODE
(MOUSE)
pUNO1-OCT4-11RHA puno1rha-hoct4 puno1rha-moct4
pUNO1-SOX2-11RHA puno1rha-hsox2 puno1rha-msox2
pUNO1-KLF4-11RHA puno1rha-hklf4 puno1rha-mklf4
pUNO1-cMYC-11RHA puno1rha-hmycb puno1rha-mmycb
InvivoGen offers a selection of small molecules that have recently been reported to either enhance reprogramming efficiencies or substitute for specific
reprogramming factors. Among these molecules, some are known to affect chromatin modifications while others influence signal transduction pathways.
PRODUCT DESCRIPTION
WORKING
CONCENTRATION
QUANTITY
CATALOG
CODE
REF. INFO
5-Aza-cytidine DNA methyltransferase inhibitor 2 μM 100 mg inh-aza 1, 2 p 157
Bix-01294 G9a histone methyltransferase inhibitor 1 μM 2 mg inh-bix 3, 4 p 157
PD0325901 MEK inhibitor 0.5 μM 2 mg inh-pd32 5, 6 p 156
SB431542 TGF-b receptor inhibitor 2 μM 5 mg inh-sb43 6, 7 p 155
Valproic Acid HDAC inhibitor 2 mM 5 g inh-vpa 2, 8 p 157
1. Mikkelsen TS. et al., 2008. Dissecting direct reprogramming through integrative genomic
analysis. Nature. 454(7200):49-55. 2. Huangfu D. et al., 2008a. Induction of pluripotent stem
cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol.
26(7):795-7. 3. Epsztejn-Litman S. et al., 2008. De novo DNA methylation promoted by
G9a prevents reprogramming of embryonically silenced genes. Nat Struct Mol Biol.
15(11):1176-83. 4. Shi Y et al., 2008. A combined chemical and genetic approach for the
generation of induced pluripotent stem cells. Cell Stem Cell. 2(6):525-8. 5. Ying QL. et al.,
2008. The ground state of embryonic stem cell self-renewal. Nature. 453(7194):519-23.
6. Lin T. et al., 2009. A chemical platform for improved induction of human iPSCs. Nat
Methods. 6(11):805-8. 7. IInman GJ. et al., 2002. SB-431542 is a potent and specific inhibitor
of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK)
receptors ALK4, ALK5, and ALK7. Mol Pharmacol. 2002 Jul;62(1):65-74. 8 . Huangfu D. et al.,
2008b. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4
and Sox2. Nat Biotechnol. 26(11):1269-75.
Contents and Storage
www.invivogen.com/ipsc
3xHA
11R
Each product is provided as a solid and shipped at room temperature. Store
at room temperature, 4°C or -20°C according to the product label.
21
CELL CULTURE & TRANSFECTION 1

2
MAMMALIAN EXPRESSION VECTORS
Lentiviral Vectors 23
Cloning Vectors 26
Genes - Open Reading Frames 36
Cellular Promoters 38
E. coli Growth & Selection 42

LENTI-Smart - Lentiviral Vector Production Kits
Lentiviral vectors derived from the human immunodeficiency virus (HIV-1) have become major tools for gene delivery in mammalian cells. The
advantageous feature of lentivirus vectors is the ability to mediate potent transduction and stable expression into dividing and non-dividing cells
both in vitro and in vivo. Currently, production of high titers of lentiviral vectors is a time consuming, multi-step procedure with low reproducibility.
LENTI-Smart has been designed to solve these problems, by creating a lyophilizate of optimized packaging plasmids combined with a DNA
transfection reagent. Upon rehydration, this lyophilizate serves as a “carrier” for your favorite lentiviral expression plasmid. Depending on your
preference, LENTI-Smart kits are available for the production of either integrating or non-integrating lentiviral (NIL) vectors.
- Consistent high titers - 1-5 x 106 transducing units (TU) per ml (unconcentrated) are commonly achieved.
- Fast - Lentiviral production time reduced and no need to produce the packaging and pseudotyping plasmids
- Simple - Everything is ready for your experiment, just add your favorite lentiviral expression plasmid.
- Versatile - Can be used with most commercially available lentiviral expression plasmids.
- Reproducible - Fluctuating parameters are kept to a minimum.
Description
LENTI-Smart is a ready-to-use product that allows for rapid and reliable
production of high titers of second generation lentiviral particles in HEK
293T cells. LENTI-Smart combines a mix of optimized packaging
plasmids precomplexed to a transfection reagent, LyoVec , selected for
its high transfection efficiency and low cell toxicity. This lyophilized complex
is provided with a control lentiviral expression plasmid.
Packaging Plasmids
The two packaging plasmids forming the LENTI-Smart lyophilizate
provide the structural and replication proteins in trans that are required
for the production of the lentiviral particles (see page 25).
• pLV-iVSV-G expresses the G glycoprotein gene from Vesicular Stomatitis
Virus (VSV-G) to allow production of a pseudotyped lentiviral vector with
a broad host range.
• pLV-HELP contains the viral gag, pol, rev and tat genes and the revresponsive
element (RRE).
- gag encodes the membrane associated and capsid proteins.
- pol encodes viral enzymes, including the reverse transcriptase and
integrase, required for replication and integration.
- tat encodes the virus transactivator protein.
- rev encodes the Rev protein which interacts with the RRE to induce
expression of the gag and pol genes.
- RRE permits Rev-dependent expression of the gag and pol genes.
Control Lentiviral Expression Plasmid
The LENTI-Smart kit includes a control lentiviral expression plasmid
designed to optimize virus production and cell transduction.
• pLV-Green expresses a green fluorescent protein (GFP) gene and
contains key viral elements for lentivirus production and safety:
- psi encodes the packaging signal.
- Long terminal repeats containing a deletion in the U3 region to ensure
self-inactivation of the lentiviral vector.
- rev and RRE (see above).
Maps and sequences of the plasmids are available
on our website:
www.invivogen.com/lentismart-maps
Lentiviral expression
plasmid
LENTI-Smart
HEK 293T
Lentiviral particles
www.invivogen.com/lentiviral-vectors
Lentiviral Vector Production
using LENTI-Smart
Invivo
LENT
1. Add lentiviral expression plasmid to
LENTI-Smart to generate complexes.
2. Add complexes to HEK 293T cells.
3. Harvest viral supernatants 36-48 hours
post-transfection.
The unique formulation of LENTI-Smart allows to prepare lentiviral expression
plasmid / packaging plasmids complexes by simply rehydrating the lyophilizate with
the lentiviral expression plasmid solution. There is no need for a transfection reagent
as it is included in the LENTI-Smart lyophilizate. Transfection of HEK 293T cells is
readily performed by adding the complexes to the cells. Lentiviral particles can be
collected 2 days after transfection.
23
MAMMALIAN EXPRESSION VECTORS 2

MAMMALIAN EXPRESSION VECTORS 2
Biosafety Features
The LENTI-Smart kit has been designed to eliminate the risk of
replication recombination through:
- the use of multiple plasmids
- the absence of HIV accessory genes and non-coding sequences
- the use of self-inactiving lentiviral expression plasmids.
To further increase the biosafety of LENTI-Smart derived lentiviral vectors,
InvivoGen provides the LENTI-Smart NIL kit for the production of nonintegrating
lentiviral vectors reducing the risk of insertional mutagenesis.
Contents and Storage
Two LENTI-Smart kits are available:
• LENTI-Smart (INT) for the generation of integrating lentiviral vectors
• LENTI-Smart Starter Kit for the generation of integrating and nonintegrating
lentiviral vectors.
LENTI-Smart (INT) is available in 2 sizes, either 5 vials or 10 vials. Each
vial allows the transfection of HEK 293T cells with a lentiviral expression
plasmid in a 10-cm culture plate or a 75 cm 2 flask.. The LENTI-Smart
Starter Kit contains 5 vials of LENTI-Smart (INT) and 5 vials of LENTI-
Smart NIL (see below).
All LENTI-Smart kits are provided with a vial of the control lentiviral
expression plasmid. The LENTI-Smart vials are provided lyophilized, the
control plasmid is provided as a liquid. Products are shipped at room
temperature and should be stored at -20°C.
24
www.invivogen.com/lentiviral-vectors
Viral titers obtained using LENTI-Smart and other transfection reagents with
different commercially available lentiviral expression plasmids.
PRODUCT QTY CAT. CODE
LENTI-Smart (INT)
5 vials
10 vials
LENTI-Smart NIL - Generation of Non-Integrating Lentiviruses
ltsint-5
ltsint-10
LENTI-Smart Starter Kit 10 vials lts-str
Integration of a transgene into the host genome poses safety concerns due to the potential risk of insertional mutagenesis. To overcome this
problem, non-integrating lentiviral vectors have been designed that combine the advantageous features of lentiviral vectors with episomal
replication alleviating the risk of insertional mutagenesis. InvivoGen provides LENTI-Smart NIL, the first kit designed for the generation of
non-integrating lentiviral vectors.
Description
LENTI-Smart NIL combines the pLV-iVSV-G plasmid (see page 23) and
the pLV-HELP-NIL D64 plasmid, precomplexed to the transfection reagent,
LyoVec . pLV-HELP-NIL D64 expresses a mutant integrase (D64V) resulting
in the generation of lentiviral vectors that are integration defective 1,2 .
Non-integrating lentiviral vectors allow transient transgene expression in
dividing cells and long-term expression in non-dividing cells, such as neurons.
LENTI-Smart NIL is provided with the control lentiviral plasmid,
pLV-Green, that expresses a GFP gene.
1. Leavitt AD. et al., 1996. Human immunodeficiency virus type 1 integrase mutants
retain in vitro integrase activity yet fail to integrate viral DNA efficiently during
infection. J Virol. 70(2):721-8. 2. Nightingale SJ. et al., 2006.Transient gene expression
by nonintegrating lentiviral vectors. Mol Ther. 13(6):1121-32.
Contents and Storage
LENTI-Smart NIL is available in 2 sizes, either 5 vials or 10 vials. Each vial
allows the transfection of HEK 293T cells with a lentiviral expression plasmid
in a 10-cm culture plate or a 75 cm 2 flask.
LENTI-Smart NIL is provided with a vial of the control lentiviral
expression plasmid. The LENTI-Smart NIL vials are provided lyophilized,
the control plasmid is provided as a liquid. Products are shipped at room
temperature and should be stored at -20°C.
Duration of GFP expression using integrating or non-integrating (NIL) lentiviral
vectors. Percentage of GFP positive HCT116 cells was assessed by FACS analysis,
following transduction with LENTI-Smart (INT)- or LENTI-Smart NIL-derived
lentiviral vectors.
PRODUCT QTY CAT. CODE
LENTI-Smart NIL Kit*
5 vials
10 vials
ltsnil-5
ltsnil-10
* LENTI-Smart NIL featuring the mutated integrase D116N is also available
upon request.

Lentiviral Vector Production
and Cell Transduction
Lentiviral vectors are typically produced in
HEK 293T cells. Essential lentiviral (HIV-1)
genes must be expressed in these cells to
allow the generation of lentiviral particles.
These genes are usually expressed by several
plasmids: (i) a lentiviral expression plasmid,
such as pLV-Green, containing the psi (Y)
packaging sequence and the transgene gene
inserted between the lentiviral LTRs for
target cell integration, (ii) a packaging
plasmid, such as pLV-HELP, encoding the pol,
gag, rev and tat viral genes and containing the
rev-response element (RRE); and (iii) a
pseudotyping plasmid, such as pLV-iVSV-G,
encoding the G protein of the Vesicular
Stomatitis Virus (VSV-G) envelope gene.
Unlike the HIV envelope, the VSV-G
envelope has a broad cell host range
extending the cell types that can be
transduced by VSV-G-expressing lentiviruses.
Two days after transfection of HEK 293T cells, the cell supernatant contains recombinant lentiviral vectors, which can be used to transduce the target cells. Once in the
target cells, the viral RNA is reverse-transcribed, imported into the nucleus and stably integrated into the host genome. One or two days after the integration of the viral
RNA, the expression of the recombinant protein can be detected.
LENTI-Smart OSKM - For the Generation of iPS Cells
Induced pluripotent stem (iPS) cells are generated from differentiated somatic cells overexpressing a set of specific transcription factors called
reprogramming factors. Typically four reprogramming factors are chosen, Oct4, Sox2, Klf4 and c-Myc (OSKM), which are introduced into the
target cells through genetic transduction using retroviral or lentiviral vectors. iPS cells offer exciting possibilities in stem cell research and
regenerative medicine. To facilitate the generation of iPS cells, InvivoGen has developed LENTI-Smart OSKM for the production of lentiviral
vectors expressing the human or murine OSKM reprogramming genes.
Description
LENTI-Smart OSKM comprises the complexing lyophilizate, formed by
the packaging plasmids and the transfection reagent, and the lentiviral
plasmid expressing the OSKM genes of human or mouse origin.
Single Reprogramming Expression Cassette
The four reprogramming genes are expressed from a single multicistronic
transcript 1 . This reprogramming cassette contains the coding sequences of
Oct4, Sox2, Klf4 and c-Myc separated by three different “self-cleaving” 2A
peptides (E2A, P2A, and T2A, respectively) 2 (figure).
Integrating or Non-Integrating Lentiviral Vectors
Two LENTI-Smart OSKM kits are available allowing the generation of
integrating or non-integrating lentiviral vectors:
• LENTI-Smart (INT) OSKM features pLV-HELP which expresses the
wild-type integrase for the production of integrating lentiviral vectors
• LENTI-Smart NIL OSKM features pLV-HELP-NIL which expresses a
mutant integrase for the production of non-integrating lentiviral (NIL)
vectors, designed for transient transgene expression.
1. Carey BW. et al., 2009. Reprogramming of murine and human somatic cells using
a single polycistronic vector. PNAS 106(1):157-62. 2. Donnelly ML. et al., 2001. The
'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and
naturally occurring '2A-like' sequences. J Gen Virol. 82(Pt 5):1027-41.
Contents and Storage
PRODUCT QTY
LENTI-Smart (INT) OSKM
LENTI-Smart NIL OSKM
www.invivogen.com/lentiviral-vectors
Schematic representation of the “reprogramming cassette”
LENTI-Smart (INT) OSKM and LENTI-Smart NIL OSKM are available
in 2 sizes, either 5 vials or 10 vials. Each vial allows the transfection of HEK
293T cells with the lentiviral OSKM expression plasmid in a 10-cm culture
plate or a 75 cm 2 flask.The LENTI-Smart vials are provided lyophilized,
the OSKM plasmid is provided as a liquid. Products are shipped at room
temperature and should be stored at -20°C.
5 vials
10 vials
5 vials
10 vials
CAT. CODE
(HUMAN)
ltsint-hoskm-5
ltsint-hoskm-10
ltsnil-hoskm-5
ltsnil-hoskm-10
CAT. CODE
(MOUSE)
ltsint-moskm-5
ltsint-moskm-10
ltsnil-moskm-5
ltsnil-moskm-10
25
MAMMALIAN EXPRESSION VECTORS 2

MAMMALIAN EXPRESSION VECTORS 2
pFUSE-CLIg and pFUSE-CHIg - Antibody Generation
pFUSE-CLIg and pFUSE-CHIg plasmids are designed to change a monoclonal antibody from one isotype to another IgG isotype therefore
enabling the generation of antibodies with the same antigen affinity but with different effector functions (increased or reduced ADCC and
CDC). Furthermore, they can be used to produce entire IgG antibodies from fragment antigen-binding (Fab) or single-chain variable fragment
(scFv) fragments that are either chimeric, humanized or fully human depending on the nature of the variable region.
➥ Isotype switch to generate IgG antibodies with different effector functions
➥ Generation of entire IgG antibodies, chimeric, humanized or fully human
26
Background
Immunoglobulin G (IgG) antibodies are large molecules composed of two
heavy chains g and two light chains, either k or l. They can be separated
in two regions: the Fab (fragment-antigen binding) that contains the
variable domain responsible for the antibody specificity, and the Fc
(fragment crystalline) that binds specific proteins to induce immune
responses such as opsonization and cell lysis.
The IgG class is divided in four isotypes: IgG1, IgG2, IgG3 and IgG4 in humans,
and IgG1, IgG2a, IgG2b and IgG3 in mice. They share more than 95%
homology in the amino acid sequences of the Fc regions but show major
differences in the amino acid composition and structure of the hinge region.
The Fc region mediates effector functions, such as antibody-dependent cellular
cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In
ADCC, the Fc region of an antibody binds to Fc receptors (FcgRs) on the
surface of immune effector cells such as natural killers and macrophages,
leading to the phagocytosis or lysis of the targeted cells. In CDC, the
antibodies kill the targeted cells by triggering the complement cascade at the
cell surface. IgG isoforms exert different levels of effector functions increasing
in the order of IgG4

Antibody generation using pFUSE-CHIg & pFUSE-CLIg
1- Obtaining VH and VL sequences
To obtain the cDNA sequence of the VH and VL regions from an antibody
producing hybridoma, total RNA or mRNA is extracted and reverse
transcribed to cDNA. PCR is performed with 5’ degenerate primers to anneal
to the unknown VH and VL regions and the 3’ primers designed to anneal to
the “known“ CH and CL regions. Alternatively 5’ RACE can be used. The
resulting amplicons are sequenced.
Contents and Storage
pFUSE-CLIg and pFUSE-CHIg plasmids are provided as 20 μg of lyophilized
DNA. Product is shipped at room temperature and should be stored at
-20°C. Each plasmid is provided with 4 pouches of E. coli Fast-Media ®
Zeo (2 TB and 2 Agar, see pages 44-45).
2- Cloning into pFUSE-CHIg and pFUSE-CLIg
Once the VH and VL sequence are known, inserts for cloning into the plasmids
can be generated. When generating the insert for VH, a Nhe I site must be
introduced at the 3’ end to maintain the integrity of the constant region.
Similarly, when generating the insert for VL, a Bsi WI (human VL) or Bst API
(mouse VL) site must be introduced at the 3’ end. There is a choice of
restriction sites at the 5’ end.
PRODUCT
pFUSE2-CLIg
ISOTYPE QUANTITY
CAT. CODE
(No IL2ss)
CAT. CODE
(With IL2ss)
pFUSE2-CLIg-hk Human kappa 20 μg pfuse2-hclk pfuse2ss-hclk
pFUSE2-CLIg-hl2 Human lambda 2 20 μg pfuse2-hcll2 pfuse2ss-hcll2
pFUSE2-CLIg-mk Mouse kappa 20 μg pfuse2-mclk pfuse2ss-mclk
pFUSE2-CLIg-ml1 NEW Mouse lambda 1 20 μg pfuse2-mcll1 pfuse2ss-mcll1
pFUSE2-CLIg-ml2 NEW Mouse lambda 2 20 μg pfuse2-mcll2 pfuse2ss-mcll2
pFUSE2-CLIg-rk1 NEW Rabbit kappa 1 20 μg pfuse2-rclk1 pfuse2ss-rclk1
pFUSE2-CLIg-rk2
pFUSE-CHIg
NEW Rabbit kappa 2 20 μg pfuse2-rclk2 pfuse2ss-rcl2
pFUSE-CHIg-hG1 Human IgG1 20 μg pfuse-hchg1 pfusess-hchg1
pFUSE-CHIg-hG2 Human IgG2 20 μg pfuse-hchg2 pfusess-hchg2
pFUSE-CHIg-hG3 Human IgG3 20 μg pfuse-hchg3 pfusess-hchg3
pFUSE-CHIg-hG4 Human IgG4 20 μg pfuse-hchg4 pfusess-hchg4
pFUSE-CHIg-mG1 Mouse IgG1 20 μg pfuse-mchg1 pfusess-mchg1
pFUSE-CHIg-mG2a Mouse IgG2a 20 μg pfuse-mchg2a pfusess-mchg2a
pFUSE-CHIg-mG2b Mouse IgG2b 20 μg pfuse-mchg2b pfusess-mchg2b
pFUSE-CHIg-mG3 Mouse IgG3 20 μg pfuse-mchg3 pfusess-mchg3
pFUSE-CHIg-rG NEW Rabbit IgG 20 μg pfuse-rchg pfusess-rchg
www.invivogen.com/antibody-generation
NEW
27
MAMMALIAN EXPRESSION VECTORS 2

MAMMALIAN EXPRESSION VECTORS 2
pFUSE-Fc - Fc-Fusion Proteins
pFUSE-Fc is a family of plasmids developed to facilitate the construction of Fc-Fusion proteins by fusing a sequence encoding a given protein
to the Fc region of an immunoglobulin. The Fc-fusion proteins are called immunoadhesins when the Fc region is combined with the functional
domain of a binding protein, such as a receptor, ligand or cell-adhesion molecule. The Fc region comprises the CH2 and CH3 domains of the
IgG heavy chain and the hinge region. The hinge serves as a flexible spacer between the two parts of the Fc-Fusion protein, allowing each part
of the molecule to function independently.
➥ Large choice of Fc regions from different species: human, mouse, rat and rabbit
➥ Fc regions without intron (pFUSE-Fc) or with introns (pINFUSE-Fc)
➥ Native Fc regions or engineered Fc regions with altered effector functions MCS
Description
pFUSE-Fc
pFUSE-Fc plasmids are designed for the generation of Fc-fusion proteins. They feature
a large choice of Fc regions from different species, with or without introns, native or
engineered. The gene encoding the Fc region is cloned under the control of a strong
and ubiquitous promoter. A multiple cloning site (MCS) located upstream of the Fc
region facilitates the cloning of the functional domain of a given protein.
pINFUSE-Fc
pINFUSE-Fc is a new family of pFUSE-Fc plasmids featuring genomic Fc regions. The
presence of introns is known to enhance the level of gene expression as splicing is
known to promote rapid and efficient mRNA export.
pFUSE-SEAP-Fc
pFUSE-SEAP-Fc plasmids are control plasmids that express the secreted embryonic
alkaline phosphatase (SEAP) reporter gene fused to each Fc region provided by
InvivoGen.
pFUSE-Fc, pINFUSE-Fc and pFUSE-SEAP-Fc plasmids are selectable with Zeocin in
E. coli and mammalian cells. They can be used for transient or stable transfection.
Features
Secretion of Fc-Fusion Proteins
Two versions of pFUSE-Fc and pINFUSE-Fc exist to allow secretion of the Fc fusion:
• No IL2ss: without the IL2 signal sequence (IL2ss) for the secretion of an Fc-fusion
protein containing the functional domain of a protein that is naturally secreted and
thus contains a native signal sequence.
• With IL2ss contains the IL2 signal sequence (IL2ss) for the generation of Fc-Fusion
proteins derived from proteins that are not naturally secreted.
Ease of Detection
Fc-Fusion proteins can be easily detected in the supernatant of pFUSE-Fc-transfected cells
by SDS-PAGE. Their functional domains can be identified by immunoblotting and ligand
blotting.
Ease of Purification
Fc-Fusion proteins can be easily purified by single-step protein A or protein G affinity
chromatography.
Contents and Storage
All pFUSE-Fc, pINFUSE-Fc and pFUSE-SEAP-Fc plasmids are provided as 20 μg of lyophilized
DNA. Product is shipped at room temperature and should be stored at -20°C. Plasmid is
stable up to one year when properly stored. Each plasmid is provided with 4 pouches of
E. coli Fast-Media ® Zeo (2 TB and 2 Agar, see pages 44-45).
28
www.invivogen.com/fc-fusions
MCS
Hinge

Engineering Fc regions for altered properties
The Fc region of an antibody mediates its serum half-life and effector functions,
such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular
phagocytosis (ADCC) and antibody-dependent cell phagocytosis (ADCP).
Engineering the Fc region of a therapeutic monoclonal antibody or Fc fusion
protein allows the generation of molecules that are better suited to the
pharmacology activity required of them 1 .
Engineered Fc regions for increased half-life
One approach to improve the efficacy of a therapeutic antibody is to increase
its serum persistence, thereby allowing higher circulating levels, less frequent
administration and reduced doses. The half-life of an IgG depends on its pHdependent
binding to the neonatal receptor FcRn. FcRn, which is expressed on
the surface of endothelial cells, binds the IgG in a pH-dependent manner and
protects it from degradation. Some antibodies that selectively bind the FcRn at
pH 6.0, but not pH 7.4, exhibit a higher half-life in a variety of animal models.
Several mutations located at the interface between the CH2 and CH3 domains,
such as T250Q/M428L 2 and M252Y/S254T/T256E + H433K/N434F 3 , have been
shown to increase the binding affinity to FcRn and the half-life of IgG1 in vivo.
However, there is not always a direct relationship between increased FcRn
binding and improved half-life 4 .
Engineered Fc regions for altered effector function
Depending on the therapeutic antibody or Fc fusion protein application, it may
be desired to either reduce or increase the effector function. For antibodies that
target cell-surface molecules, especially those on immune cells, abrogating effector
functions is required. Conversely, for antibodies intended for oncology use,
increasing effector functions may improve the therapeutic activity.
The four human IgG isotypes bind the activating Fcg receptors (FcgRI, FcgRIIa,
FcgRIIIa), the inhibitory FcgRIIb receptor, and the first component of complement
(C1q) with different affinities, yielding very different effector functions 5 . Binding of
IgG to the FcgRs or C1q depends on residues located in the hinge region and the
CH2 domain. Two regions of the CH2 domain are critical for FcgRs and C1q
binding, and have unique sequences in IgG2 and IgG4. Substitutions into human
IgG1 of IgG2 residues at positions 233-236 and IgG4 residues at positions 327,
330 and 331 were shown to greatly reduce ADCC and CDC 6, 7 . Furthermore,
Idusogie et al. demonstrated that alanine substitution at different positions, including
K322, significantly reduced complement activation 8 . Similarly, mutations in the CH2
domain of murine IgG2A were shown to reduce the binding to FcgRI, and C1q 9 .
Numerous mutations have been made in the CH2 domain of human IgG1 and
their effect on ADCC and CDC tested in vitro 7-10 . Notably, alanine substitution
at position 333 was reported to increase both ADCC and CDC 7, 9 . Lazar et al.
described a triple mutant (S239D/I332E/A330L) with a higher affinity for FcgRIIIa
and a lower affinity for FcgRIIb resulting in enhanced ADCC 11 . The same
mutations were used to generate an antibody with increased ADCC 12 . Richards
et al. studied a slightly different triple mutant (S239D/I332E/G236A) with
Features of Engineered Fc
Engineered Fc Isotype Mutations FcR/C1q Binding Effector Function Ref.
hIgG1e1-Fc Human IgG1 T250Q/M428L Increased binding to FcRn Increased half-life 2
hIgG1e2-Fc Human IgG1 M252Y/S254T/T256E + H433K/N434F Increased binding to FcRn Increased half-life 3
hIgG1e3-Fc Human IgG1 E233P/L234V/L235A/DG236 + A327G/A330S/P331S Reduced binding to FcγRI Reduced ADCC and CDC 6, 7
hIgG1e4-Fc Human IgG1 E333A Increased binding to FcγRIIIa Increased ADCC and CDC 8, 10
hIgG1e5-Fc Human IgG1 S239D/I332E/A330L Increased binding to FcγRIIIa Increased ADCC 11, 12
hIgG1e6-Fc Human IgG1 P257I/Q311I Increased binding to FcRn Unchanged half-life 4
hIgG1e7-Fc Human IgG1 K326W/E333S Increased binding to C1q Increased CDC 9
hIgG1e9-Fc Human IgG1 S239D/I332E/G236A Increased FcγRIIa/FcγRIIb ratio Increased macrophage phagocytosis 13
hIgG2e1-Fc Human IgG2 K322A Reduced binding to C1q Reduced CDC 8
hIgG4e1-Fc Human IgG4 S228P - Reduced Fab-arm exchange 14
mIgG2ae1-Fc Mouse IgG2a L235E + E318A/K320A/K322A Reduced binding to FcγRI and C1q Reduced ADCC and CDC 9
www.invivogen.com/fc-fusions
improved FcgRIIIa affinity and FcgRIIa/FcgRIIb ratio that mediates enhanced
phagocytosis of target cells by macrophages 13 .
Due to their lack of effector functions, IgG4 antibodies represent the preferred
IgG subclass for receptor blocking without cell depletion. IgG4 molecules can
exchange half-molecules in a dynamic process termed Fab-arm exchange. This
phenomenon can occur between therapeutic antibodies and endogenous IgG4.
The S228P mutation has been shown to prevent this recombination process
allowing the design of less unpredictable therapeutic IgG4 antibodies 14 .
1. Strohl WR., 2009. Optimization of Fc-mediated effector functions of monoclonal antibodies.
Curr Opin Biotechnol. 20(6):685-91. Review. 2. Hinton PR. et al., 2004. Engineered human
IgG antibodies with longer serum half-lives in primates. J Biol Chem. 279(8):6213-6. 3. Vaccaro
C. et al., 2005. Engineering the Fc region of immunoglobulin G to modulate in vivo antibody
levels. Nat Biotechnol. 23(10):1283-8. 4. Datta-Mannan A. et al., 2007. Humanized IgG1 Variants
with Differential Binding Properties to the Neonatal Fc Receptor: Relationship to
Pharmacokinetics in Mice and Primates. Drug Metab. Dispos. 35: 86 - 94. 5. Bruhns P. et al.,
2009. Specificity and affinity of human Fcgamma receptors and their polymorphic variants for
human IgG subclasses. Blood. 113(16):3716-25. 6. Armour KL. et al., 1999. Recombinant human
IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities. Eur J
Immunol. 29(8):2613-24. 7. Shields RL. et al., 2001. High resolution mapping of the binding
site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of
IgG1 variants with improved binding to the Fc gamma R. J Biol Chem. 276(9):6591-604.
8. Idusogie EE. et al., 2000. Mapping of the C1q binding site on rituxan, a chimeric antibody
with a human IgG1 Fc. J Immunol. 164(8):4178-84. 9. Steurer W. et al., 1995. Ex vivo coating
of islet cell allografts with murine CTLA4/Fc promotes graft tolerance. J Immunol. 155(3):1165-
74. 10. Idusogie EE. et al., 2001. Engineered antibodies with increased activity to recruit
complement. J Immunol. 166(4):2571-5. 11. Lazar GA. et al., 2006. Engineered antibody Fc
variants with enhanced effector function. PNAS 103(11): 4005–4010. 12. Ryan MC. et al.,
2007. Antibody targeting of B-cell maturation antigen on malignant plasma cells. Mol. Cancer
Ther., 6: 3009 - 3018. 13. Richards JO,. et al., 2008. Optimization of antibody binding to
FcgammaRIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther. 7(8):2517-
27. 14. Labrijn AF. et al., 2009. Therapeutic IgG4 antibodies engage in Fab-arm exchange with
endogenous human IgG4 in vivo. Nat Biotechnol. 27(8):767-71.
29
MAMMALIAN EXPRESSION VECTORS 2

MAMMALIAN EXPRESSION VECTORS 2
30
Choose a pFUSE-Fc plasmid accordingly to your application:
• Protein purification - All pFUSE-Fc can be used for Protein A or Protein G affinity chromatography (see table below).
• Long term expression in vivo - Choose a pFUSE-Fc with an Fc engineered to display an increased half-life
• Therapeutic use with cell depletion activity - Choose a pFUSE-Fc with an Fc engineered to display increased ADCC and CDC
• Therapeutic use without cell depletion activity - Choose a pFUSE-Fc with an Fc engineered to display reduced ADCC and CDC
PRODUCT ISOTYPE QUANTITY CAT. CODE (No IL2ss) CAT. CODE (With IL2ss)
pFUSE-Fc - Native Fc Regions
pFUSE-hIgG1-Fc Human IgG1 20 μg pfuse-hg1fc1 pfuse-hg1fc2
pFUSE-hIgG2-Fc Human IgG2 20 μg pfuse-hfc1 pfuse-hfc2
pFUSE-hIgG3-Fc Human IgG3 20 μg pfuse-hg3fc1 pfuse-hg3fc2
pFUSE-hIgG4-Fc Human IgG4 20 μg pfuse-hg4fc1 pfuse-hg4fc2
pFUSE-mIgG1-Fc* Mouse IgG1 20 μg pfuse-mg1fc1 pfuse-mg1fc2
pFUSE-mIgG2a-Fc Mouse IgG2a 20 μg pfuse-mg2afc1 pfuse-mg2afc2
pFUSE-mIgG2b-Fc Mouse IgG2b 20 μg pfuse-mg2bfc1 pfuse-mg2bfc2
pFUSE-mIgG3-Fc Mouse IgG3 20 μg pfuse-mg3fc1 pfuse-mg3fc2
pFUSE-rIgG-Fc Rabbit IgG 20 μg pfuse-rfc1 pfuse-rfc2
pFUSE-rtIgG2b-Fc Rat IgG2b 20 μg pfuse-rtg2bfc1 pfuse-rtg2bfc2
pINFUSE - Native Fc Regions with introns
pINFUSE-hIgG1-Fc Human IgG1 20 μg pfc1-hgin1 pfc2-hgin1
pINFUSE-hIgG2-Fc Human IgG2 20 μg pfc1-hgin2 pfc2-hgin2
pINFUSE-hIgG3-Fc Human IgG3 20 μg pfc1-hgin3 pfc2-hgin3
pINFUSE-hIgG4-Fc Human IgG4 20 μg pfc1-hgin4 pfc2-hgin4
pINFUSE-mIgG2b-Fc Mouse IgG2b 20 μg pfc1-mgin2 pfc2-mgin2
pFUSE-Fc - Engineered Fc Regions (see description page 33)
pFUSE-hIgG1e1-Fc Human IgG1 20 μg pfc1-hg1e1 pfc2-hg1e1
pFUSE-hIgG1e2-Fc Human IgG1 20 μg pfc1-hg1e2 pfc2-hg1e2
pFUSE-hIgG1e3-Fc** Human IgG1 20 μg pfc1-hg1e3 pfc2-hg1e3
pFUSE-hIgG1e4-Fc Human IgG1 20 μg pfc1-hg1e4 pfc2-hg1e4
pFUSE-hIgG1e5-Fc Human IgG1 20 μg pfc1-hg1e5 pfc2-hg1e5
pFUSE-hIgG1e6-Fc Human IgG1 20 μg pfc1-hg1e6 pfc2-hg1e6
pFUSE-hIgG1e7-Fc Human IgG1 20 μg pfc1-hg1e7 pfc2-hg1e7
pFUSE-hIgG1e9-Fc Human IgG1 20 μg pfc1-hg1e9 pfc2-hg1e9
pFUSE-hIgG2e1-Fc Human IgG2 20 μg pfc1-hg2e1 pfc2-hg2e1
pFUSE-hIgG4e1-Fc Human IgG4 20 μg pfc1-hg4e1 pfc2-hg4e1
pFUSE-mIgG2ae1-Fc Mouse IgG2a 20 μg pfc1-mg2ae1 pfc2-mg2ae1
pFUSE-SEAP-Fc - Control Plasmids
pFUSE-SEAP-hG1Fc Human IgG1 20 μg pfuse-hg1sp -
pFUSE-SEAP-hG2Fc Human IgG2 20 μg pfuse-hsp -
pFUSE-SEAP-hG3Fc Human IgG3 20 μg pfuse-hg3sp -
pFUSE-SEAP-hG4Fc Human IgG4 20 μg pfuse-hg4sp -
pFUSE-SEAP-mG2aFc Mouse IgG2a 20 μg pfuse-mg2asp -
pFUSE-SEAP-mG2bFc Mouse IgG2b 20 μg pfuse-mg2bsp -
pFUSE-SEAP-mG3Fc Mouse IgG3 20 μg pfuse-mg3sp -
pFUSE-SEAP-rFc Rabbit IgG 20 μg pfuse-rsp -
pFUSE-SEAP-rtFc Rat IgG2b 20 μg pfuse-rtsp -
* The Fc region of mouse IgG1 appears to have a very low affinity for Protein G unlike the entire IgG1 molecule. Therefore, protein G may not be suitable for the
purification of mIgG1-Fc-fusion proteins. Protein A may be used after optimization of the purification conditions. High salt concentration and alkaline pH are expected
to increase affinity (Nagaoka T. et al, 2003 Protein Eng. 16(4):243:245).
** pFUSE-hIgG1e3 plasmids featuring the IgG1e3 engineered Fc are sold by InvivoGen under a limited license from Cambridge Enterprise Limited (International
Publication Number WO 99/58572).
www.invivogen.com/fc-fusions

pBROAD & pWHERE - Optimized Vector for Rodent Transgenesis
• pBROAD plasmids were designed for the expression of your favorite gene in virtually all tissues of new transgenic mice and rats. pBROAD
plasmids feature the ubiquitous ROSA26 promoter.
• pWHERE plasmid was specifically designed for the determination of temporal expression and tissue distribution of your promoter of
interest, cloned within an insulated LacZ cassette, in transgenic mice and rats.
Features and Benefits
pBROAD
pBROAD plasmids feature the ROSA26 promoter, a high CpG content
and TATA-less promoter. It directs in vivo expression of the transgene in
most tissue types including cells of the developing germ line and
hematopoietic lineage 1, 2 . It is also very effective in vitro in a very broad
range of mammalian cell lines.
• pBROAD2 features the human ROSA26 promoter.
• pBROAD3 features the mouse ROSA26 promoter.
pBROAD plasmids are available with a multiple cloning site (MCS)
containing several commonly used restriction sites, for convenient cloning
of your gene of interest. pBROAD plasmids are also available with the
synthetic lacZDCpGnls gene, a CpG-free allele of the LacZ gene with a
nuclear localization signal.
The E. coli region can be easily removed by using the Pac I restriction
enzyme.
pWHERE
pWHERE plasmid features two murine H19 insulators 3, 4 , that flank on
each side the LacZ transcription unit. These insulators are expected to
protect the integrated transcriptional LacZ unit from negative as well as
positive influences from neighboring sequences. Thus pWHERE provides
an optimized environment to study the temporal and tissue distribution
of a promoter of interest in mice and rats.
pWHERE contains the lacZDCpGnls gene, a CpG-free allele to eliminate
interference of CpG methylation on expression (see page 138). A multiple
cloning site with many restriction sites is located upstream of the
lacZDCpGnls gene for convenient cloning of your own promoter or a
promoter chosen from InvivoGen’s list (see pages 40-41).
The E. coli region is flanked on either side by Pac I restriction sites to
facilitate its excision.
1. Kisseberth WC. et al. 1999. Ubiquitous expression of marker transgenes in mice and
rats. Dev Biol.214:128-38. 2. Farley FW. et al. 2000. Widespread recombinase expression
using FLPeR (flipper) mice. Genesis. 28:106-10. 3. Kaffer CR. et al. 2000. A transcriptional
insulator at the imprinted H19/Igf2 locus. Genes Dev. 14:1908-19. 4 Bell AC. & Felsenfeld
G. 2000. Methylation of a CTCF-dependent boundary controls imprinted expression of
the Igf2 gene. Nature. 405:482-485.
Contents and Storage
pBROAD and pWHERE plasmids are provided as 20 μg of lyophilized
DNA. Plasmids are shipped at room temperature. Store at -20°C.
pBROAD and pWHERE plasmids are also available in a kit that contains:
- pBROAD kit: 20 μg pBROAD-mcs plasmid, 20 μg pBROAD-LacZ
containing the lacZDCpGnls gene, 4 pouches of E. coli Fast-Media ® Amp
- pWHERE kit: - 20 μg pWHERE plasmid, 10 μg pWHERE-rEF1 containing
the rat EF-1a promoter, 4 pouches of E. coli Fast-Media ® Amp (2 TB and
2 Agar, see pages 44-45)
Pac I
Pac I
www.invivogen.com/rodent-transgenesis
Pac I
MCS
or
LacZnls
Pac I
MCS
PRODUCT QTY CAT. CODE
pBROAD2-mcs 20 μg pbroad2
pBROAD2-LacZnls 20 μg pbroad2-lacz
pBROAD2 Kit 1 kit kbroad2
pBROAD3-mcs 20 μg pbroad3
pBROAD3-LacZnls 20 μg pbroad3-lacz
pBROAD3 Kit 1 kit kbroad3
pWHERE 20 μg pwhere
pWHERE Kit 1 kit k-where
31
MAMMALIAN EXPRESSION VECTORS 2

MAMMALIAN EXPRESSION VECTORS 2
pMONO & pSELECT - Expression of One Gene of Interest
pMONO & pSELECT plasmids are specifically designed for strong and constitutive expression of a gene of interest in a wide variety of mammalian
cell lines. They allow the selection of stable transfectants and offer a choice of selectable markers for both E. coli and mammalian cells.
• pMONO plasmids contain a unique transcription unit that drives the expression of the gene of interest and the selectable marker through
an internal ribosome entry site (IRES). This dual gene expression system ensures that stable clones express the gene of interest.
• pSELECT plasmids contain two transcription units, the first drives the expression of the gene of interest and the second drives the
expression of the resistance gene.
Features and Benefits
Strong and Ubiquitous Expression of the Transgene
- pMONO plasmids feature the SV40/FerH/mEF-1a promoter composed
of the ferritin heavy chain (FerH) core promoter fused at its 5’ end to
the SV40 enhancer, and at its 3’ end to the intron-containing 5’UTR of
the mouse elongation factor 1 alpha gene.
- pSELECT plasmids feature the strong EF-1a/HTLV composite promoter
that combines the elongation factor 1 alpha core promoter and the
5’untranslated region of the Human T-cell Leukemia Virus.
Variety of Selection Options
- pMONO plasmids are selectable with blasticidin, hygromycin,
kanamycin/G418 and Zeocin . All resistance genes are cloned
downstream of the IRES from the Foot and Mouth Disease Virus (FMDV),
eliminating the need of a second mammalian promoter. The FMDV IRES
is in tandem with the constitutive bacterial EM7 promoter conferring
resistance both in E. coli and mammalian cells.
- pSELECT plasmids are selectable with blasticidin, hygromycin,
kanamycin/G418, puromycin and Zeocin . These selectable markers are
driven by the CMV promoter in tandem with the bacterial EM7 promoter
for selection in both E. coli and mammalian cells. pSELECT is also available
with the GFPzeo fusion gene that combines the GFP reporter gene and
the Zeocin resistance gene.
Multiple Cloning Site or Reporter Gene
- pMONO plasmids carry a multiple cloning site (MCS) downstream of
the SV40/FerH/mEF-1a promoter for convenient cloning of a gene of
interest. pMONO plasmids are also available with a GFP allele and can
be used as control vectors.
- pSELECT plasmids contain an MCS downstream of the EF-1a/HTLV
promoter or the LacZ reporter gene as a control.
Contents and Storage
pMONO and pSELECT plasmids are provided as 20 μg of lyophilized DNA.
Product is shipped at room temperature and should be stored at -20°C.
Plasmid is stable up to one year when properly stored.
pMONO and pSELECT plasmids are provided with 4 pouches of E. coli Fast-
Media ® containing the appropriate selective antibiotic (2 TB and 2 Agar).
32
Related Products
Blasticidin, page 17 HygroGold , see page 18
Puromycin, page 17 Zeocin , page 18
Fast-Media ® , pages 44-45
PRODUCT QTY
www.invivogen.com/gene-expression
CAT. CODE
(MCS)
MCS
or
GFP
CAT. CODE
(GFP)
pMONO-blasti-mcs 20 μg pmonob-mcs pmonob-gfp
pMONO-hygro-mcs 20 μg pmonoh-mcs pmonoh-gfp
pMONO-neo-mcs 20 μg pmonon-mcs pmonon-gfp
pMONO-zeo-mcs 20 μg pmonoz-mcs pmonoz-gfp
PRODUCT QTY
CAT. CODE
(MCS)
MCS
or
LacZ
CAT. CODE
(LacZ)
pSELECT-blasti 20 μg psetb-mcs psetb-lacz
pSELECT-hygro 20 μg pseth-mcs pseth-lacz
pSELECT-neo 20 μg psetn-mcs psetn-lacz
pSELECT-puro 20 μg psetp-mcs psetp-lacz
pSELECT-zeo 20 μg psetz-mcs psetz-lacz
pSELECT-GFPzeo 20 μg psetgz-mcs psetgz-lacz

pSELECT-Tag - Expression of a GFP-, HA- or His-Tagged Gene
pSELECT-Tag is a new family of expression plasmids designed to generate tagged proteins in order to facilitate their detection and/or purification.
pSELECT-Tag features three well-known tags: the green fluorescent protein (GFP) gene, the human influenza hemagglutinin (HA) epitope and
the polyhistidine (His) tag. pSELECT-Tag plasmids allow to add the tag either at the N or C terminus of the protein of interest.
Features and Benefits
Three Commonly-Used Tags
• GFP-Tag
• HA-Tag
• His-Tag
N-Tagged or C-Tagged Protein
- N-terminal tag: the tag encompasses the Start Codon and is followed
by a multiple cloning site (MCS).
- C-terminal tag: the tag is cloned downstream of a multiple cloning site
and followed by a Stop codon.
Many Selectable Markers Available
pSELECT plasmids come with a variety of selectable markers that confer
antibiotic resistance in both E. coli and mammalian cells.
Applications
• GFP-Tag:- Visualization of the spatial and temporal localization of the
tagged protein by fluorescence microscopy
• HA-Tag: - Detection of the tagged protein by immunocytochemistry,
immunoprecipitation or Western blotting
- Purification of the tagged protein by affinity chromatography
• His-Tag: - Purification of the tagged protein using an NI-NTA column
Contents and Storage
pSELECT-Tag plasmids are provided as 20 μg of lyophilized DNA. Product
is shipped at room temperature and should be stored at -20°C. Plasmid is
stable up to one year when properly stored.
pSELECT-Tag plasmids are provided with 4 pouches of E. coli Fast-Media ®
containing the appropriate selective antibiotic (2 TB and 2 Agar).
Related Products
Blasticidin, page 17 Anti-HA Tag Antibody, page 73
Zeocin , page 18 Fast-Media ® , pages 44-45
www.invivogen.com/gene-expression
EM7 Antibio
R pAn pAn
PRODUCT
GFP Tag
QTY
CAT. CODE
(N-Tag)
CAT. CODE
(C-Tag)
pSELECT-GFP-blasti 20 μg psetb-ngfp psetb-cgfp
pSELECT-GFP-zeo
HA Tag
20 μg psetz-ngfp psetz-cgfp
pSELECT-HA-blasti 20 μg psetb-nha psetb-cha
pSELECT-HA-zeo
His Tag
20 μg psetz-nha psetz-cha
pSELECT-His-blasti 20 μg psetb-nhis psetb-chis
pSELECT-His-zeo 20 μg psetz-nhis psetz-chis
33
MAMMALIAN EXPRESSION VECTORS 2

MAMMALIAN EXPRESSION VECTORS 2
pVITRO - Expression of Two Genes of Interest in vitro
pVITRO is a family of plasmids developed mainly for in vitro studies. They allow the ubiquitous and constitutive co-expression of two genes of
interest. pVITRO plasmids can be stably transfected in mammalian cells and the genes of interest are expressed at high levels. Each pVITRO
plasmid is available with either two multiple cloning sites or two reporter genes.
Features and Benefits
Gene Combinations in a Single Plasmid
Co-expression of two or more genes from a single vector is more efficient
and convenient than using two separate vectors. pVITRO are multigenic
plasmids that contain two distinct transcription units (TU).
Strong and Constitutive Expression of Two Transgenes
Each pVITRO plasmid features two constitutive promoters that drive high
levels of expression from two separate TU in a large number of
mammalian cell lines. Transcriptional interference is minimized by using
promoters of different origins or promoters that are coordinately
activated (i.e. ferritin promoters), and strong polyadenylation signals
(polyA).
• pVITRO1 plasmids carry two elongation factor 1 alpha (EF-1a)
promoters, from rat and mouse origins combined to the CMV and SV40
enhancers respectively.
• pVITRO2 plasmids contain two human ferritin composite promoters,
FerH (heavy chain) and FerL (light chain), combined to the SV40 and
CMV enhancers respectively. To eliminate the iron regulation, their 5’UTRs
have been replaced by the 5’UTR of the mouse and chimpanzee EF-1a
genes.
Single Selection Marker for E. coli and Mammalian Cells
pVITRO plasmids are available with different selectable markers that are
active both in E. coli and mammalian cells: bsr (blasticidin resistance), hph
(hygromycin resistance), neo (kanamycin/G418 resistance). In bacteria, the
resistance gene is expressed from the E. coli EM7 promoter. In mammalian
cells, it is transcribed from the promoter located 3’ of the Ori, as a
polycistronic mRNA and translated through the IRES of the Foot and
Mouth Disease Virus.
Available with Two MCS or Two Reporter Genes
pVITRO-mcs plasmids contain two multiple cloning sites (MCS) for the
convenient cloning of cDNAs.
pVITRO-GFP/LacZ express the GFP and LacZ reporter genes, and can
be used as controls. As the reporter genes are flanked by unique
restriction sites, they can be used for the cloning of open reading frames
that can be chosen from InvivoGen’s Gene A-List (see page 36 or visit
our website www.invivogen.com/orfs).
Contents and Storage
Each pVITRO plasmid is provided as 20 μg of lyophilized DNA. Product
is shipped at room temperature and should be stored at -20°C. Plasmid
is stable up to one year when properly stored.
Each pVITRO is provided with 4 pouches of the appropriate E. coli Fast-
Media ® (2 TB and 2 Agar).
34
Related Products
Blasticidin, page 17 G418, page 17
HygroGold , see page 18 Puromycin, page 17
Zeocin , page 18 E. coli Fast-Media ® , pages 44-45
MCS1
or
GFP
MCS1
or
GFP
www.invivogen.com/gene-expression
MCS 1 5’- Bsp EI, Bst 1107I, Bam HI, Bsi WI, Avr II -3’
MCS 2 5’- Age I, Eco RV, Bgl II, Bsr GI, Nhe I -3’
MCS2
or
LacZ
MCS2
or
LacZ
MCS 1 5’- Age I, Eco RV, Bam HI, Mlu I, Cla I, Sal I, Avr II -3’
MCS 2 5’- Sgr AI, Fsp I, Bgl II, Eco RI, Bst BI, Xho I, Nhe I-3’
PRODUCT QTY CAT. CODE
pVITRO1-blasti-mcs 20 μg pvitro1-bmcs
pVITRO1-blasti-GFP/LacZ 20 μg pvitro1-bgfplacz
pVITRO1-hygro-mcs 20 μg pvitro1-mcs
pVITRO1-hygro-GFP/LacZ 20 μg pvitro1-gfplacz
pVITRO1-neo-mcs 20 μg pvitro1-nmcs
pVITRO1-neo-GFP/LacZ 20 μg pvitro1-ngfplacz
pVITRO2-blasti-mcs 20 μg pvitro2-bmcs
pVITRO2-blasti-GFP/LacZ 20 μg pvitro2-bgfplacz
pVITRO2-hygro-mcs 20 μg pvitro2-mcs
pVITRO2-hygro-GFP/LacZ 20 μg pvitro2-gfplacz
pVITRO2-neo-mcs 20 μg pvitro2-nmcs
pVITRO2-neo-GFP/LacZ 20 μg pvitro2-ngfplacz

pVIVO - Expression of Two Genes of Interest in vivo
pVIVO is a family of plasmids specifically designed for in vivo experiments. They allow strong and sustained co-expression of two genes of
interest in many tissues and organs. Each pVIVO is available with a pair of multiple cloning sites or a pair of reporter genes.
Features and Benefits
Concomitant Expression of Two Genes of Interest
pVIVO plasmids contain two separate transcription units (TU), each
consisting of a strong promoter and an efficient polyadenylation signal. Both
promoters in each pVIVO plasmid were chosen for their ability to work
simultaneously, eliminating the potential risk of transcriptional interferences
between the two TUs.
• pVIVO1 plasmids contain two glucose regulated protein (GRP)
promoters coupled with either the SV40 or CMV enhancer. The human
GRP94 and hamster GRP78 promoters drive high levels of expression in
normal conditions and are induced in stress conditions prevailing inside
tumors, such as glucose deprivation and hypoxia.
• pVIVO2 plasmids contain two human ferritin composite promoters,
FerH (heavy chain) and FerL (light chain), combined to the SV40 and
CMV enhancers respectively. To eliminate the iron regulation, their 5’UTRs
have been replaced by the 5’UTR of the mouse and chimpanzee EF-1a
genes.
pVIVO plasmids carry the hygromycin resistance gene for selection and
amplification in E. coli. pVIVO plasmids do not contain a selectable marker
for mammalian cells.
Sustained Expression of the Transgenes
Both promoters in pVIVO plasmids are of mammalian origin and can yield
longer lasting expression of the transgenes in vivo compared to viral
promoters. Indeed, viral DNA sequences are recognized by the host
immune system leading to their inactivation usually through methylation.
To further increase the duration of expression, immunostimulatory
sequences known as CpG motifs, which are present in high numbers in
bacterial DNA, were removed from the plasmid backbone. These specific
motifs, found in E. coli hygromycin resistance (hph) and b-galactosidase
(lacZ) genes, were eliminated by chemically synthesizing a new allele of
these genes without altering the protein sequence (see page 138).
Available with Two MCS or Two Reporter Genes
pVIVO-mcs plasmids contain two multiple cloning sites (MCS) for the
convenient cloning of cDNAs.
pVIVO-GFP/LacZ express the GFP and LacZ reporter genes, and can be
used as controls or for the cloning of open reading frames.
Contents and Storage
Each pVIVO plasmid is provided as 20 μg of lyophilized DNA. Product
is shipped at room temperature and should be stored at -20°C.
Plasmid is stable up to one year when properly stored.
Each pVIVO is provided with 4 pouches of E. coli Fast-Media ® Hygro
(2 TB and 2 Agar).
Related Products
Hygromycin B, see page 18 E. coli Fast-Media ® , pages 44-45
MCS1
or
GFP
MCS 1 5’- Age I, Bsp HI, Xba I, Hind III, Bst 1107I, Avr II -3’
MCS 2 5’- Ngo MI, Nco I, Bgl II, Eco RI, Nhe I-3’
MCS1
or
GFP
www.invivogen.com/gene-expression
SV40 hph-∆CpG Ori pAn
MCS 1 5’- Bsp HI, Xba I, Bsr GI, Bst 1107I, Avr II -3’
MCS 2 5’- Nco I, Bam HI, Eco RI, Eco RV, Nhe I-3’
MCS2
or
LacZ
MCS2
or
LacZ
PRODUCT QTY CAT. CODE
pVIVO1-mcs 20 μg pvivo1-mcs
pVIVO1-GFP/LacZ 20 μg pvivo1-gfplacz
pVIVO2-mcs 20 μg pvivo2-mcs
pVIVO2-GFP/LacZ 20 μg pvivo2-gfplacz
35
MAMMALIAN EXPRESSION VECTORS 2

MAMMALIAN EXPRESSION VECTORS 2
Gene A-List - Human and Murine ORFs
Gene A-List is a collection of high quality, full-length sequenced human and mouse open reading frames (ORFs) available in an expression
vector ready-to-use for protein expression. All ORFs are cloned downstream of a strong and ubiquitous mammalian promoter which makes
these clones suitable for expression and functional studies in various mammalian cell lines.
Features and Benefits
Full-length Sequenced Open Reading Frames
InvivoGen provides a large collection of human and murine genes, fully sequenced. These genes are provided as open reading frames (ORFs) from the ATG
to the Stop codon, excluding introns and untranslated regions. As the ORFs are amplified from cDNA banks, a variant form is sometimes obtained. Most
of the variations have been reported in Genbank. Furthermore for cloning convenience, a neutral amino acid is sometimes added immediately after the
Start codon in order to create a suitable cloning site.
Among the Gene A-List , some genes encode proteins that are naturally secreted. Their sequence include their native signal sequence which is generally
located at the 5' end of the coding sequence. Other genes that code for engineered proteins, such as particular fragments of longer proteins (i.e. angiostatic
proteins) may include the signal sequence of the human IL-2 gene between the ATG and the second codon to allow their secretion.
Suitable for Expression in Mammalian Cells
Each ORF is cloned in a mammalian expression cassette consisting of a potent and ubiquitous composite promoter and the strong SV40 polyadenylation
signal. Each ORF is flanked by a unique restriction site at the 5’ end and 3’ end to facilitate its subcloning into another vector.
The ORFs are supplied in two different plasmid backbones:
• pORF, a plasmid selectable only in E. coli with ampicillin
• pBLAST/pUNO, a plasmid selectable in mammalian cells and in E. coli with blasticidin.
Search online the Gene A-List
You can easily find your ORF of interest in the Gene A-List at www.invivogen.com/ORF.php. You can browse the entire list based on HUGO-approved
gene symbol (and alias) and function. All sequences are available online or can be emailed upon request.
36
MAJOR GENE FAMILIES EXAMPLES
Adaptor Genes MyD88, TIRAP, TRAM, TRIF/TICAM1
Angiogenic & Angiostatic Genes ANGPT-1, NRP1, ANGIO, ENDO, FLK1, FLT1
Apoptotic & Anti-Apoptotic Genes BAD, Bcl-XS, BIK, Bcl-XL, Bcl-2, Survivin
Autophagy Genes Atg5, Beclin-1, LC3b
Cellular Matrix Genes Caveolin-1, Decorin
Chemokine & Chemokine Receptor Genes IP-10, LIX, MIG, MIP-1, PF4, SDF1, CCR4, CCR5, CXCR3
Chromatin-Remodeling Genes DNMT1, HDAC1, KAISO, SUV39H1
Connexin Genes Cx26, Cx32, Cx43
Co-Receptor Genes CD14, LBP, MD1, MD2
Co-Stimulatory / CD Genes 4-1BB, B7.1, B7.2, OX40L, CD4, CD8, CD40, CD70
Cytokine & Cytokine Suppressor Genes GM-CSF, IL-2, IL-10, IL-12, LIF, TNF-a, SOCS-1, SOCS-2
Cytolytic Genes Granulysin, Granzyme, Perforin
Cytotoxic / Suicide Genes CD, UPRT, HSV1-TK
DAMP & DAMP Receptor Genes HMGB1, S100A8, S100A9, SAA1, Mincle, RAGE, TREM-1
Growth Factors EGF, FGF, HGF, IGF, TGF-b, VEGF
Hematopoietic Genes EPO, HOXB4, TPO
Immune Receptors FcgR, IL-1R, IL-6R, IL-10R, TNFRSF
Inhibitors of Differentiation Id1, Id2, Id3, Id4
Interferon & Interferon Signaling Genes IFN-a, IFN-b, IFN-g, IRF1, IRF3, IRF5, IRF7
Pattern Recognition Receptors TLR1-13, NOD1, NOD2, NLRP3, MDA-5, RIG-I, Dectin-1
Reprogramming Factors (IPSC) Klf4, Lin28, c-Myc, Nanog, Oct4, Sox2
Signaling Genes AKT, FADD, IPS-1, IRAK, RIP1, STAT, TAK1, TBK1, TRAF6
Signaling Inhibitors A20, Bcl-3, IRAK-M, SIGGIR, SIKE, Tollip
Transcription Factors c-REL, IRF, NF-kB, SMAD, STAT
Tumor Antigens MAGE
Tumor Suppressors HRAS, KRAS, p21, p53, PTEN
www.invivogen.com/orfs

pORF
Description
pORF plasmids feature a large collection of native and fusion genes as
open reading frames (ORF). The expression of each ORF is under the
control of a strong and ubiquitous mammalian promoter. This promoter
consists of the elongation factor 1 alpha (EF-1a) core promoter combined
to the 5’UTR of either the human T cell leukemia virus (HTLV) type 1
long terminal repeat or the human eukaryotic initiation factor 4g (eIF-4g).
These two composite promoters yield similar expression levels.
pORF plasmids are selectable in bacteria with ampicillin. The bacterial
selection cassette can be replaced upon request with a selection cassette
that functions both in bacteria and mammalian cells. This cassette subcloned
from the pSELECT can express the resistance gene of your choice.
pORF plasmids containing a multiple cloning site (MCS) in place of an ORF
were designed to serve as controls and/or cloning vectors. The MCS features
commonly used restriction sites to allow convenient insertion of an ORF.
Contents and Storage
Each pORF plasmid is provided as a lyophilized transformed E. coli strain
on a paper disk. Transformed strains are shipped at room temperature and
should be stored at -20°C. Lyophilized E. coli cells are stable for at least one
year when properly stored.
Each pORF is provided with 4 pouches of E. coli Fast-Media ® Amp (2 TB
and 2 Agar).
pBLAST & pUNO
Description
pBLAST/pUNO plasmids feature a wide choice of native and fusion
genes, mainly involved in innate immunity. The gene of interest is under
the control of the same promoter as the pORF plasmids, that is EF-
1a/HTLV or EF-1a/eIF-4g, allowing for strong expression in mammalian
cells.
pBLAST/pUNO plasmids contain the blasticidin resistance gene (bsr)
under the control of a mammalian promoter in tandem with a
bacterial promoter. This allows the amplification of the plasmid in E.
coli and the selection of stable clones in mammalian cells. Blasticidin is
a potent antibiotic that permits the selection of transfectants in only
a few days.
pBLAST-mcs and pUNO-mcs plamids were designed to serve as control
and/or cloning vectors. They carry a multiple cloning site (MCS) that
features commonly used restriction sites to allow convenient insertion of
an open reading frame.
Contents and Storage
pBLAST plasmids are provided as 20 μg of lyophilized DNA. pUNO
plasmids are provided as lyophilized transformed E. coli strains on a
paper disk. Products are shipped at room temperature. Store at -20°C.
Plasmids and lyophilized E. coli cells are stable up to one year when
properly stored.
Each pBLAST and pUNO is provided with 4 pouches of E. coli
Fast-Media ® Blas (2 TB and 2 Agar).
www.invivogen.com/orfs
Related Products
Fast-Media ® Amp, page 45
pSELECT, page 32
PRODUCT QTY CAT. CODE
pORF- E. coli disk porf-
pORF-mcs E. coli disk porf-mcs
* Visit www.invivogen.com/ORF.php for complete ordering information
Related Products
Fast-Media ® Blas, page 45
Blasticidin, page 17
PRODUCT QTY CAT. CODE
pBLAST- 20 μg pbla-
pBLAST-mcs 20 μg pbla-mcs
pUNO- E. coli disk puno-
pUNO-mcs E. coli disk puno-mcs
* Visit www.invivogen.com/ORF.php for complete ordering information
37
MAMMALIAN EXPRESSION VECTORS 2

MAMMALIAN EXPRESSION VECTORS 2
PromTest - Ten Promoters to Choose From
Promoters regulate differently gene expression depending on the cellular context. To help you determine easily and rapidly the best promoter
for your cell line, InvivoGen offers PromTest , a collection of ten ubiquitous promoters driving the GFP reporter gene.
➥ Ten ready-to-use promoters with different levels of expression
➥ Drive GFP expression for convenient monitoring of promoter strength
➥ Cost-effective
Description
PromTest is a collection of ten ubiquitous composite promoters provided in the pDRIVE5-GFP
plasmid. These composite promoters were generated by assembling enhancers, core promoters
and 5’UTRs of different origins. The activity of each combination depends on the cellular context
(see graphs).
pDRIVE5-GFP features a GFP reporter gene for convenient monitoring of promoter activity. The
strength of each promoter can be assessed qualitatively by fluorescence microscopy and
quantitatively using a fluorometer or flow cytometry.
pDRIVE5-GFP plasmids are selectable in E. coli with Zeocin .
PromTest contains 5 μg of each plasmid, enough for multiple transfections using your favorite
reagent or technique. pDRIVE5-GFP plasmids can be amplified in any common E. coli laboratory
strains. They are also available individually as 20 μg high-quality endotoxin-free DNA.
Once you have determined the best promoter for your cell line and application, you can either
replace the GFP gene with your gene of interest, or subclone the promoter into another plasmid
with mammalian selection, such as InvivoGen’s pSELECT.
Evaluation of PromTest in different cell lines: HeLa (human cervical cancer) and CHO (chinese hamster ovary) cells were transiently transfected with each of the 10 pDRIVE5-
GFP plasmids of PromTest using LyoVec (cat. code: lyec-12). The strength of the various promoters was analyzed by flow-cytometry 48h after transfection.
Abbreviations: chEF1, chimpanzee elongation factor 1 alpha; hAlda, human aldolase A; hCMV, human cytomegalovirus; hEF1, human elongation factor 1 alpha; hFerL, human ferritin
light chain; HTLV, human T lymphocyte virus; mCMV, mouse cytomegalovirus; mTyr, mouse tyrosinase; SV40, simian virus 40.
Contents and Storage
PromTest contains 10 plasmids, each provided as 5 μg lyophilized DNA
Plasmids can also be purchased individually as 20 μg high-quality
endotoxin-free DNA. Plasmids are shipped at room temperature.
Store at -20°C. Plasmids are stable up to one year when properly stored.
38
Plasmid Enhancer Core
Promoter
pDRIVE5-GFP-1
pDRIVE5-GFP-2
pDRIVE5-GFP-3
pDRIVE5-GFP-4
pDRIVE5-GFP-5
pDRIVE5-GFP-6
pDRIVE5-GFP-7
pDRIVE5-GFP-8
pDRIVE5-GFP-9
pDRIVE5-GFP-1 0
hCMV
mCMV
SV40
mTyr
hCMV
SV40
hCMV
mCMV
SV40
hAldA
hEF1
hEF1
hEF1
hEF1
hCMV
hCMV
hFerL
hFerL
hFerL
hFerL
5’ UTR
HTLV
HTLV
HTLV
HTLV
HTLV
HTLV
chEF1
chEF1
chEF1
chEF1
www.invivogen.com/promoters
PRODUCT QTY CAT. CODE
PromTest 10 x 5 μg prom-test
pDRIVE5-GFP-n 20 μg pdv5-gfp

Prom A-List - Human and Rodent Promoters
Prom A-List is an expanding collection of native and composite promoters provided in the pDRIVE plasmid. Promoters are valuable tools to
study the expression of a given gene both in vitro and in vivo. Now with pDRIVE, you can choose to express your gene of interest at high or
low levels, ubiquitously or specifically, and in a constitutive or inducible manner.
Features and Benefits
An Expanding Collection of Promoters
Each promoter is fully sequenced and its expression tested in different
cell lines.
Native or Composite Promoters
• Native promoters, also called minimal promoters, consist of a single
fragment from the 5’ region of a given gene. Each of them comprises a
core promoter and its natural 5’UTR. In some cases, the 5’UTR contains
an intron.
• Composite promoters combine promoter elements of different origins
(e.g. SV40 enhancer/AFP promoter) or were generated by assembling a
distal enhancer with a minimal promoter of the same origin (e.g. CEA
enhancer/promoter).
Various Expression Patterns
• Ubiquitous Promoters, strongly active in a wide range of cells, tissues
and cell cycles.
• Tissue Specific Promoters, active in a specific type of cells or tissues.
• Tumor Specific Promoters, active specifically in tumor cells.
Easy Subcloning of the Promoters
In every pDRIVE, several unique restriction sites flank the promoter (a
multiple cloning site at the 5’ end and Nco I or Bsp HI at the 3’ end) in
order to excise the promoter easily. These restriction sites are compatible
with many other enzymes, thus facilitating cloning. Other restriction sites
may be introduced by performing PCR using primers containing the
restriction sites of interest.
Two Backbones with Different Reporter Genes
In each pDRIVE, the promoter drives the expression of a reporter gene
to facilitate the evaluation of its activity. Two different reporter genes are
available in two different backbones:
• LacZ in pDRIVE: pDRIVE plasmids contain the LacZ reporter gene
which expression can be determined using chromogenic (see page 12),
luminescent or histochemical detection. They carry the Sh ble gene
conferring resistance to Zeocin downstream of the EM7 bacterial
promoter.
• SEAP in pDRIVE5-SEAP: pDRIVE5-SEAP plasmids feature an engineered
secreted embryonic alkaline phosphatase (SEAP) reporter gene. SEAP
expression levels can be assayed with chromogenic (see pages 13-15) or
luminescent methods.
Transient Expression in vitro
pDRIVE plasmids are selectable with the antibiotic Zeocin in E. coli.They
can be used for transient transfection experiments in mammalian cells in
vitro or for expression studies in vivo.
Related Products
Custom-pDRIVE, page 162 Fast-Media ® Zeo, page 45
Contents and Storage
www.invivogen.com/promoters
Each pDRIVE plasmid is provided as lyophilized transformed E. coli strain.
Transformed bacteria are shipped at room temperature. Store at -20°C.
Transformed bacteria are stable for at least 1 year when properly stored.
Each pDRIVE is provided with 4 pouches of E. coli Fast-Media ® Zeo (2 TB
and 2 Agar).
PRODUCT QTY CAT. CODE*
pDRIVE-LacZ E. coli disk pdrive-
pDRIVE-LacZ E. coli disk pdrive-
pDRIVE5-SEAP E. coli disk pdrive5s-
pDRIVE5-SEAP
* See tables pages 40-41
E. coli disk pdrive5s-
39
MAMMALIAN EXPRESSION VECTORS 2

MAMMALIAN EXPRESSION VECTORS 2
b-Act Beta-Actin Ubiquitous Human 2387 bp pdrive-hbact pdrive5s-hbact
EF-1a Elongation Factor 1 alpha Ubiquitous Chimpanzee 1365 bp pdrive-chef1 pdrive5s-chef1
Mouse 1315 bp pdrive-mef1 pdrive5s-mef1
Rat
1314 bp pdrive-ref1 pdrive5s-ref1
EGR1 Early Growth Response 1 Ubiquitous, inducible by radiation Human 1062 bp pdrive-hegr1 pdrive5s-hegr1
eIF4A1 Eukaryotic Initiation Factor 4A1 Ubiquitous Human 523 bp pdrive-heif4a1 pdrive5s-heif4a1
Mouse 482 bp pdrive-meif4a1 pdrive5s-meif4a1
FerH Ferritin Heavy Chain Ubiquitous Human 370 bp pdrive-hferh pdrive5s-hferh
FerL Ferritin Light Chain Ubiquitous Human 429 bp pdrive-hferl pdrive5s-hferl
GAPDH Glyceraldehyde 3-phosphate Dehydrogenase Ubiquitous Human 794 bp pdrive-hgapdh pdrive5s-hgapdh
GRP78 Glucose-Regulated Protein 78 Ubiquitous, inducible by stress Human 531 bp pdrive-hgrp78 pdrive5s-hgrp78
Mouse 545 bp pdrive-mgrp78 pdrive5s-mgrp78
Rat
679 bp pdrive-rgrp78 pdrive5s-rgrp78
GRP94 Glucose-Regulated Protein 94 Ubiquitous, inducible by stress Human 701 bp pdrive-hgrp94 pdrive5s-hgrp94
Mouse 696 bp pdrive-mgrp94 pdrive5s-mgrp94
Rat
731 bp pdrive-rgrp94 pdrive5s-rgrp94
HSP70 Heat Shock Protein 70 Ubiquitous, inducible by heat Human 464 bp pdrive-hhsp70 pdrive5s-hhsp70
Mouse 1041 bp pdrive-mhsp70 pdrive5s-mhsp70
b-Kin Beta-Kinesin Ubiquitous Human 539 bp pdrive-hbkin pdrive5s-hbkin
PGK1 Phosphoglycerate Kinase 1 Ubiquitous Mouse 1428 bp pdrive-mpgk pdrive5s-mpgk
ROSA Rosa Ubiquitous Human 2815 bp pdrive-hrosa pdrive5s-hrosa
Mouse 1926 bp pdrive-mrosa pdrive5s-mrosa
Ubi B Ubiquitin B Ubiquitous Human 1092 bp pdrive-hubiquitinb pdrive5s-hubiquitinb
Ubiquitous Composite Promoters
b-Act/RU5’ Beta-Actin prom / HTLV 5’UTR Ubiquitous Human 1785 bp pdrive-hbactru5 pdrive-hbactru5
EF-1a/RU5’ Elongation Factor 1 prom / HTLV 5’UTR Ubiquitous Chimpanzee 672 bp pdrive-chef1ru5 pdrive5s-chef1ru5
Rat
630 bp pdrive-ref1ru5 pdrive5s-ref1ru5
FerH/RU5’ Ferritin H prom / HTLV 5’UTR Ubiquitous Human 457 bp pdrive-hferhru5 pdrive5s-hferhru5
SV40/FerH SV40 enh / Ferritin H prom / EF-1a 5’UTR Ubiquitous Human / mouse 1423 bp pdrive-sv40ferhef1 pdrive5s-sv40ferhef1
FerL/RU5’ Ferritin L prom / HTLV 5’UTR Ubiquitous Human 537 bp pdrive-hferlru5 pdrive5s-hferlru5
CMV/FerL CMV enh / Ferritin L prom / EF-1a 5’UTR Ubiquitous Human / chimp. 1666 bp pdrive-cmvferlef1 pdrive5s-cmvferlef1
Tissue-Specific Native Promoters
B29 Immunoglobulin Beta B cells Human 1219 bp pdrive-hb29 pdrive5s-hb29
Mouse 1235 bp pdrive-mb29 pdrive5s-mb29
CD14 Monocyte Receptor for Bacterial LPS Monocytic cells Human 612 bp pdrive-hcd14 pdrive5s-hcd14
CD43 Leukosialin, Sialophorin Leukocytes and platelets Human 775 bp pdrive-hcd43 pdrive5s-hcd43
CD45 Leukocyte Common Antigen (LCA) Hematopoietic cells Human 856 bp pdrive-hcd45 pdrive5s-hcd45
CD68 Human Homolog of Macrosialin Macrophages Human 658 bp pdrive-hcd68 pdrive5s-hcd68
Mouse 811 bp pdrive-mcd68 pdrive5s-mcd68
Desmin Desmin Muscle Mouse 983 bp pdrive-mdesmin pdrive5s-mdesmin
Elastase Elastase1 Pancreatic acinar cells Rat 264 bp pdrive-relastase pdrive5s-relastase
Endoglin Endoglin Endothelial cells Human 886 bp pdrive-hendoglin pdrive5s-hendoglin
FN Fibronectin Differentiating cells, healing tissues Human 1037 bp pdrive-hfibronectin pdrive5s-hfibronectin
Flt-1 VEGF Receptor 1 Endothelial cells Human 786 bp pdrive-hflt1 pdrive5s-hflt1
GFAP Glial Fibrillary Acidic Protein Astrocytes Human 1675 bp pdrive-hgfap pdrive5s-hgfap
Mouse 1679 bp pdrive-mgfap pdrive5s-mgfap
Rat
1588 bp pdrive-rgfap pdrive5s-rgfap
40
PROMOTER GENE TISSUE DISTRIBUTION SPECIES SIZE
Ubiquitous Native Promoters
www.invivogen.com/promoters
CAT. CODE
(LacZ)
CAT. CODE
(SEAP)

PROMOTER GENE TISSUE DISTRIBUTION SPECIES SIZE
Tissue-Specific Native Promoters
CAT. CODE
(LacZ)
CAT. CODE
(SEAP)
GPIIb Integrin alpha IIb Megakaryocytes Human 928 bp pdrive-hgp2b pdrive5s-hgp2b
ICAM-2 Intercellular Adhesion Molecule 2 Endothelial cells Human 397 bp pdrive-hicam2 pdrive5s-hicam2
IFN-b Interferon beta Hematopoietic cells Mouse 214 bp pdrive-mifnb pdrive5s-mifnb
Mb Myoglobin Muscle Human 450 bp pdrive-hmb pdrive5s-hmb
Mouse 404 bp pdrive-mmb pdrive5s-mmb
NphsI Nephrin Podocytes Human 1233 bp pdrive-hnphs pdrive5s-hnphs
OG-2 Osteocalcin 2 Osteoblasts Mouse 225 bp pdrive-mog2 pdrive5s-mog2
SP-B Surfactant Protein B Lung Human 633 bp pdrive-hspb pdrive5s-hspb
Synapsin Synapsin Neurons Human 556 bp pdrive-hsynapsin pdrive5s-hsynapsin
WASP Wiskott-Aldrich Syndrome Protein Hematopoietic cells Human 506 bp pdrive-hwasp pdrive5s-hwasp
Tissue-Specific Composite Promoters
SV40/Alb SV40 enh / Albumin prom Liver Bovine 443 bp pdrive-sv40balb pdrive5s-sv40balb
Human 449 bp pdrive-sv40halb pdrive5s-sv40halb
SV40/CD43 SV40 enh / Leukosialin prom Leukocytes and platelets Human 1014 bp pdrive-sv40hcd43 pdrive5s-sv40hcd43
SV40/CD45 SV40 enh / Leukocyte Common Antigen prom Hematopoietic cells Human 1096 bp pdrive-sv40hcd45 pdrive5s-sv40hcd45
NSE-RU5’ Neuron-Specific Enolase prom / HTLV 5’UTR Mature neurons Rat 2043 bp pdrive-rnseru5 pdrive5s-rnseru5
Tumor-Specific Native Promoters
AFP Alpha-Fetoprotein Hepatocellular carcinoma Human 274 bp pdrive-hafp pdrive5s-hafp
CCKAR Cholecystokinin type A Receptor Pancreatic cancer Human 724 bp pdrive-hcckar pdrive5s-hcckar
CEA Carcinoembryonic Antigen Epithelial cancers Human 428 bp pdrive-hcea pdrive5s-hcea
c-erbB2 C-erbB2/neu Oncogen Breast and pancreas cancer Human 891 bp pdrive-herbb2 pdrive5s-herbb2
COX-2 Cyclo-oxygenase 2 Tumor
Human
Mouse
1568 bp
1104 bp
pdrive-hcox2
pdrive-mcox2
pdrive5s-hcox2
pdrive5s-mcox2
CXCR4 Receptor for Chemokine SDF1 Tumor Human 278 bp pdrive-hcxcr4 pdrive5s-hcxcr4
E2F-1 E2F Transcription Factor 1 Tumor Human 399 bp pdrive-he2f1 pdrive5s-he2f1
HE4 Human Epididymis Protein 4 Tumor Human 652 bp pdrive-hhe4 pdrive5s-hhe4
LP L-Plastin Tumor Human 2393 bp pdrive-hlp pdrive5s-hlp
MUC1 Mucin-like Glycoprotein Carcinoma cells Human 757 bp pdrive-hmuc1 pdrive5s-hmuc1
PSA Prostate Specific Antigen Prostate and prostate cancers Human 670 bp pdrive-hpsa pdrive5s-hpsa
Survivin Survivin Tumor Human 266 bp pdrive-hsurvivin pdrive5s-hsurvivin
TRP Tyrosinase Related Protein Melanocytes and melanoma Mouse 1201 bp pdrive-mtrp pdrive5s-mtrp
Tyr Tyrosinase Melanocytes and melanoma Mouse 335 bp pdrive-mtyr pdrive5s-mtyr
Tumor-Specific Composite Promoters
AFP/AFP Alpha-Fetoprotein enh-prom Hepatocellular carcinoma Human 2144 bp pdrive-afphafp pdrive5s-afphafp
SV40/AFP SV40 enh /Alpha-Fetoprotein prom Hepatocellular carcinoma Human 514 bp pdrive-sv40hafp pdrive5s-sv40hafp
CEA/CEA Carcinoembryonic Antigen enh-prom Epithelial cancers Human 1861 bp pdrive-ceahcea pdrive5s-ceahcea
PSA/PSA Prostate Specific Antigen enh-prom Prostate and prostate cancers Human 2261 bp pdrive-psahpsa pdrive5s-psahpsa
SV40/Tyr SV40 enh / Tyrosinase prom Melanocytes and melanoma Mouse 576 bp pdrive-sv40mtyr pdrive5s-sv40mtyr
Tyr/Tyr Tyrosinase enh-prom Melanocytes and melanoma Mouse 540 bp pdrive-tyrmtyr pdrive5s-tyrmtyr
www.invivogen.com/promoters
41
MAMMALIAN EXPRESSION VECTORS 2

MAMMALIAN EXPRESSION VECTORS 2
E. coli Competent Cells
InvivoGen’s competent E. coli strains have been designed to include genotypes that make them efficient and
convenient. These isogenic strains contain genetic modifications which are introduced using an elaborate
technique of homologous recombination. This technique allows the generation of targeted deletions or insertions
in the E. coli genome. The resulting strains are made chemically competent by an optimized procedure followed
by transformation efficiency verification and provided frozen (ChemiComp) or lyophilized (LyoComp).
Different Strains for Different Applications
InvivoGen provides two different E. coli competent strains that offer distinct
characteristics suitable for a particular application. These strains derive from
a common parental strain that features many useful genetic markers
making them also versatile strains.
• GT115 Ddcm, uidA::pir-116, DsbcC-sbcD mutant strain
F - mcrA D(mrr-hsdRMS-mcrBC) Φ80lacZDM15 DlacX74 recA1 rspL (StrA)
endA1 Ddcm uidA(DMluI)::pir-116 DsbcC-sbcD
• GT116 Ddcm, DsbcC-sbcD mutant strain
F - mcrA D(mrr-hsdRMS-mcrBC) Φ80lacZDM15 DlacX74 recA1 rspL (StrA)
endA1 Ddcm DsbcC-sbcD
The dcm gene encodes a DNA methylase that methylates the internal cytosine residues in the recognition sequence 5´-C*CAGG-3´ or 5´-C*CTGG-3´.This
methylation which is not found in vertebrates confers a bacterial signature to DNA prepared in E. coli making it prone to recognition as foreign DNA by
the host immune system. Mutation of dcm gene diminishes the immunostimulatory effects of plasmid DNAs.
The pir gene encodes the R6K specific initiator protein π which is required for the replication of plasmids harboring the R6K gamma origin of replication,
such as the pCpG plasmid family. The uidA::pir-116 mutant is the result of the integration of a mutant pir gene in the uidA locus. This mutant gene increases
plasmid copy number several fold.
The sbcC and sbcD genes encode a dimeric protein called SbcCD that recognizes and cleaves hairpins rendering plasmids with hairpin structures particularly
unstable in E. coli. Deletion of the sbcCD operon significantly improves the number of recombinant clones in plasmids containing hairpin structures.
Significant Genetic Markers
Marker Description
Significance
dcm
endA1
hsdR
lacZ∆M15
mcrA, mcrBC
mrr
recA
uidA::pir-116
sbcC-sbcD
Deletion eliminating Dcm methylase activity
Mutation inactivating endonuclease 1, a potent and non-specific enzyme
Restriction endonuclease mutation abolishing restriction and modification
(r-, m-)
Defective Φ80 prophage carrying a partial deletion of LacZ that allows
a-complementation
Mutation abolishing degradation of DNA with methylated cytosine
Mutation eliminating restriction of DNA containing 6-methyladenine or
6-methylcytosine
Homologous recombination deficiency
pir gene integrated in the uidA sequence
Deletion eliminating the recognition and cleavage of hairpin structures
GT100
F- mcrA D(mrr-hsdRMS-mcrBC)
Φ80lacZDM15 DlacX74 recA1 rspL (StrA) endA1
Ddcm
uidA::pir-116 DsbcC-sbcD
DsbcC-sbcD
42 www.invivogen.com/competent-cells
GT115 GT116
Allows for the production of high quality plasmid DNA
Improves the quality of purified plasmid DNA
Permits introduction of DNA coming from non-E. coli sources such as PCR
amplified DNA
Allows for blue/white color selection of clones when supplemented with
X-Gal
Allows more efficient cloning of 5-methylcytosine containing DNA
Allows more efficient cloning of DNA containing methyladenine residues
Transformed DNA will not recombine with host DNA ensuring the stability
of inserts
Allows for high copy number of plasmids containing the R6Kg origin of
replication
Increases the stability of hairpin structures and improves the number of
recombinant clones

ChemiComp E. coli
ChemiComp E. coli are chemically competent bacteria provided frozen. Their high transformation efficiency makes them ideal for cloning and
plasmid propagation. ChemiComp E. coli are shipped on dry ice.
ChemiComp GT115
(Ddcm, uidA::pir-116, sbcCD)
ChemiComp GT115 cells are high efficiency chemically competent cells
specifically designed for cloning and propagation of plasmids containing
hairpin structures and the R6K gamma origin of replication, such as pCpGsiRNA
plasmids.
Transformation efficiency: 0.1-1 x 10 9 cfu/μg
ChemiComp GT116
(Ddcm, sbcCD)
ChemiComp GT116 cells are high efficiency chemically competent cells
specifically designed for cloning and propagation of shRNA-expressing
plasmids which contain hairpin structures, such as psiRNA plasmids.
Transformation efficiency: 0.1-1 x 10 9 cfu/μg
LyoComp E. coli
LyoComp E. coli are chemically competent bacteria that have been lyophilized using a proprietary method. LyoComp E. coli are easy to handle
and more stable than standard competent cells. They are shipped at room temperature eliminating the need of costly dry ice shipping. Their
competency is sufficient for cloning and propagation of siRNA-expressing plasmids.
LyoComp GT115
(Ddcm, uidA::pir-116, sbcCD)
LyoComp GT115 cells are recommended for cloning and propagation
of plasmids containing hairpin structures and the R6K gamma origin of
replication, such as pCpG-siRNA plasmids.
Transformation efficiency: 1 x 10 6 cfu/μg
LyoComp GT116
(Ddcm, sbcCD)
LyoComp GT116 cells are recommended for cloning and propagation
of plasmids containing hairpin structures, such as psiRNA plasmids.
Transformation efficiency: 1 x 10 6 cfu/μg
Contents and Storage
ChemiComp E. coli cells are provided as 100 μl or 200 μl aliquots.
ChemiComp cells are shipped on dry ice. Upon receipt, store
ChemiComp cells at -80°C. ChemiComp cells are stable for at least 6
months when properly stored.
PRODUCT QUANTITY CAT. CODE
ChemiComp GT115
ChemiComp GT116
Contents and Storage
5 x 0.1 ml (5-10 transf.)
5 x 0.2 ml (10-20 transf.)
5 x 0.1 ml (5-10 transf.)
5 x 0.2 ml (10-20 transf.)
gt115-11
gt115-21
gt116-11
gt116-21
LyoComp E. coli are provided as 4 or 5 vials, each containing the
equivalent of 0.1 or 0.2 ml competent cells for GT115 and 0.5 ml or 1
ml competent cells for GT116. Cells are shipped at room temperature.
Upon receipt, store LyoComp E. coli at -20°C. Cells are stable for at least
6 months when properly stored.
PRODUCT QUANTITY CAT. CODE
LyoComp GT115
LyoComp GT116
5 x 0.1 ml (5-10 transf.)
5 x 0.2 ml (10-20 transf.)
4 x 0.5 ml (5-10 transf.)
4 x 1 ml (10-20 transf.)
lyo-115-11
lyo-115-21
lyo-116-11
lyo-116-21
www.invivogen.com/competent-cells 43
MAMMALIAN EXPRESSION VECTORS 2

MAMMALIAN EXPRESSION VECTORS 2
Fast-Media ® - The Hassle-free Way to Prepare E. coli Selection Media
All you need to make liquid or solid selective E. coli medium are five minutes, a microwave and Fast-Media ® . This time-saving product, developed
by InvivoGen, comes in individually sealed pouches, each with enough reagents to prepare 200 ml of sterile liquid or agar medium at the
appropriate antibiotic concentration. Fast-Media ® is extensively tested to guarantee sterility, antibiotic activity and E. coli growth. We subject
every ready-to-use Fast-Media ® pouch to rigorous quality control to ensure consistent results.
➥ 5-minute prep time
➥ Performance with control
➥ Large antibiotic selection
Features and Benefits
Fast-Media ® Eliminates Time Consuming Medium Preparation
- Rapid preparation: only 5 minutes
- Ready-made: no weighing or mixing of media components
- Presterilized: no autoclaving - just microwave
- Already contains the antibiotic of choice
Fast-Media ® is also available with no selective antibiotics.
Fast-Media ® is Ready-to-Use
Fast-Media ® is provided in individual sealed pouches. Each pouch contains
sufficient reagents to prepare 200 ml of sterile liquid or agar medium in just 5
minutes by using a microwave.
As Easy as 1, 2, 3
1- Empty pouch into a glass bottle
2- Mix pouch contents with 200 ml water
3- Microwave for 3 minutes
Performance and Control
Quality Control
To ensure constant quality and great reproducibility, each new batch of
Fast-Media ® is rigorously controlled according to approved procedures.
Each Fast-Media ® lot is tested with widely used E. coli K12 strains. Adequate
plasmids conferring appropriate resistance to E. coli strains are used as positive
controls.
Stability and Storage
After preparation, Fast-Media ® keeps all its intrinsic properties 48 hours at
37°C or 4 weeks at 4°C.
Sterility is guaranteed when Fast-Media ® is properly prepared and stored.
Contents and Storage
Each type of E. coli Fast-Media ® is provided in a 20- or 30-pouch unit. Each pouch
enables the preparation of 200 ml liquid medium or 8-10 agar plates. Store at
room temperature. Pouches are stable 12 months at room temperature. After
preparation, poured plates or reconstituted media are stable 4 weeks when
stored at 4°C.
44 www.invivogen.com/fast-media
Available in a wide variety
of antibiotics
Ampicillin
Blasticidin S
Hygromycin B
Kanamycin
Puromycin
Zeocin

Fast-Media ® Preparation
1. Empty pouch contents into a clean
borosilicate glass bottle. Mix Fast-Media ®
with 200 ml of distilled water.
2. Heat in microwave oven on medium
power (400 watts) for 3 minutes, mix and
reheat for 30 seconds*.
* InvivoGen has developed a process that guarantees the antibiotic activity after microwave heating.
3. Let cool and pour 8-10 plates.
PRODUCT ANTIBIOTIC QUANTITY CAT. CODE
Preparation of Liquid Medium
Fast-Media ® Base TB None 30 pouches
500 pouches
fas-l500
Fast-Media ® Amp TB Ampicillin 30 pouches
fas-am-l
500 pouches
fas-am-l500
Fast-Media ® Blas TB Blasticidin S 20 pouches fas-bl-l
Fast-Media ® Hygro TB Hygromycin B 20 pouches fas-hg-l
Fast-Media ® Kan TB Kanamycin 30 pouches
fas-kn-l
500 pouches
fas-kn-l500
Fast-Media ® Puro TB Puromycin 20 pouches fas-pr-l
Fast-Media ® Zeo TB Zeocin 20 pouches fas-zn-l
Fast-Media ® Base Agar None 30 pouches
fas-s
500 pouches
fas-s500
Fast-Media ® Amp Agar Ampicillin 30 pouches
fas-am-s
500 pouches
fas-am-s500
Fast-Media ® Blas Agar Blasticidin S 20 pouches fas-bl-s
Fast-Media ® Hygro Agar Hygromycin B 20 pouches fas-hg-s
Fast-Media ® Kan Agar Kanamycin 30 pouches
fas-kn-s
500 pouches
fas-kn-s500
Fast-Media ® Puro Agar Puromycin 20 pouches fas-pr-s
Fast-Media ® Zeo Agar Zeocin 20 pouches fas-zn-s
Fast-Media ® Amp Agar X-Gal Ampicillin 20 pouches fas-am-x
Fast-Media ® Blas Agar X-Gal Blasticidin S 20 pouches fas-bl-x
Fast-Media ® Hygro Agar X-Gal Hygromycin B 20 pouches fas-hg-x
Fast-Media ® Kan Agar X-Gal Kanamycin 20 pouches fas-kan-x
Fast-Media ® Zeo Agar X-Gal Zeocin 20 pouches fas-zn-x
Fast-Media ® Zeo Agar X-Gluc Zeocin Preparation of Agar Plates
Preparation of Agar Plates with X-Gal (+IPTG)
Preparation of Agar Plates with X-Gluc
10 pouches fas-zn-g
www.invivogen.com/fast-media 45
fas-l
MAMMALIAN EXPRESSION VECTORS 2

3
INNATE IMMUNITY
Toll-like Receptors 49
TLR & TLR-Related Genes 56-63
TLR-Expressing Cell Lines 64-71
TLR Antibodies 72
Reporter Plasmids 74
TLR Agonists & Antagonists 76-79
TLR Agonist Kits 81
LPS Detection Kit 90
TLR Ligand Screening 91
TLR shRNAs 92
Inhibitors of TLR Signaling 93
NOD-like Receptors 52
NLR & NLR-Related Genes 56-60
NLR-Expressing Cell Lines 65-67
Reporter Plasmids 74
NLR Agonists 80
NOD Ligand Screening 91
NLR shRNAs 92

RIG-I-like Receptors 54
RLR & RLR-Related Genes 56-60
RLR-Expressing Cell Lines 64, 71
RLR Agonist 80
RLR shRNAs 92
C-Type Lectin Receptors 55
CLR & CLR-Related Genes 56-60
Dectin-1-Expressing Cell Line 70
Dectin-1 Agonists 81
CLR shRNAs 92
Inflammasomes 96
Inflammasome Inducers & Inhibitors 98
IL-1b Sensor Cells 100
Autophagy and TLRs 101
Autophagy Genes 101
Autophagy Inducers & Inhibitors 102

INNATE IMMUNITY 3
PATTERN RECOGNITION
RECEPTORS
The innate immune system is an evolutionally conserved mechanism that provides an early and effective response
against invading microbial pathogens. It relies on a limited set of pattern recognition receptors (PRRs) that
recognize specific pathogen-associated molecular patterns (PAMPs) commonly present in microbes but not in
host. Upon detection of PAMPs, the PRRs trigger an inflammatory response that recruits phagocytic cells, induce
antimicrobial peptides and cytokine/chemokine secretion, leading to efficient destruction of the invading
pathogens. Three main families of PRRs have been shown to initiate proinflammatory signaling pathways: the
Toll-Like receptors (TLRs), the NOD-Like receptors (NLRs) and RIG-I-Like receptors (RLRs).
Toll-Like Receptors (TLRs)
TLRs are the first identified and best characterized receptors among the signaling PRRs. They initiate key inflammatory responses and also shape adaptative
immunity. All TLRs (10 in humans and 11 in mice) are type I transmembrane proteins characterized by an extracellular leucine-rich domain and a cytoplasmic
tail that contains a conserved region called the Toll/IL-1 receptor (TIR) domain. They recognize a variety of PAMPs from bacteria, fungi, parasites, and viruses,
including lipid-based bacterial cell wall components such as lipopolysaccharide (LPS) and lipopeptides, microbial protein components such as flagellin, and
nucleic acids such as single-stranded or double-stranded RNA and CpG DNA. TLRs initiate shared and distinct signaling pathways by recruiting different
combinations of four TIR-domain-containing adaptor molecules: MyD88, TIRAP (MAL), TRIF (TICAM1) and TRAM (TICAM2). These signaling pathways
activate the transcription factors NF-kB and AP-1 leading to the production of inflammatory cytokines and chemokines. They also activate interferon
regulatory factors (IRFs) leading to the production of type I interferons.
Nod-Like Receptors (NLRs)
NOD-Like Receptors (NLRs, also known as CATERPILLERs) constitute a recently identified family of intracellular pattern recognition receptors (PRRs),
which contains more than 20 members in mammals. Although the ligands and functions of many of these receptors are not known, their primary role is to
recognize cytoplasmic pathogen-associated molecular patterns (PAMPs) and/or endogenous danger signal, inducing immune responses. NLRs are
characterized by a tripartite-domain organization with a conserved nucleotide binding oligomerization domain (NOD) and leucine-rich repeats (LRRs). The
general domain structure consists of C-terminal LRRs involved in microbial sensing, a centrally located NOD domain and an N-terminal effector region
comprising a protein-protein interaction domain such as the CARD, Pyrin or BIR domain. NLRs have been grouped into several subfamilies on the basis of
their effector domains: NODs, NALPs, CIITA, IPAF, and NAIPs. NODs and IPAF contain CARD effector domains, whereas NALPs and NAIPs contain pyrin
(PYD) effector domains and three BIR domains, respectively.
RIG-I-Like Receptors (RLRs)
RIG-I-like receptors (RLRs) constitute a family of cytoplasmic RNA helicases that are critical for host antiviral responses. RIG-I (retinoic-acid-inducible
protein 1, also known as Ddx58) and MDA-5 (melanoma-differentiation-associated gene 5, also known as Ifih1 or Helicard) sense double-stranded RNA
(dsRNA), a replication intermediate for RNA viruses, leading to production of type I interferons (IFNs) in infected cells. A third RLR has been described:
laboratory of genetics and physiology 2 (LGP2). LGP2 contains a RNA binding domain but lacks the CARD domains and thus acts as a negative feedback
regulator of RIG-I and MDA-5.
C-Type Lectin Receptors (CLRs) and Other Pathogen Sensors
Besides TLRs, NLRs and RLRs, other receptors can also recognize molecules present on pathogenic organisms. Those receptors include members of the
PGRP (peptidoglycan recognition proteins) and C-type lectin families. C-type lectins, also called C-type lectin receptors (CLRs), encompass a large family of
proteins that act as phagocytic receptors that bind carbohydrate moieties of various pathogens. This family comprises MBL (mannose binding lectin),
Dectin-1, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin) and the structurally related receptors SIGNRs.
48 www.invivogen.com/innate-immunity

Toll-Like Receptors
Toll-Like Receptors (TLRs) play a critical role in the early innate immune
response to invading pathogens by sensing microorganisms. These
evolutionarily conserved receptors, homologues of the Drosophila Toll gene,
recognize highly conserved structural motifs only expressed by microbial
pathogens, called pathogen-associated microbial patterns (PAMPs). PAMPs
include various bacterial cell wall components such as lipopolysaccharide
(LPS), peptidoglycan (PGN) and lipopeptides, as well as flagellin, bacterial
DNA and viral double-stranded RNA. Stimulation of TLRs by PAMPs
initiates signaling cascades that involves a number of proteins, such as
MyD88, TRIF and IRAK 1 . These signaling cascades lead to the activation of
transcription factors, such as AP-1, NF-kB and IRFs inducing the secretion
of pro-inflammatory cytokines and effector cytokines that direct the
adaptive immune response.
The TLR Family
TLRs are type I transmembrane proteins characterized by an extracellular
domain containing leucine-rich repeats (LRRs) and a cytoplasmic tail that
contains a conserved region called the Toll/IL-1 receptor (TIR) domain. The
structure of the extracellular domain of TLR3 was recently revealed by
crystallography studies as a large horseshoe-shape 2 . TLRs are predominantly
expressed in tissues involved in immune function, such as spleen and
peripheral blood leukocytes, as well as those exposed to the external
environment such as lung and the gastrointestinal tract. Their expression
profiles vary among tissues and cell types. TLRs are located on the plasma
membrane with the exception of TLR3, TLR7, TLR9 which are localized
intracellularly 3 .
Ten human and twelve murine TLRs have
been characterized, TLR1 to TLR10 in humans,
and TLR1 to TLR9, TLR11, TLR12 and TLR13
in mice, the homolog of TLR10 being a
pseudogene. TLR2 is essential for the
recognition of a variety of PAMPs from Grampositive
bacteria, including bacterial
lipoproteins, lipomannans and lipoteichoic
acids. TLR3 is implicated in virus-derived
double-stranded RNA. TLR4 is predominantly
activated by lipopolysaccharide. TLR5 detects
bacterial flagellin and TLR9 is required for
response to unmethylated CpG DNA. Finally,
TLR7 and TLR8 recognize small synthetic antiviral molecules 4 , and recently
single-stranded RNA was reported to be their natural ligand 5 . TLR11(12)
has been reported to recognize uropathogenic E. coli 6 and a profilin-like
protein from Toxoplasma gondii 7 .
The repertoire of specificities of the TLRs is apparently extended by the
ability of TLRs to heterodimerize with one another. For example, dimers of
TLR2 and TLR6 are required for responses to diacylated lipoproteins while
TLR2 and TLR1 interact to recognize triacylated lipoproteins 8 . Specificities
of the TLRs are also influenced by various adapter and accessory molecules,
such as MD-2 and CD14 that form a complex with TLR4 in response to
LPS 9 .
TLR Signaling (see pathway next page)
TLR signaling consists of at least two distinct pathways: a MyD88-dependent
pathway that leads to the production of inflammatory cytokines, and a
MyD88-independent pathway associated with the stimulation of IFN-b and
the maturation of dendritic cells. The MyD88-dependent pathway is
common to all TLRs, except TLR3 10 . Upon activation by microbial antigens,
TLRs induce the recruitment of MyD88 via its TIR domain which in turn
recruits IRAK1 and IRAK4. IRAK4 then activates IRAK-1 by phosphorylation.
Both IRAK-1 and IRAK4 leave the MyD88-TLR complex and associate
temporarily with TRAF6 leading to its ubiquitination. Bcl10 and MALT1 form
oligomers that bind to TRAF6 promoting TRAF6 self-ubiquitination 11 .
Recently, IRAK-2 was shown to play a central role in TRAF6 ubiquitination 12 .
Following ubiquitination, TRAF6 forms a complex with TAB2/TAB3/TAK1
inducing TAK1 activation 13 . TAK1 then couples to the IKK complex, which
includes the scaffold protein NEMO, leading to the phosphorylation of IkB
and the subsequent nuclear localization of NF-kB. Activation of NF-kB
triggers the the production of pro-inflammatory cytokines such as TNF-a,
IL-1 and IL-12.
Differences between signaling pathways induced by individual TLRs are
emerging. TLR4 and TLR2 signaling requires the adaptor TIRAP/Mal, which
is involved in the MyD88-dependent pathway 14 . TLR3 triggers the
production of IFN-b in response to double-stranded RNA, in a MyD88independent
manner, through the adaptor TRIF/TICAM-1 15 . TRAM/TICAM-
2 is another adaptor molecule involved in the MyD88-independent
pathway 5 which function is restricted to the TLR4 pathway 16 .
TLR3, TLR7, TLR8 and TLR9 recognize viral nucleic acids and induce type I
IFNs. The signaling mechanisms leading to the induction of type I IFNs differ
depending on the TLR activated (see Chapter “Cytokine Signaling”). They
involve the interferon regulatory factors (IRFs), a growing family of
transcription factors known to play a critical role in antiviral defense, cell
growth and immune regulation. Three IRFs (IRF3, IRF5 and IRF7) function
as direct transducers of virus-mediated TLR signaling. TLR3 and TLR4 activate
IRF3 and IRF7 17 , while TLR7 and TLR8 activate IRF5 and IRF7 18 . Furthermore,
type I IFN production stimulated by TLR9 ligand CpG-A has been shown
to be mediated by PI(3)K and mTOR 19 .
Current knowledge of the TLRs indicates that these receptors are essential
elements in host defense against pathogens by activating the innate
immunity, a prerequisite to induction of adaptive immunity. The growing
interest in TLRs should bring a more complete understanding of the role of
TLR-mediated responses and increase our range of weapons to treat
infectious and immune diseases.
1. Medzhitov R. et al., 1997. A human homologue of the Drosophila Toll protein signals
activation of adaptive immunity. Nature, 388(6640):394-7. 2. Choe J. et al., 2005. Crystal
structure of human Toll-like receptor 3 (TLR3) ectodomain. Science 309; 581-585. 3. Nishiya
T. & DeFranco AL., 2004. Ligand-regulated chimeric receptor approach reveals distinctive
subcellular localization and signaling properties of the Toll-like receptors. J Biol Chem.
279(18):19008-17. 4. Jurk M. et al., 2002. Human TLR7 or TLR8 independently confer
responsiveness to the antiviral compound R-848. Nat Immunol, 3(6):499. 5. Heil F. et al., 2004.
Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science.
303(5663):1526-9. 6. Zhang D. et al., 2004. A toll-like receptor that prevents infection by
uropathogenic bacteria. Science. 303:1522-1526. 7. Lauw FN. et al., 2005. Of mice and man:
TLR11 (finally) finds profilin. Trends Immunol. 26(10):509-11. 8. Ozinsky A. et al., 2000. The
repertoire for pattern recognition of pathogens by the innate immune system is defined by
cooperation between toll-like receptors. PNAS USA, 97(25):13766-71. 9. Miyake K., 2003.
Innate recognition of lipopolysaccharide by CD14 and toll-like receptor 4-MD-2: unique roles
for MD-2. Int Immunopharmacol. 3(1):119-28. 10. Adachi O. et al., 1998.Targeted disruption
of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity. 9(1):143-
50. 11. Sun L. et al., 2004. The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation
by BCL10 and MALT1 in T lymphocytes. Mol Cell. 12. Keating SE. et al., 2007. IRAK-2
participates in multiple Toll-like receptor signaling pathways to NFkB via activation of TRAF6
ubiquitination. J Biol Chem. 282: 33435-33443. 13. Kanayama A. et al., 2004. TAB2 and TAB3
activate the NF-kappaB pathway through binding to polyubiquitin chains. Mol Cell. 15(4):535-
48. 14. Horng T. et al., 2002. The adaptor molecule TIRAP provides signalling specificity for
Toll-like receptors. Nature. 420(6913):329-33. 15. Yamamoto M. et al., 2002. Cutting edge: a
novel Toll/IL-1 receptor domain-containing adaptor that preferentially activates the IFN-beta
promoter in the Toll-like receptor signaling. J Immunol. 169(12):6668-72. 16. Yamamoto M. et
al., 2003. TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent
signaling pathway. Nat Immunol. 4(11):1144-50. 17. Doyle S. et al., 2002. IRF3 mediates a
TLR3/TLR4-specific antiviral gene program. Immunity. 17(3):251-63. 18. Schoenemeyer A. et
al., 2005. The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7
signaling. J Biol Chem. 280(17):17005-12. 19. Costa-Mattioli M. & Sonenberg N. 2008. RAPping
production of type I interferon in pDCs through mTOR. Nature Immunol. 9: 1097-1099.
www.invivogen.com/innate-immunity 49
INNATE IMMUNITY 3

INNATE IMMUNITY 3
50 www.invivogen.com/innate-immunity

www.invivogen.com/innate-immunity 51
INNATE IMMUNITY 3

INNATE IMMUNITY 3
Nod-Like Receptors
NOD-Like Receptors (NLRs, also known as CATERPILLERs) constitute a
recently identified family of intracellular pattern recognition receptors
(PRRs), which contains more than 20 members in mammals. Although the
ligands and functions of many of these receptors are not known, their
primary role is to recognize cytoplasmic pathogen-associated molecular
patterns (PAMPs) and/or endogenous danger signals also called damageassociated
molecular patterns (DAMPs), inducing immune responses. NLRs
are characterized by a tripartite-domain organization with a conserved
nucleotide binding oligomerization domain (NOD) and leucine-rich repeats
(LRRs). The general domain structure consists of C-terminal LRRs involved
in microbial sensing, a centrally located NOD domain and an N-terminal
effector region comprising a protein-protein interaction domain such as the
CARD, Pyrin or BIR domain. NLRs have been grouped into several
subfamilies on the basis of their effector domains: NODs, NALPs, CIITA,
IPAF, and NAIPs. NODs and IPAF contain CARD effector domains, whereas
NALPs and NAIPs contain pyrin (PYD) effector domains and three BIR
domains, respectively.
NOD1 and NOD2
The first mammalian NLRs reported to sense intracellular microbial PAMPs
are NOD1 (CARD4) and NOD2 (CARD15), which contain one and two
N-terminal CARD domains, respectively. They recognize peptidoglycan
(PGN), an essential constituent of the bacterial cell wall. NOD1 and NOD2
detect specific motifs within the PGN. NOD1 senses the D-g-glutamyl-meso-
DAP dipeptide (iE-DAP) which is found in PGN of all Gram-negative and
certain Gram-positive bacteria 1,2 whereas NOD2 recognizes the muramyl
dipeptide (MDP) structure found in almost all bacteria 3 . Thus NOD2 acts as
a general sensor of PGN and NOD1 is involved in the recognition of a specific
subset of bacteria. Both iE-DAP and MDP must be delivered intracellularly
either by bacteria that invade the cell or through other cellular uptake
mechanisms to be detected by NOD1 and NOD2 respectively. NOD1 and
NOD2 signal via the serine/threonine RIP2 (RICK,CARDIAK) kinase through
CARD-CARD homophilic interactions 4 . Once activated, RIP2 mediates
ubiquitination of NEMO/IKKg leading to the activation of NF-kB and the
production of inflammatory cytokines such as TNF-a and IL-6 5 . In addition to
the NF-kB pathway, NOD1 and NOD2 stimulation induces the activation of
MAPKs 6 . Recent work has implicated CARD9 in the selective control of
NOD2-dependent p38 and JNK signaling 7 . The physiological importance of
NOD1 and NOD2 in immune responses is evident from the linkage of their
mutations with inflammatory diseases in humans. Genetic variation in NOD2
is associated with Crohn’s disease, one of the major forms of inflammatory
bowel diseases 8 . Several NOD1 polymorphisms are linked to the
development of atopic eczema and asthma 9 .
Schematic structure of Lys-PGN (found in Gram-positive bacteria) and DAP-PGN
(found in Gram-negative bacteria)
52 www.invivogen.com/innate-immunity
NALPs
The NALP subfamily consists of 14 members that are characterized by the
presence of PYD effector domains. Although the functions of many of the
NALPs are largely unknown, several NALPs play a key role in the regulation
of caspase-1 by forming a mutiprotein complex known as the
‘inflammasome’. Caspase-1 participates in the processing and subsequent
release of proinflammatory cytokines, such as IL-1b and IL-18. At least two
types of NALP inflammasomes have been identified: the NALP1
inflammasome comprising NALP1 (NLRP1, CARD7), ASC, Caspase-1 and
Caspase-5, and the NALP3 inflammasome containing NALP3 (NLRP3,
cryopyrin, CIAS1), ASC, Cardinal and Caspase-1 10 . NALP1 and NALP3
recruit through their PYD domain the adaptor protein ASC which in turn
interacts with Caspase-1 via a CARD-CARD interaction. NALP1 also
recruits Caspase-5 via its additional CARD effector domain at the C
terminus, whereas NALP3, lacking such a CARD, interacts with the CARDcontaining
adaptor Cardinal to recruit additional Caspase-1.
Two molecules have been recently described to activate NALP1: MDP and
the anthrax toxin. In vitro reconstitution experiments revealed that NALP1
oligomerization is a two step mechanism requiring MDP and ATP 11 . In vivo
studies with NALP1 knock-out mice are necessary to confirm that MDP is
a true NALP1 ligand. A murine variant of NALP1 (NALP1b) was shown to
respond to the anthrax toxin suggesting an engagement of the NALP1
inflammasome in the immune response to Bacillus anthracis infection 12 .
NALP3 mediates caspase-1 activation in response to a wide variety of
stimuli: whole bacteria (Listeria monocytogenes, Staphylococcus aureus),
bacterial RNA, synthetic purine-like compounds (R848, R837), uric acid
crystals, extracellular ATP and pore-forming toxins (nigericin, maitotoxin) 13-
15 . Activation of Caspase-1 induced by NALP3 appears to be TLRindependent,
whereas secretion of mature IL-1b seems to require two
stimuli involving the TLRs and NALP3. The first stimulus, a TLR ligand such
as LPS, triggers the generation of pro-IL-1b, while the second, a stimulus
such as ATP, induces oligomerization and inflammasome assembly 16-17 . In
addition, studies suggest that NALP3 does not sense ATP directly but rather
intracellular potassium depletion resulting from ATP signaling. Bacterial
toxins, such as nigericin and maitotoxin that cause a change in intracellular
ion composition activate the NALP3 inflammasome. Thus, NALP3 appears
to serve as an activator of the inflammasome in response to specific toxins,
endogenous danger signals released by damaged cells or tissues (uric acid
crystal, elevated ATP), or microbial pathogens. Mutations in NALP3 are the
cause for several human diseases such as familial cold autoinflammatory
syndrome and Muckle-Wells syndrome 18 .

IPAF and NAIP5
IPAF (NLRC4, CLAN/CARD12) and NAIP5 constitute another set of NLRs.
IPAF belongs to the CARD subfamily whereas NAIP5 is a member of the
BIR subfamily. Both NLRs have been shown to respond to flagellin, the main
component of the bacterial flagellum, restricting the proliferation of
intracellular bacteria such as Salmonella typhimurium, Shigella flexneri and
Legionella pneumophila. IPAF senses flagellin from S. typhimurium and S. flexneri
secreted by the bacterial type III secretion system (TTSS). SipB in S.
typhimurium and IpaB in S. flexneri are part of a TTSS and are required for
Caspase-1 activation. These proteins participate in the translocation of
flagellin in the cytosol by forming a pore in the host-cell membrane.
Caspase-1 activation induced by cytosolic flagellin has been shown to be
IPAF-dependent, but TLR5-independent 19 . Thus TLR5 and IPAF appear to
be two different sensors that respond to extracellular and cytosolic flagellin,
respectively 20 .The adaptor ASC seems to be involved in Caspase-1 activation
in response to infection with S. typhimurium and S. flexneri 21 . Sensing of L.
pneumophila flagellin which is secreted by the bacterial type IV secretion
system is mediated by NAIP5. Mutations affecting the NAIP5 locus (also
known as Birc1e) have been implicated in increased susceptibility of A/J
macrophages to infection with L. pneumophila. NAIP5 appears to form an
inflammasome complex containing IPAF (but not ASC) and Caspase-1 22 .
NAIP5-mediated signaling pathway and IPAF-mediated Caspase-1 activation
may act in concert to restrict L. pneumophila growth in macrophages 23,24 .
1. Chamaillard M. et al., 2003. An essential role for NOD1 in host recognition of bacterial
peptidoglycan containing diaminopimelic acid. Nat. Immunol. 4: 702-707. 2. Girardin SE. et al.,
2003. Nod1 detects a unique muropeptide from Gram-negative bacterial peptidoglycan. Science
300: 1584-1587. 3. McDonald C., N. Inohara, G. Nunez. 2005. Peptidoglycan signaling in innate
immunity and inflammatory disease. J. Biol. Chem. 280: 20177-20180. 4. Kobayash, K. et al., 2002.
RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune
systems. Nature 416: 194-199. 5. Inohara N. et al., 2000. An induced proximity model for NF-
B activation in the Nod1/RICK and RIP signaling pathways. J. Biol. Chem. 275: 27823-27831.
6. Kobayashi KS. et al., 2005. Nod2-dependent regulation of innate and adaptive immunity in
the intestinal tract. Science 307: 731-734. 7. Hsu YM. et al., 2007. The adaptor protein CARD9
is required for innate immune responses to intracellular pathogens. Nat Immunol. 8(2):198-205.
8. Ogura Y. et al., 2001. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s
disease. Nature 411: 603-606. 9. Hysi P. et al., 2005. NOD1 variation, immunoglobulin E and
asthma. Hum. Mol. Genet. 14: 935-941. 10. Martinon F. & Tschopp J., 2004. Inflammatory
caspases: linking an intracellular innate immune system to autoinflammatory diseases. Cell.
117(5):561-74. 11. Faustin B, et al., 2006. In Vitro Reconstitution of the NALP1 Inflammasome
Reveals Requirements for Caspase Activation. Blood (ASH Annual Meeting Abstracts), 108:
3658. 12. Boyden ED. & Dietrich WF., 2006. Nalp1b controls mouse macrophage susceptibility
to anthrax lethal toxin. Nat Genet. 38(2):240-4. 13. Martinon F. et al., 2006. Gout-associated
uric acid crystals activate the NALP3 inflammasome. Nature 440(7081)237-241. 14. Kanneganti
TD. et al., 2006. Bacterial RNA and small antiviral compounds activate caspase-1 through
cryopyrin/Nalp3. Nature 440(7081):233-236. 15. Mariathasan S. et al., 2006. Cryopyrin activates
the inflammasome in response to toxins and ATP. Nature. 440(7081):228-32. 16. Sutterwala FS.
et al., 2006. Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through
its regulation of caspase-1. Immunity. 24(3):317-27. 17. Kanneganti TD. et al., 2007. Pannexin-1mediated
recognition of bacterial molecules activates the cryopyrin inflammasome independent
of Toll-like receptor signaling. Immunity. 26(4):433-43. 18. Ting JP. et al., 2006. CATERPILLERs,
pyrin and hereditary immunological disorders. Nat. Rev. Immunol. 6: 183-195. 19. Miao EA. et
al., 2008. Pseudomonas aeruginosa activates caspase 1 through Ipaf. PNAS.105:2562-2567. 20.
Yu HB.& Finlay BB. 2008. The Caspase-1 inflammasome: a pilot of innate immune responses.
Cell Host Microbe. 4:198-207. 21. Mariathasan S & Monack DM., 2007. Inflammasome adaptors
and sensors: intracellular regulators of infection and inflammation. Nat Rev Immunol. 7(1):31-
40. 22. Zamboni DS. et al., 2006. The Birc1e cytosolic pattern-recognition receptor contributes
to the detection and control of Legionella pneumophila infection. Nat Immunol. 7(3):318-25.
23. Lamkanfi M. et al., 2007. The Nod-like receptor family member Naip5/Birc1e restricts
Legionella pneumophila growth independently of caspase-1 axctivation. J Immunol. 178:8022-
8027. 24. Vinzing M. et al. 2008. NAIP and IPAF control legionella pneumophila replication in
human cells. J Immunol. 180:6808-15.
www.invivogen.com/innate-immunity 53
INNATE IMMUNITY 3

INNATE IMMUNITY 3
RIG-I-Like Receptors
RIG-I-like receptors (RLRs), also known as RIG-I-like helicases (RLHs)
constitute a family of cytoplasmic RNA helicases that are critical for host
antiviral responses. RIG-I (retinoic-acid-inducible protein 1, also known as
Ddx58) and MDA-5 (melanoma-differentiation-associated gene 5, also
known as Ifih1 or Helicard) sense double-stranded RNA (dsRNA), a
replication intermediate for RNA viruses, leading to production of type I
interferons (IFNs) in infected cells 1 . Viral dsRNA is also recognized by Toll-
Like receptor 3 (TLR3) which is expressed on the cell surface membrane
or endosomes. Recognition of dsRNA by RIG-I/MDA-5 or TLR3 is cell-type
dependent. Studies of RIG-I- and MDA-5-deficient mice have revealed that
conventional dendritic cells (DCs), macrophages and fibroblasts isolated
from these mice have impaired IFN induction after RNA virus infection,
while production of IFN is still observed in plasmacytoid DCs (pDCs) 2 . Thus
in cDCs, macrophages and fibroblasts, RLRs are the major sensors for viral
infection, while in pDCs, TLRs play a more important role.
RIG-I and MDA-5 contain a DExD/H box RNA helicase and two caspase
recruiting domain (CARD)-like domains. The helicase domain interacts with
dsRNA, whereas the CARD domains are required to relay the signal.
Despite the overall structural similarity between these two sensors, they
detect distinct viral species. RIG-I participates in the recognition of
Paramyxoviruses (Newcastle disease virus (NDV), Sendai virus (SeV)),
Rhabdoviruses (vesicular stomatitis virus (VSV)), Flaviviruses (hepatitis C
(HCV)) and Orthomyxoviruses (Influenza), whereas MDA-5 is essential for
the recognition of Picornaviruses (encephalo-myocarditis virus (EMCV))
and poly(I:C), a synthetic analog of viral dsRNA 3 . Notably, RIG-I binds
specifically to single stranded RNA containing 5’-triphosphate such as viral
RNA and in vitro-transcribed long dsRNA 4 . Mammalian RNA is either
capped or contains base modifications suggesting that RIG-I is able to
discriminate between self and non-self RNA. Recently Kato et al., showed
by using poly(I:C) treated with RNase III that RIG-I binds preferentially to
54
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short dsRNA while MDA-5 recognizes preferentially long dsRNA 5 .
Although RIG-I and MDA-5 recognize different ligands, they share common
signaling features. Upon recognition of dsRNA, they are recruited by the
adaptor IPS-1 (also known as MAVS, CARDIF or VISA) to the outer
membrane of the mitochondria leading to the activation of several
transcription factors including IRF3, IRF7 and NF-kB 6 . IRF3 and IRF7 control
the expression of type I IFNs, while NF-kB regulates the production of
inflammatory cytokines. IRF3 and IRF7 activation involves TNF (tumor
necrosis factor) receptor-associated factor 3 (TRAF3), NAK-associated
protein 1 (NAP1), TANK and the protein kinase TANK-binding kinase 1
(TBK1) or IkB kinase epsilon (IKKε) 6-8 . Recently, DDX3, a DEAD box
helicase, was shown to interact with TBK1/IKKε 9 . IPS-1 interacts also with
Fas-associated-death-domain (FADD) and receptor interacting protein 1
(RIP1) which induces the activation of the NF-kB pathway 6-8, 10 .
Because the production of cytokines is closely controlled, the RIG-I/
MDA-5 pathway is also strictly regulated. A third RLR has been described:
laboratory of genetics and physiology 2 (LGP2). LGP2 contains a RNA
binding domain but lacks the CARD domains and thus acts as a negative
feedback regulator of RIG-I and MDA-5. LGP2 appears to exert this activity
at multiple levels by i) competitively sequestering dsRNA, ii) forming a
protein complex with IPS-1, and/or iii) binding directly to RIG-I through a
repressor domain 11-13 . Many other molecules seem to be involved in the
negative control of RIG-I/MDA-5-induced IFN production. Dihydroxyacetone
kinase (DAK), A20, ring-finger protein 125 (RNF125), suppressor
of IKKε (SIKE), and peptidyl-propyl isomerase 1 (Pin1), have been recently
described as physiological suppressors of the RIG-I/MDA-5 signaling
pathway. Furthermore, some viral proteases have been identified, such as
NS3/NS4A of the hepatitis C virus, that inactivate RIG-I/MDA-5 signaling
by targeting IPS-1 as effective means to evade innate immunity.
1. Yoneyama M. & Fujita T., 2007. Function of RIG-I-like Receptors in Antiviral Innate
Immunity. J. Biol. Chem. 282: 15315-15318. 2. Kato H. et al., 2005. Cell type-specific
involvement of RIG-I in antiviral response. Immunity. 23(1):19-28. 3. Kawai T. & Akira S.,
2007. Antiviral signaling through pattern recognition receptors. J Biochem. 141(2):137-45.
4. Pichlmair A. et al., 2006. RIG-I-mediated antiviral responses to single-stranded RNA
bearing 5'-phosphates. Science 314:997-1001. 5. Kato H. et al., 2008. Length-dependent
recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and
melanoma differentiation-associated gene 5. J Exp Med. 205(7):1601-10. 6. Kawai T. et al.,
2005. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.
Nat Immunol. 6(10):981-988 7. Saha SK. et al., 2006. Regulation of antiviral responses by a
direct and specific interaction between TRAF3 and Cardif. Embo J. 25:3257-3263. 8. Sasai
M. et al., 2006. NAK-associated protein 1 participates in both the TLR3 and the cytoplasmic
pathways in type I IFN induction. J Immunol. 177:8676-8683. 9. Schröder M, et al, 2008. Viral
targeting of DEAD box protein 3 reveals its role in TBK1/IKKepsilon-mediated IRF activation.
EMBO J. 27(15):2147-57. 10. Takahashi K. et al., 2006. Roles of caspase-8 and caspase-10 in
innate immune responses to double-stranded RNA. J Immunol. 176:4520-4524. 11.
Yoneyama M. et al., 2005. Shared and unique functions of the DExD/H-box helicases RIG-
I, MDA5, and LGP2 in antiviral innate immunity. J Immunol. 175:2851-58. 12. Komuro A. &
Horvath CM., 2006. RNA- and virus-independent inhibition of antiviral signaling by RNA
helicase LGP2. J Virol. 80(24): 12332-12342. 13. Saito T. et al., 2007. Regulation of innate
antiviral defenses through a shared repressor domain in RIG-I and LGP2. PNAS. 104(2):582-
587.

C-Type Lectin Receptors
C-type lectin receptors (CLRs) comprise a large family of receptors that
bind to carbohydrates in a calcium-dependent manner. The lectin activity of
these receptors is mediated by conserved carbohydrate-recognition
domains (CRDs). CLRs include Dectin-1, macrophage-inducible C-type
lectin (Mincle), the dendritic cell-specific ICAM3-grabbing nonintegrin (DC-
SIGN), DC-SIGN related (DC-SIGNR) and the circulating mannose binding
lectin (MBL). These CLRs share one or more CRD that were originally found
in the mannose-binding lectin and are evolutionarily conserved. These
receptors are involved in fungal recognition and the modulation of the
innate immune response. CLRs are expressed by most cell types including
macrophages and dendritic cells (DCs), which internalize various
glycoproteins and microbes for the purposes of clearance and antigen
presentation to T lymphocytes.
Dectin-1 plays an important role in antifungal innate immunity. Dectin-1,
which is expressed on phagocytes, is a specific receptor for b-glucans 1 . b-
Glucans are glucose polymers found in the cell walls of fungi, including the
yeasts Saccharomyces cerevisiae and Candida albicans. Dectin-1 is a type II
transmembrane protein with a CRD connected by a stalk to the
transmembrane region, followed by a cytoplasmic tail containing an ITAMlike
motif. Upon binding to its ligand, Dectin-1 triggers phagocytosis and
activation of Src and Syk kinases, through its ITAM-like motif. Syk, in turn,
induces the CARD9-Bcl10-Malt1 complex leading to the production of
reactive oxygen species (ROS), activation of NF-kB and subsequent
secretion of proinflammatory cytokines 2, 3 . Dectin-1 signaling has been
shown to collaborate with TLR2 signaling to enhance the responses
triggered by each receptor 3, 4 . Furthermore, Dectin-1 can modulate cytokine
expression by inducing NFAT through the Ca 2+ -calcineurin-NFAT pathway 5 .
Mincle is another CLR expressed in phagocytes which signaling leads to
ITAM-dependent activation of NF-kB and NFAT. Mincle recognizes a variety
of endogenous and exogenous stimuli, such as necrotic cells, mycobacteria
and certain fungi, including C. albicans and Malassezia species 6 . Mincle senses
damaged cells by recognizing spliceosome-associated protein 130 (SAP130),
a soluble factor released by necrotic cells 7 . Mincle recognizes also fungal
a-mannose, and the mycobacterial glycolipid, trehalose-6’6’-dimycolate
(TDM, also known as cord factor), the most studied immunostimulatory
component of Mycobacterium tuberculosis 8 . Mincle interacts with the Fc
receptor common g-chain (FcRg), which triggers intracellular signaling
through Syk leading to CARD9-dependent NF-kB activation. Syk induces
www.invivogen.com/innate-immunity
also the mobilization of intracellular calcium (Ca 2+ ) and the activation of the
calcineurin-NFAT pathway.
Human DC-SIGN is of special interest because it is involved in the interaction
of certain DCs with several viruses (HIV-1, HCV, dengue virus, CMV, ebola
virus) and other microbes, Leishmania and Candida species. This type II
transmembrance protein has a single C-type lectin domain and is expressed
on immature monocyte-derived DCs. Five mouse DC-SIGN homologues
exist. Only one of these named mDC-SIGN is expressed in a restricted
fashion in DCs, while the others have a different tissue distribution and are
termed SIGNR1, SIGNR2, SIGNR3 and SIGNR4 for SIGN-related proteins 9 .
Despite similarities in the carbohydrate recognition domains (CRDs) of the
mouse homologues to the hDC-SIGN, the membrane proximal neck
domains were much shorter.
A molecular signaling pathway induced by the C-type lectin DC-SIGN that
modulates TLR signaling at the level of the transcription factor NF-kB has been
identified. It has been demonstrated that pathogens trigger DC-SIGN on
human DCs to activate the serine and threonine kinase Raf-1, which
subsequently leads to acetylation of the NF-kB subunit p65, but only after TLRinduced
activation of NF-kB. Acetylation of p65 both prolonged and increased
IL-10 transcription to enhance anti-inflammatory cytokine response. It has been
demonstrated that different pathogens such as Mycobacterium tuberculosis, M.
leprae, Candida albicans, measles virus, and HIV-1 interacted with DC-SIGN to
activate the Raf-1-acetylation-dependent signaling pathway to modulate
signaling by different TLRs 10 . Thus, this pathway is involved in regulation of
adaptive immunity by DCs to bacterial, fungal, and viral pathogens.
MBL, a soluble C-type lectin, is an effector molecule of the innate immune
system. MBL plays a crucial role in innate immunity against yeast by enhanced
complement activation and enhanced uptake of polymorphonuclear cells 11 .
MBL binds to repetitive mannose and/or N-acetylglucosamine residues on
microorganisms, leading to opsonization and activation of the lectin
complement pathway. MBL also interacts with carbohydrates on the
glycoprotein (gp)120 of HIV-1. MBL may inhibit DC-SIGN-mediated uptake
and spread of HIV 12 . Differences exist in polysaccharide recognition, endocytic
capacities and microbe capture among CLRs. Furthermore, the ligands and
physiological functions of many of the CLRs are still unknown. Mincle is one
such orphan receptor. Mincle is the first example of a CLR that recognizes an
endogenous nuclear ligand and necrotic cells 13 .
1.Brown GD. et al. , 2003. Dectin-1 mediates the biological effects of beta glucans. J Exp
Med. 197: 1119- 24. 2. Gross O. et al., 2006. Card9 controls a non-TLR signaling pathway
for innate anti-fungal immunity. Nature. 442:651- 6. 3 Dennehy KM. & Brown GD., 2007.
The role of the beta-glucan receptor Dectin-1 in control of fungal infection . J Leukoc
Biol.;82(2):253-8. 4. Gantner BN. et al., 2003. Collaborative induction of inflammatory
responses by dectin-1 and Toll- like receptor 2. J Exp Med. 197: 1107-17. 5. Goodridge HS.
et al., 2007. Dectin-1 stimulation by Candida albicans yeast or zymosan triggers NFAT
activation in macrophages and dendritic cells. J Immunol. 178(5):3107-15. 6. Yamasaki S. et
al., 2009. C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia.
PNAS 106(6): 1897–1902. 7. Yamasaki S. et al., 2008. Mincle is an ITAM-coupled activating
receptor that senses damaged cells. Nat Immunol. 9(10):1179-88. 8. Ishikawa E. et al., 2009.
Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin
Mincle. J Exp Med. 206(13):2879-88. 9. Takahara K et al., 2004. Functional comparison of
the mouse DC-SIGN, SIGNR1, SIGNR3 and Langerin C-type lectins. Int Immunol. 16:819-829.
10. den Dunnen J. et al., 2008. Innate signaling by C-type lectin DC-SIGN dictates immune
responses. Cancer Immunol Immunother. 26:605-610. 11. Van Asbeck et al., 2008. Mannose
binding lectin plays a crucila role in innate immunity against yeast by enhanced comploment
activation and enhanced uptake of polymorphonuclear cells. BMC Microbiol. 8:229. 12. Ji X.
et al., 2005. Mannose-binding lectin binds to Ebola and Marburg envelope glycoproteins,
resulting in blocking of virus interaction with DC-SIGN and complement-mediated virus
neutralization. J Gen Virol. 86; 2535-2542. 13. Brown GD. 2008. Sensing necrosis with Mincle.
Nature Immunol. 9:1099-1100.
55
INNATE IMMUNITY 3

INNATE IMMUNITY 3
TLR, NLR, RLR & Related Genes
TLR, NLR, RLR & Related Genes is an expanding collection of genes encoding Toll-like receptors (TLRs), NOD-
like receptors (NLRs) and RIG-I-like receptors (RLRs) as well as proteins involved in their signaling pathways.
These genes are either native or modified by addition of a tag and/or deletion to generate dominant negative
(DN) variants. They are provided as full-length, entirely sequenced open reading frames (ORFs) cloned into
plasmidic expression vectors.
Native Genes (pUNO)
• Toll-Like receptor (TLR) & TLR co-receptor genes • Adaptor genes
• NOD-Like receptor (NLR) genes • Signaling genes
• RIG-I-Like receptor (RLR) genes • Signaling inhibitors
• Other pathogen sensor genes • Transcription factors
pUNO plasmids express native genes under the control of the strong and ubiquitous EF1a/HTLV composite promoter, comprised of the elongation
factor 1 alpha (EF-1a) core promoter and the R-U5’ of the human T cell leukemia virus (HTLV). pUNO plasmids are selectable with blasticidin allowing the
selection of stable mammalian clones in only a few days.
HA-Tagged Genes (pUNO-HA)
56
• TLR genes • Interferon regulatory factor (IRF) genes
pUNO-HA plasmids, which possess the same backbone as the pUNO plasmids, feature genes fused at their 3’ end to the influenza hemaglutinine (HA) tag.
This tag provides a simple and convenient method to detect genes by Western blot using the HA-tag antibody (see page 73).
Gene Associations (pDUO)
• TLR/TLR genes • Co-receptor/co-receptor genes
• TLR/co-receptor genes
pDUO plasmids contain two transcription units allowing the co-expression of two TLR or TLR-related genes. They feature two strong composite promoters
derived from the ferritin light chain (FerL) and heavy chain (FerH) core promoters. Most pDUO plasmids are selectable with blasticidin in both E. coli and
mammalian cells. Some are selectable with hygromycin (pDUO2).
Dominant Negative Variants (pZERO-TLR & pDeNy)
• TLR, NLR, RLR genes • Adaptor and signaling genes
pZERO-TLR and pDeNy plasmids express dominant negative variants created by insertion of a mutation and/or deletion of a key region such as the TIR
domain of the TLR genes. The variants are cloned under the control of the EF1a/HTLV composite promoter. pZERO-TLR and pDeNy are selectable with
puromycin and Zeocin , respectively.
HA-Tagged Dominant Negative Variants (pZERO-TLR-HA)
• TLR genes
pZERO-TLR-HA plasmids express HA-tagged TIR-deleted TLR genes that act as dominant negative variants. They can be detected by Western blot using
the HA-tag antibody (see page 73).
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pUNO
pUNO-HA
Toll-Like Receptors (TLRs)
pDUO
pZERO/pDeNy
TLR1 h, m, p h, m h, m h, m h, m
TLR2 h, m, p, b h, m h, m h, m h, m
TLR3 h, m h, m - h, m h, m
TLR4 h, m h, m h, m h, m h, m
TLR5 h, m, p, b h, m - h, m h, m
TLR6 h, m, p, b h, m h, m h, m h, m
TLR7 h, m, p, b h, m - h, m h, m
TLR8 h, m h, m - h, m h, m
TLR9 h, m, p, b h, m - h, m h, m
TLR10 h, p h - h h
TLR11 m - - - -
TLR11/12 m m - - -
TLR13 m - - - -
NOD-Like Receptors (NLRs)
IPAF / CARD12 h - - - -
NAIP5 / BIRC1E m - - - -
NALP1 h - - - -
NALP2 h - - - -
NALP3 / NLRP3 h - - - -
NALP12 / Monarch-1 h - - - -
NOD1 / CARD4 h, m - - h -
NOD2 / CARD15 h, m - - h -
NOD9 / NLRX1 h, m - - -
RIG-I-Like Receptors (RLRs)
LGP2 h, m - - - -
MDA-5 h - - - -
RIG-I / DDX58 h, m - - h -
Other Pathogen Sensors
AIM2 / IFI210 h, m - - - -
CLEC9A h - - - -
DAI / ZBP h, m - - - -
DC-SIGN / CD209 h, m - - - -
Dectin-1 h, m - - - -
IFI16 h, m - - - -
L-SIGN h - - - -
MBL1, 2 h, m - - - -
Mincle / CLEC4E h, m - - - -
PGRP-L, -S, -Ia h, m - - - -
SIGNR1, 2, 3, 4
Adaptors
m - - - -
ASC / PYCARD / CARD5 h, m - - - -
Cardinal / CARD8 h - - - -
IPS1 / MAVS / VISA h, m - - - -
MyD88 h, m - - h, m -
RAC1 m - - - -
SARM1 h, m - - - -
TIRAP / Mal h, m - - h -
TRAM / TICAM2 h, m - - h -
TRIF / TICAM1
Co-receptors
h, m - - h -
CD14 h, m - h, m - -
sCD14 (soluble) h - - - -
CD36 h, m - - - -
LBP h, m - - - -
MD2 / Ly96 h, m - h, m - -
PRAT4A, 4B h, m - - - -
pZERO-TLR-HA
Signaling Effectors
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pUNO
pUNO-HA
pDUO
pZERO/pDeNy
BCL10 / CLAP h, m - - - -
CARD9 h, m - - - -
DDX3 m - - - -
FADD / MORT1 h, m - - - -
IKKa, b h, m - - - -
IKKε h, m - - - -
IRAK-1, -4 h, m - - - -
ITCH h, m - - - -
MAPK1 h - - - -
MAPK2 h, m - - - -
NAP1 / AZI2 h, m - - - -
NEMO / IKKg h, m - - - -
Pellino1, 2, 3 h, m - - - -
PRKD1 h - - - -
PKR h, m - - h -
PRKRA / PACT h, m - - - -
RIP1 / RIPK1 h, m - - - -
RIP2 / RIPK2 h, m - - - -
RIP3 / RIPK3 h, m - - - -
STING h, m - - - -
SUGT1 h, m - - - -
Syk h, m - - - -
TAB1, 2, 3 h - - - -
TAK1 / MAP3K7 h, m - - - -
TANK h, m - - - -
TBK1 h, m - - - -
TIFA h, m - - - -
TRADD h, m - - - -
TRAF3 h, m - - - -
TRAF6 h, m - - - -
UNC93B1 h, m - - - -
Signaling Inhibitors
ABIN1, 2, 3 h, m - - - -
ATF3 h, m - - - -
AXL / UFO h, m - - - -
Bcl-3 h, m - - - -
DAK h, m - - - -
DUBA / OTUD5 m - - - -
FLII / Fliih h, m - - - -
IRAK-M h, m - - - -
MD-1 h, m - h, m - -
MFN2 h, m - - - -
MULAN / Dublin h, m - - - -
PIN1 / DOB h, m - - - -
PPP3CA, CB h, m - - - -
PPP3R1 h, m
RNF125 / TRAC-1 h, m - - - -
RP105 h, m - h, m - -
SHP-1 / PTPN6 h, m - - - -
SIGIRR / TIR8 h, m - - - -
SIKE h, m - - - -
T1 / ST2 / IL1RL1 h - - - -
TNFAIP3 / A20 h, m - - - -
Tollip / IL-1RAcPIP h, m - - - -
TRAFD1 / FLN29 h, m - - - -
TRIAD3A h, m - - - -
Tyro3 / BYK h, m - - - -
pZERO-TLR-HA
57
INNATE IMMUNITY 3

INNATE IMMUNITY 3
pUNO - Native Genes
Features and Benefits
Numerous genes encoding pattern recognition receptors (PRRs), co-receptors, adaptors,
signaling effectors and signaling inhibitors are available in the pUNO plasmid. The genes are
cloned from the ATG to the Stop codon, excluding introns and untranslated regions. All
genes are fully sequenced, the sequences are available online at www.invivogen.com or can
be emailed upon request.
PRR and related genes are cloned under the control of the strong and ubiquitous mammalian
promoter, EF1a/HTLV. This composite promoter is comprised of the elongation factor 1
alpha (EF-1a) core promoter and the R-U5’ of the human T cell leukemia virus (HTLV).
pUNO plasmids can be used directly for in vitro or in vivo transfection experiments. They are
selectable with blasticidin in both E. coli and mammalian cells. To facilitate the excision and
subcloning of the gene of interest into another vector, each gene is flanked by unique
restriction sites that are compatible with many others.
Some genes are provided in the pUNO2 (Zeocin -resistant) or pUNO3 (hygromycinresistant)
backbone
Contents and Storage
Each pUNO plasmid is provided as a lyophilized transformed E. coli strain on a paper disk.
Transformed strains are shipped at room temperature and should be stored at -20ºC.
Lyophilized E. coli cells are stable for at least 1 year when properly stored. Each plasmid is
provided with 4 pouches of E. coli Fast-Media ® Blas (2 TB and 2 Agar).
58
Toll-Like Receptors (TLRs)
TLR genes from different species are available:
- human
- mouse
- pig
- bovine
Human TLR genes are available in two different plasmid backbones:
- pUNO/pUNO1 selectable with blasticidin
- pUNO3, selectable with hygromycin
GENE NAME DESCRIPTION
TOLL-Like Receptors (TLRs)
CAT. CODE
(human)
CAT. CODE
(human, hygro)
CAT. CODE
(mouse)
TLR1 Toll-like Receptor 1 puno-htlr1 puno3-htlr1 puno-mtlr1 puno1-ptlr1 NEW -
TLR2 Toll-like Receptor 2 puno-htlr2 puno3-htlr2 puno-mtlr2 puno1-ptlr2 NEW puno1-btlr2 NEW
TLR3 Toll-like Receptor 3 puno-htlr3 puno3-htlr3 puno-mtlr3 -
TLR4 Toll-like Receptor 4 puno1-htlr4a puno3-htlr4a puno-mtlr4 -
TLR5 Toll-like Receptor 5 puno-htlr5 puno3-htlr5 puno-mtlr5 puno1-ptlr5 NEW puno1-btlr5 NEW
TLR6 Toll-like Receptor 6 puno-htlr6 puno3-htlr6 puno-mtlr6 puno1-ptlr6 NEW puno1-btlr6 NEW
TLR7 Toll-like Receptor 7 puno-htlr7 puno3-htlr7 puno-mtlr7 puno1-ptlr7 NEW puno1-btlr7 NEW
TLR8 Toll-like Receptor 8 puno1-htlr8b puno3-htlr8b puno-mtlr8 -
TLR9 Toll-like Receptor 9 puno1-htlr9a puno3-htlr9a puno-mtlr9 puno1-ptlr9 NEW puno1-btlr9 NEW
TLR10 Toll-like Receptor 10 puno-htlr10 puno3-htlr10 - puno1-ptlr10 NEW
TLR11 Toll-like Receptor 11 - - puno-mtlr11t -
TLR11/12 Toll-like Receptor 11/12 - - puno-mtlr11z -
TLR13 Toll-like Receptor 13 - - puno-mtlr13 -
www.invivogen.com/innate-immunity-puno
Related Products
Blasticidin, page 17
Hygromycin B, HygroGold , page 18
Fast-Media ® Blas, page 45
Fast-Media ® Hygro, page 45
CAT. CODE
(pig)
CAT. CODE
(bovine)

GENE NAME/ALIASES DESCRIPTION CAT. CODE (HUMAN) CAT. CODE (MOUSE)
NOD-Like Receptors (NLRs)
IPAF / CARD12 Flagellin receptor - Caspase-1-activating protein puno-hcard12 -
NAIP5 / BIRC1E Flagellin receptor - Caspase-1-activating protein - puno1-mnaip5
NALP1 / CARD7 Anthrax toxin receptor - Caspase-1-activating protein puno-hnalp1a -
NALP2 Caspase-1-activating protein puno-hnalp2 -
NALP3 / NLRP3 Receptor of various ligands - Caspase-1-activating protein puno-hnalp3a -
NALP12 / Monarch-1 Negative regulator of inflammation puno-hnalp12 -
NOD1 / CARD4 PGN receptor puno-hnod1 puno-mnod1
NOD2 / CARD15 PGN receptor puno-hnod2a puno-mnod2a
NOD9 / NLRX1 Mitochondrial PRR modulator puno1-hnod9a puno1-mnod9
RIG-I-Like-Receptors (RLRs)
LGP2 Negative regulator of RIG-I and MDA-5 puno1-hlgp2 puno1-mlgp2
MDA5 / IFIH1 dsRNA receptor puno1-hmda5 puno1-mmda5
RIG-I / DDX58 dsRNA receptor puno-hrigi puno-mrigi
Other Pathogen Sensors
AIM2 / IFI210 Cytoplasmic DNA receptor - Caspase-1-activating protein puno1-haim2 puno1-maim2
CLEC9A C-type lectin-like receptor - Ligand unknown puno1-hclec9a puno1-mclec9a
DAI / ZBP1 B-DNA binding protein puno1-hzbp1 puno1-mzbp1a
DC-SIGN / CD209 Mannose-binding C-type lectin puno-hdcsign1a puno-mdcsign
Dectin-1 Beta-glucan receptor puno-hdectin1b puno-mdectin1
IFI16 NEW Cytosolic DNA sensor puno1-hifi16 puno1-mifi16
L-SIGN Mannose-binding C-type lectin puno-hdcsign2a -
MBL1, 2 Mannose binding lectins - puno-mmbl1/2
Mincle / CLEC4E Macrophage-inducible C-type lectin puno1-hmincle puno1-mmincle
PGPR-L, -S Peptidoglycan recognition protein, long or short puno-hpgrps puno-mpgrpl/s
PGPR-1A Peptidoglycan Recognition protein 1 a puno-hpgrp1a -
SIGNR1, 2, 3, 4 DC-SIGN related proteins 1, 2, 3, 4 - puno-msignr1/2/3/4
Adaptors
ASC / PYCARD / CARD5 NLR adaptor protein puno-hasca puno1-masc
Cardinal / CARD8 NALP3 adaptor protein porf-hcard8 -
IPS1 / MAVS / VISA RLR adaptor protein puno-hips1 puno-mips1
MyD88 TLR/IL-1R adaptor protein puno-hmyd88 puno-mmyd88
RAC1 Regulator of TLR2 and TLR4 signaling - puno-mrac1
SARM1 Specific inhibitor of TRIF-dependent TLR signaling puno-hsarm1a puno-msarm1b
TIRAP / Mal Adaptor protein of MyD88-dependent TLR signaling puno-htirap puno-mtirap
TRAM / TICAM2 Adaptor protein of MyD88-independent TLR4 signaling puno-htram puno-mtram
TRIF / TICAM1 Adaptor protein of MyD88-independent TLR3/4 signaling puno2-htrif puno-mtrif
Co-receptors
CD14 Membrane-associated co-receptor of TLR4 puno-hcd14 puno-mcd14
CD36 Scavenger receptor interacting with TLR2 puno3-hcd36 puno3-hcd36
LBP LPS Binding Protein puno-hlbp puno-mlbp
MD2 / Ly96 Accessory molecule for LPS-induced TLR4 signaling puno-hmd2 puno-mmd2
PRAT4A, 4B Regulators of cell surface expression of TLR4 puno1-hprat4a/4b puno1-mprat4a/4b
Signaling Effectors
BCL10 / CLAP NF-kB activator puno1-hbcl10 puno1-mbcl10
CARD9 Mediator of NOD2 & Dectin-1 signaling puno-hcard9 puno-mcard9
DDX3 / DDX3X Mediator of TBK1/IKKε signaling - puno-mddx3x
FADD / MORT1 Effector of Fas-mediated apoptosis puno-hfadd puno-mfadd
IKKa / IKBKA Subunit alpha of IkappaB Kinase puno-hikka puno-mikka
IKKb / IKBKB Subunit beta of IkappaB Kinase puno-hikkb puno-mikkb
IKKε / IKBKE Effector of RIG-I/MDA-5-induced activation of IRF puno-hikke puno-mikke
IRAK-1 Signal transduction mediator for TLR signaling puno-hirak1 puno-mirak1
IRAK-4 Signal transduction mediator for TLR signaling puno-hirak4 puno-mirak4
www.invivogen.com/innate-immunity-puno
59
INNATE IMMUNITY 3

INNATE IMMUNITY 3
60
GENE NAME/ALIASES DESCRIPTION CAT. CODE (HUMAN) CAT. CODE (MOUSE)
Signaling Effectors
ITCH NEW p38 and JNK activator and NF-kB inhibitor puno1-hitch puno1-mitch
MAPK1 NEW p38 and JNK activator puno1-hmapk1 -
MAPK2 NEW p38 and JNK activator puno1-hmapk2 puno1-mmapk2
NAP1 / AZI2 Regulatory subunit of TBK1/IKKε puno-hnap1 puno-mnap1
NEMO / IKKg Regulatory subunit of IKK complex puno-hnemo puno-mnemo
Pellino1 Co-factor of IL-1-mediated signaling puno-hpeli1 puno-mpeli1
Pellino2 Modulator of IL-1 and LPS signaling puno-hpeli2 puno-mpeli2
Pellino3 Promoter of c-Jun and Elk-1 activation puno-hpeli3a -
PKD1 / PKRD Protein kinase D1 puno1-hprkd1 -
PKR Mediator in dsRNA-induced TLR3 and LPS-induced TLR4 signaling puno-hpkr puno-mpkr
PRKRA / PACT Modulator of PKR activity puno-hprkra puno-mprkra
RIP1 / RIPK1 Mediator of TLR3-induced NF-kB activation puno-hripk1 puno-mripk1
RIP2 / RIPK2 Signal transducer for TLRs and NODs puno-hrick puno-mrick
RIP3 / RIPK3 Modulator of NF-kB activation puno-hripk3 puno-mripk3
STING NEW Stimulator of interferon genes puno1-hsting puno1-msting
SUGT1 NEW Regulator of NOD1 activation puno1-hsugt1b puno1-msugt1
Syk Mitochondrial PRR modulator puno1-hsyk puno1-msyk
TAB1, 2, 3 TAK1 binding proteins 1, 2 ,3 - Mediators of NF-kB activation puno-htab1/2a/3 -
TAK1 / MAP3K7 Modulator of TLR3/TLR4-mediated NF-kB and AP-1 activation puno-hmap3k7b puno-mmap3k7b
TANK Mediator of RLR- and TLR-induced IFN production puno1-htank puno1-mtank
TBK1 Mediator of RLR- and TLR-induced IFN production puno-htbk1 puno-mtbk1
TIFA Mediator of TRAF6/IRAK1 complexes puno-htifa puno-mtifa
TRADD TNF-R1-Associated via Death Domain puno1-htradd puno1-mtradd
TRAF3 Mediator of TBK1 signaling puno-htraf3 puno-mtraf3
TRAF6 Key signal transducer of TLR pathways puno-htraf6 puno-mtraf6
UNC93B1 Multitransmembrane endoplasmic reticulum protein puno1-hunc93b1 puno1-munc93b1
Signaling Inhibitors
ABIN1, 2, 3 A20-associating protein inhibitor of NF-kB puno1-htnip1a/2a/3 puno1-mtnip1a/2/3
ATF3 Negative regulator of TLR signaling puno-hatf3 puno-matf3
AXL / UFO Negative regulator of TLR signaling puno1-haxl puno1-maxl
BCL3 Inhibitor of NF-kB p50 ubiquitination puno1-hbcl3 puno1-mbcl3
DAK Negative regulator of MDA-5 puno1-hdak puno1-mdak
DUBA / OTUD5 TRAF3 inhibitor - puno1-motud5
FLII / Fliih TLR4/MyD88 signaling complex inhibitor puno-hflii puno-mflii
IRAK-M Inhibitor of IRAK-1/TRAF6 complexes puno-hirakm puno-mirakm
MD1 RP105 co-receptor and inhibitor TLR4/MD2 complex puno-h md1 puno-m md1
MFN2 NEW IPS-1 inhibitor puno1-hmfn2 puno1-mmfn2
MULAN/ Dublin Inhibitor of RIG-I/MDA-5 signaling puno1-hmul1 puno1-mmul1
PIN1 / DOB IRF3 inhibitor puno-hpin1 puno-mpin1
PPP3CA NEW MyD88 and TRIF inhibitor - Calcineurin A (NFAT pathway) puno1-hppp3cab puno1-mppp3ca
PPP3CB NEW MyD88 and TRIF inhibitor - Calcineurin B (NFAT pathway) puno1-hppp3cb puno1-mppp3cb
PPP3R1 NEW MyD88 and TRIF inhibitor puno1-hppp3r1 puno1-mppp3r1
RNF125 / TRAC-1 Inhibitor of RIG-I signaling puno1-hrnf125 puno1-mrnf125
RP105 Inhibitor TLR4/MD2 complex puno-hrp105 puno-mrp105
SHP-1 / PTPN6 Negative regulator of TLR signaling puno1-hptpn6 puno1-mptpn6
SIGIRR / TIR8 Negative regulator of TLR/IL-1R signaling puno-hsigirr puno-msigirr
SIKE Physiological repressor of IKKε and TBK1 puno1-hsike puno1-msike
T1 / ST2 / IL1RL1 Negative regulator of IL-1R and TLR2, 4, 9 signaling puno-hil1rl1b -
TNFAIP3 / A20 Inhibitor of TLR and RLR-induced NF-kB activation puno-htnfaip3 puno-mtnfaip3
Tollip / IL-1RAcPIP Inhibitor of NF-kB activation induced by IL-1R, TLR2 and TLR4 puno-htollip puno-mtollip
TRAFD1 / FLN29 Inhibitor of LPS-induced TRAF6 function puno-htrafd1 puno1-mtrafd1
TRIAD3A Negative regulator of TIRAP, TRIF and RIP1 puno-htriad3a puno-mtriad3a
Tyro3 / BYK Negative regulator of TLR signaling puno1-htyro3 puno1-mtyro3
www.invivogen.com/innate-immunity-puno

pDUO - Gene Associations
Features and Benefits
TLR Associations in a Single Plasmid
pDUO is an expression vector containing two transcription units allowing
the co-expression of two TLR or TLR-related genes.
Strong & Similar Levels of Expression of Both Genes
pDUO plasmids feature two strong composite promoters derived from
the ferritin light chain (FerL) and heavy chain (FerH) core promoters.
Both promoters work concomitantly to express Ferritin, an ubiquitous
protein, therefore eliminating potential transcriptional interferences.
Rapid Selection of Stable Transfectants
Each pDUO can be used for transient or stable transfection experiments.
Most pDUO plasmids are selectable with blasticidin in both E. coli and
mammalian cells. Some are selectable with hygromycin (pDUO2).
Contents and Storage
Each pDUO plasmid is provided as a lyophilized transformed E. coli strain
on a paper disk. Transformed strains are shipped at room temperature and
should be stored at -20ºC. Lyophilized E. coli cells are stable for at least 1
year when properly stored.
Each pDUO plasmid is provided with 4 pouches of the appropriate E.coli
Fast-Media ® (2 TB and 2 Agar).
PRODUCT QTY CAT. CODE*
pDUO- (Blasti) E. coli disk pduo-
pDUO2- (Hygro)
*See table
E. coli disk pduo2-
Related Products
Blasticidin, page 17 Hygromycin B, page 18
Fast-Media ® Blas, page 45 Fast-Media ® Hygro, page 45
TLR Associations Available in pDUO
Plasmids
TLR/TLR Associations
- TLR1/TLR2
- TLR6/TLR2
TLR/Co-receptor Associations
- CD14/TLR2
- CD14/TLR4
- MD2/TLR4
Co-receptor/Co-receptor Associations
- MD2/CD14 (available in pDUO2)
- RP105/MD1
GENES PLASMID DESCRIPTION CAT. CODE (human) CAT. CODE (mouse)
CD14/TLR2 pDUO-CD14-TLR2 CD14 with Toll-like receptor 2 pduo-hcd14tlr2 pduo2-mcd14tlr2
CD14/TLR4 pDUO-CD14-TLR4 CD14 with Toll-like receptor 4 pduo-hcd14tlr4 pduo-mcd14tlr4
MD2/CD14 pDUO2-MD2-CD14 (hygro) MD2 with CD14 pduo2-hmd2cd14 pduo2-mmd2cd14
MD2/TLR4 pDUO-MD2-TLR4 MD2 with Toll-like receptor 4 pduo-hmd2tlr4 pduo-mmd2tlr4
RP105/MD1 pDUO-RP105/MD1 RP105 (CD180) with MD1 pduo-h rp105md1 pduo-mrp105md1
TLR1/TLR2 pDUO-TLR1/TLR2 Toll-like receptors 1 and 2 pduo-htlr12 pduo-mtlr12
TLR6/TLR2 pDUO-TLR6/TLR2 Toll-like receptors 6 and 2 pduo-htlr62 pduo-mtlr62
www.invivogen.com/innate-immunity-pduo
61
INNATE IMMUNITY 3

INNATE IMMUNITY 3
pUNO-HA - TLR-HA Genes
Features and Benefits
pUNO-HA is a family of expression vectors featuring HA-tagged TLR and IRF (Interferon
Regulatory Factor) genes. The genes have been fused at the 3’end to the influenza
hemaglutinine (HA) tag. This short sequence (YPYDVPDYA) encodes a peptide which is the
epitope of a very efficient and specific monoclonal antibody. The use of HA-tagged TLR genes
provides a simple and convenient method to detect the expression of the TLR genes by
Western blot. All TLR genes can be detected using the same primary antibody, the HA tag
monoclonal antibody (see page 73).
pUNO-HA plasmids are selectable with blasticidin in both E. coli and mammalian cells.
pUNO-TLR-GFP - TLR-GFP Fusion Genes
TLR-GFP fusion proteins can be used to study the localization of the TLRs. Transfected
cells can be analyzed for GFP expression by flow cytometry and by Western-blotting
using GFP antibodies.
Features and Benefits
TLR-GFP fusion genes were generated by fusing the GFP gene to the C terminus of various
human TLR genes (TLR1 to TLR6). The TLR-GFP fusion genes are cloned in the pUNO
plasmid under the control of the strong and ubiquitous mammalian promoter EF1a/HTLV.
The pUNO plasmid is selectable with blasticidin in both E. coli and mammalian cells.
All TLR::GFP fusion genes have been fully sequenced, their fluorescence confirmed and their
function tested in HEK293 cells coexpressing an NF-kB reporter plasmid and stimulated with
the appropriate ligand.
Contents and Storage
62
GENE PLASMID QTY
CAT. CODE
(human)
CAT. CODE
(mouse)
TLR1-HA pUNO-TLR1-HA E. coli disk punoha-htlr1 punoha-mtlr1
TLR2-HA pUNO-TLR2-HA E. coli disk punoha-htlr2 punoha-mtlr2
TLR3-HA pUNO-TLR3-HA E. coli disk punoha-htlr3 punoha-mtlr3
TLR4-HA pUNO-TLR4-HA E. coli disk punoha-htlr4a punoha-mtlr4
TLR5-HA pUNO-TLR5-HA E. coli disk punoha-htlr5 punoha-mtlr5
TLR6-HA pUNO-TLR6-HA E. coli disk punoha-htlr6 punoha-mtlr6
TLR7-HA pUNO-TLR7-HA E. coli disk punoha-htlr7 punoha-mtlr7
TLR8-HA pUNO-TLR8-HA E. coli disk punoha-htlr8a punoha-mtlr8
TLR9-HA pUNO-TLR8-HA E. coli disk punoha-htlr9a punoha-mtlr9
TLR10-HA pUNO-TLR10-HA E. coli disk punoha-htlr10 -
TLR11-HA pUNO-TLR11-HA E. coli disk - punoha-mtlr11
Each pUNO-TLR-GFP plasmid is provided as a lyophilized transformed
E. coli strain on a paper disk. Transformed strains are shipped at room
temperature and should be stored at -20ºC. Lyophilized E. coli cells are
stable for at least 1 year when properly stored.
Each plasmid is provided with 4 pouches of E. coli Fast-Media ® Blas (2 TB
and 2 Agar, see pages 44-45).
www.invivogen.com/innate-immunity
Contents and Storage
Each pUNO-HA plasmid is provided as a lyophilized
transformed E. coli strain on a paper disk. Transformed
strains are shipped at room temperature and should
be stored at -20ºC. Lyophilized E. coli cells are stable
for at least 1 year when properly stored.
Each plasmid is provided with 4 pouches of E.coli
Fast-Media ® Blas (2 TB and 2 Agar). For more
information about Fast-Media ® , see pages 44-45.
GENE PLASMID QTY CAT. CODE
hTLR1-GFP pUNO-hTLR1-GFP E. coli disk phtlr1-gfp
hTLR2-GFP pUNO-hTLR2-GFP E. coli disk phtlr2-gfp
hTLR3-GFP pUNO-hTLR3-GFP E. coli disk phtlr3-gfp
hTLR4-GFP pUNO-hTLR4-GFP E. coli disk phtlr4-gfp
hTLR5-GFP pUNO-hTLR5-GFP E. coli disk phtlr5-gfp
hTLR6-GFP pUNO-hTLR6-GFP E. coli disk phtlr6-gfp

pZERO-TLR & pDeNy - Dominant Negative TLR & TLR Signaling Genes
Features and Benefits
DN TLR Genes
Dominant negative (DN) TLR genes were generated by deleting a
fragment of approximately 500 bp located at the 3’ end of each TLR gene
corresponding to the TIR domain (TLR-DTIR genes). These truncated
genes are still able to recognize their ligands but are unable to induce the
signaling cascade.
DN TLR genes are provided in the pZERO plasmid. pZERO-TLR plasmids
are selectable with puromycin.
HA-Tagged DN TLR Genes
DN TLR genes are available with the influenza hemaglutinine (HA) tag
fused at their 3’ end to facilitate their detection by Western blot. The HAtagged
DN TLR proteins can be detected using the anti-HAtag antibody
(see page 73).
HA-tagged DN TLR genes are also provided in the pZERO plasmid.
pZERO-TLR-HA plasmids are selectable with puromycin.
DN TLR Signaling Genes
DN forms of TLR signaling genes were created by inserting a mutation
within the gene and/or deleting a region of the gene. These modifications
have been described elsewhere (see table) and shown to block the wildtype
form of these genes.
DN TLR signaling genes are provided in the pDeNy plasmid which is
selectable with Zeocin .
pZERO or pDeNy plasmids can be cotransfected with a pUNO plasmid
(see page 58). Selection of stable clones expressing both plasmids,
pZERO/pUNO or pDeNy/pUNO, is achieved with addition of puromycin
and blasticidin or Zeocin and blasticidin, respectively.
Contents and Storage
pZERO-TLR, pZERO-TLR-HA and pDeNy plasmids are provided as
lyophilized transformed E. coli strains on paper disk. Transformed strains
are shipped at room temperature and should be stored at -20ºC.
Lyophilized E. coli cells are stable for at least 1 year when properly stored.
pZERO-TLR and pZERO-TLR-HA plasmids are provided with 4 pouches
of E. coli Fast-Media ® Puro, while pDeNy plasmids are provided with 4
pouches of E.coli Fast-Media ® Zeo (2 TB and 2 Agar).
Related Products
Puromycin, page 17 Zeocin , page 18
Anti-HAtag antibody, page 73 Fast-Media ® , page 45
PLASMID QTY
www.invivogen.com/innate-immunity
CAT. CODE
(human)
CAT. CODE
(mouse)
DN TLR Genes
pZERO-TLR1 E. coli disk pzero-htlr1 pzero-mtlr1
pZERO-TLR2 E. coli disk pzero-htlr2 pzero-mtlr2
pZERO-TLR3 E. coli disk pzero-htlr3 pzero-mtlr3
pZERO-TLR4 E. coli disk pzero-htlr4 pzero-mtlr4
pZERO-TLR5 E. coli disk pzero-htlr5 pzero-mtlr5
pZERO-TLR6 E. coli disk pzero-htlr6 pzero-mtlr6
pZERO-TLR7 E. coli disk pzero-htlr7 pzero-mtlr7
pZERO-TLR8 E. coli disk pzero-htlr8 pzero-mtlr8
pZERO-TLR9 E. coli disk pzero-htlr9 pzero-mtlr9
pZERO-TLR10 E. coli disk pzero-htlr10 -
HA-Tagged DN TLR Genes
pZERO-TLR1-HA E. coli disk pzero-htlr1-ha pzero-mtlr1-ha
pZERO-TLR2-HA E. coli disk pzero-htlr2-ha pzero-mtlr2-ha
pZERO-TLR3-HA E. coli disk pzero-htlr3-ha pzero-mtlr3-ha
pZERO-TLR4-HA E. coli disk pzero-htlr4-ha pzero-mtlr4-ha
pZERO-TLR5-HA E. coli disk pzero-htlr5-ha pzero-mtlr5-ha
pZERO-TLR6-HA E. coli disk pzero-htlr6-ha pzero-mtlr6-ha
pZERO-TLR7-HA E. coli disk pzero-htlr7-ha pzero-mtlr7-ha
pZERO-TLR8-HA E. coli disk pzero-htlr8-ha pzero-mtlr8-ha
pZERO-TLR9-HA E. coli disk pzero-htlr9-ha pzero-mtlr9-ha
pZERO-TLR10-HA E. coli disk pzero-htlr10-ha -
PLASMID SPECIES
MUTATION/
DELETION
CAT. CODE
DN TLR Signaling Genes
pDeNy-hIRAK human aa1-211 pdn-hirak
pDeNy-hMyD88 human aa161-296 pdn-hmyd88
pDeNy-mMyD88 mouse aa161-296 pdn-mmyd88
pDeNy-hNOD1 human aa127-518 pdn-hnod1
pDeNy-hNOD2 human L145P pdn-hnod2
pDeNy-hPKR human aa361-366 pdn-hpkr
pDeNy-hRIG-I human aa1-217 pdn-hrigi
pDeNy-hTIRAP human P125H pdn-htirap
pDeNy-hTRAM human C117H pdn-htram
pDeNy-hTRAF6 human aa289-522 pdn-traf6
pDeNy-hTRIF human aa387-566
+ P434H
pdn-htrif
63
INNATE IMMUNITY 3

Reporter Cell Lines
Engineered HEK293 Cells
Cells that constitutively express a given functional TLR gene are valuable tools for many applications, such as the study of the mechanisms
involved in TLR recognition or signaling, and the development of new potential therapeutic drugs. InvivoGen provides HEK293 cells stably
expressing human or murine TLR or NOD genes.
• 293/TLR & 293/NOD Clones
• HEK-Blue TLR and HEK-Blue NOD Cells
Immune Reporter Cells
InvivoGen provides THP1-XBlue , Ramos-Blue and RAW-Blue Cells, derived from a human monocytic cell line, a human B lymphocyte and
murine macrophages, respectively. They naturally express a large repertoire of different classes of pattern recognition receptors (PRRs), such
as the TLRs, NLRs and RLRs and stably express an NF-kB-inducible SEAP reporter gene to facilitate the study of these PRRs.
• THP1-XBlue
Cells
• Ramos-Blue Cells
• RAW-Blue
Cells
MEF Reporter Cell Lines
InvivoGen provides murine embryonic fibroblasts (MEFs) isolated from various mouse strains and immortalized with the SV40 large antigen.
They express a large repertoire of pattern recognition receptors. InvivoGen’s MEFs express an NF-kB-inducible reporter (SEAP) system
allowing the convenient monitoring of NF-kB activation following TLR or RLR stimulation.
• C3H/TLR4mut & C3H/WT MEFs
• C57/WT MEFs
Expression of TLR, NOD1/2 and RIG-I/MDA-5 mRNAs and their activity in InvivoGen’s Reporter Cell Lines*
INNATE IMMUNITY 3+/- - +/- - +/- +/- - - - - +/- - - -
64
REPORTER CELL LINES TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9 TLR10 NOD1 NOD2 RIG-I MDA-5
HEK293-Null cells
THP1-XBlue Cells
Ramos-Blue Cells
RAW-Blue Cells
C3H MEFs
C57 MEFs
+ - + - + + - - - - + - - -
+ + +/- + + + + + + + + + + +
+ + - + + + - + - ND + ? ND ND
+ + + + + + + + + + + - + +
+/- +/- + - - +/- + - + + + - ND ND
+ + + + +/- + + + + - +/- + + +
+ + +/- + - + + ND + - - + ND ND
+ + + + - + - - - - + + + +
+ + + + - + - - - - ND ND ND ND
+ + + + - + + + + - + + + +
ND ND ND ND - ND ND ND ND - ND ND + +
Rows with no background correspond to mRNA expression,rows with a grey background correspond to the activity
B16-Blue IFN-a/b Cells
InvivoGen provides B16-Blue IFN-a/b, a cell line that in addition to detect bioactive murine type I IFNs can also be used to study
RIG-I/MDA-5 agonists, such as transfected poly(I:C) or 5’ppp-dsRNA (see page 106).
www.invivogen.com/reporter-cells

293/TLR & 293/NOD Clones
293/TLR Clones
293/TLR clones are HEK293 cells stably transfected with a pUNO-TLR or
pDUO-TLR plasmid. They can be used to determine TLR activation upon ligand
stimulation by assessing IL-8 production or NF-kB activation. The latter can be
evaluated by transfecting transiently or stably 293/TLR clones with pNiFty, a
family of NF-kB inducible reporter plasmids expressing either the secreted
alkaline phosphatase or luciferase reporter gene (see page 74).
293/TLR-HA Clones
293/hTLR-HA clones were obtained by stably transfecting HEK293 cells with a
pUNO-TLR-HA plasmid. pUNO-TLR-HA plasmids express TLR genes that have
been fused at the 3’end to the influenza hemaglutinine (HA) tag. Addition of this
tag has no deleterious effect on the expression and function of the TLR genes.
The HA tag is the epitope of a very efficient and specific monoclonal antibody
(see page 73).
293/NOD Clones
293/NOD clones are transfected HEK293 cells that express stably the NOD1
or NOD2 genes. These clones can be used to study NOD1 and NOD2
activation pathways following stimulation with their cognate ligand by assessing
IL-8 production or NF-kB activation. NF-kB activation can be assessed using
an NF-kB-inducible reporter system such as pNiFty.
293/Control Clones
293/Control clones were generated by stably transfecting HEK293 cells with a
pUNO control plasmid expressing either the lacZ reporter gene (293/LacZ)
or a multiple cloning site (293/null).
Pam3CSK4 (100 ng/ml)
Poly(I:C) (100 ng/ml)
LPS-EB (100 ng/ml)
Flagellin (10 ng/ml)
Imiquimod (10 μg/ml)
ssRNA40 (5 μg/ml)
ODN 2006 (10 μg/ml)
iEDAP (100 ng/ml)
MDP (100 ng/ml)
TNF-a (50 ng/ml)
NI
293/null
293/hNOD1
293/hNOD2
293/hTLR2
293/hTLR3
293/hTLR4-MD2-CD14
293/hTLR5
293XL/null
293XL/hTLR7
293XL/hTLR8
293XL/hTLR9
TLR and NOD induction profile of 293 clones : 293/TLR, NOD and control clones were
transfected transiently with pNiFty-SEAP and stimulated with TLR and NOD ligands. After
16 hour stimulation, NF-kB-induced SEAP activity was assessed using QUANTI-Blue , a
SEAP-detection medium.
Note: 293 clones and in particular 293XL clones express endogenous levels of TLR3 and
TLR5, as well as TLR1, TLR6 and NOD1.
CELL LINE
293/TLR Clones
CAT. CODE
(HUMAN)
CAT. CODE
(MOUSE)
293/MD2-CD14 293-hmd2cd14 -
293/TLR1 - 293-mtlr1
293/TLR1/2 - 293-mtlr1/2
293/TLR2 293-htlr2 293-mtlr2
293/TLR2-CD14 293-htlr2cd14 -
293/TLR2/6 293-htlr2/6 293-mtlr2/6
293/TLR3 293-htlr3 293-mtlr3
293/TLR4 293-htlr4a 293-mtlr4
293/TLR4-MD2-CD14 293-htlr4md2cd14 293-mtlr4md2cd14
293/TLR5 293-htlr5 293-mtlr5
293/TLR5-CD14 293-htlr5cd14 -
293/TLR6 - 293-mtlr6
293XL/TLR7* 293xl-htlr7 293xl-mtlr7
293XL/TLR8A* 293xl-htlr8 -
293/TLR9 - 293-mtlr9
293XL/TLR9A* 293xl-htlr9 -
293/hTLR-HA Clones
293/hTLR1-HA 293-htlr1ha -
293/hTLR2-HA 293-htlr2ha -
293/hTLR3-HA 293-htlr3ha -
293/hTLR4-HA 293-htlr4ha -
293/hTLR5-HA 293-htlr5ha -
293/hTLR6-HA 293-htlr6ha -
293XL/hTLR7-HA* 293xl-htlr7ha -
293XL/hTLR8-HA* 293xl-htlr8ha -
293XL/hTLR9-HA* 293xl-htlr9ha -
293/hTLR10-HA
293/NOD Clones
293-htlr10ha -
293/NOD1 293-hnod1 293-mnod1
293/NOD2 293-hnod2 293-mnod2
293/Control Clones
293/LacZ 293-lacz -
293/null 293-null -
293XL/null* 293xl-null -
*293XL clones express the human anti-apoptotic Bcl-XL gene.
Contents
All 293 clones are grown in standard DMEM medium with 10% FBS,
2mM L-glutamine supplemented with blasticidin (10 μg/ml). Cells are
provided frozen in a cryotube containing 5-7 x 10 6
cells and supplied
with 100 μl of blasticidin at 10 mg/ml. Cells are shipped on dry ice.
The cells are guaranteed mycoplasma-free.
Related Products
www.invivogen.com/reporter-cells
Blasticidin, page 17 TLR Ligands, pages 77-80
pNiFty, see page 74 NOD Ligands, see page 80
65
INNATE IMMUNITY 3

INNATE IMMUNITY 3
HEK-Blue TLR & HEK-Blue NOD Cells
InvivoGen introduces HEK-Blue TLR and HEK-Blue NOD cells, a collection of engineered cell lines designed to provide a rapid, sensitive and reliable method
to screen and validate TLR and NOD agonists or antagonists. They express an NF-kB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene
that can be conveniently monitored using the SEAP detection media QUANTI-Blue or HEK-Blue Detection.
HEK-Blue TLR cells
HEK-Blue TLR cells are engineered HEK293 cells that stably co-express a
human or murine TLR gene and an NF-kB-inducible SEAP (secreted
embryonic alkaline phosphatase) reporter gene. To increase the sensitivity
to their cognate agonists, HEK-Blue TLR2 and HEK-Blue TLR4 cells were
further transfected with the co-receptors CD14 and MD2/CD14,
respectively.
HEK-Blue TLR cells are resistant to the selective antibiotics blasticidin
and Zeocin . HEK-Blue TLR2 and HEK-Blue TLR4 cells are additionally
resistant to hygromycin.
HEK-Blue NOD cells
HEK-Blue NOD cells are engineered HEK293 cells that stably co-express
the human and murine NOD1 or NOD2 gene and an NF-kB-inducible SEAP
reporter gene.
HEK-Blue NOD cells are resistant to blasticidin and Zeocin .
HEK-Blue Null cells
HEK-Blue Null cells are the parental cell lines used to generate
HEK-Blue TLR and HEK-Blue NOD cells.
• HEK-Blue Null1 cells express the SEAP reporter gene under the
control of the IFN-b minimal promoter fused to five NF-kB binding
sites.This cell line is the parental cell line of HEK-Blue hTLR2, hTLR3,
hTLR5, hTLR8, h/mTLR9 and h/mNOD1 cells.
• HEK-Blue Null1-k cells express the same reporter system than HEK-
Blue Null1 cells but are slightly different genotypically. This cell line is the
parental cell line of HEK-Blue mTLR3 and hTLR7 cells.
• HEK-Blue Null1-v cells express the same reporter system than HEK-
Blue Null1 cells but are slightly different genotypically. This cell line is the
parental cell line of HEK-Blue mTLR4 and mTLR8 cells.
• HEK-Blue Null2 cells express the SEAP reporter gene under the
control of the IL-12 p40 minimal promoter fused to five NF-kB binding
sites.This cell line is the parental cell line of HEK-Blue mTLR2, hTLR4
and h/mNOD2 cells.
• HEK-Blue Null2-k cells express the same reporter system than HEK-
Blue Null2 cells but are slightly different genotypically. This cell line is the
parental cell line of HEK-Blue mTLR5 and mTLR7 cells.
HEK-Blue Null cells are resistant to Zeocin .
Principle
Recognition of a TLR or NOD agonist by its cognate receptor triggers a
signaling cascade leading to the activation of NF-kB and the production
of SEAP (figure 1). SEAP levels can be determined spectrophotometrically
using HEK-Blue Detection or QUANTI-Blue , both are SEAP detection
media that turn purple/blue in the presence of alkaline phosphatase.
Quality Control
Expression of exogenous TLRs, NOD,1/2, CD14 and/or MD2 was
confirmed by RT-PCR. The functionality of each cell line is validated through
stimulation assays performed with a selection of TLR and NOD agonists.
Note: HEK-Blue TLR, HEK-Blue NOD and HEK-Blue Null cells
express endogenous levels of TLR3, TLR5 and NOD1.
66
PRODUCT
www.invivogen.com/reporter-cells
TLR- and NOD-induced NF-kB signaling pathway
CAT. CODE
(human)
CAT. CODE
(mouse)
HEK-Blue MD2-CD14 Cells hkb-hmdcd NEW -
HEK-Blue TLR2 Cells hkb-htlr2 hkb-mtlr2 NEW
HEK-Blue TLR3 Cells hkb-htlr3 hkb-mtlr3 NEW
HEK-Blue TLR4 Cells hkb-htlr4 hkb-mtlr4 NEW
HEK-Blue TLR5 Cells hkb-htlr5 hkb-mtlr5 NEW
HEK-Blue TLR7 Cells hkb-htlr7 hkb-mtlr7 NEW
HEK-Blue TLR8 Cells hkb-htlr8 hkb-mtlr8 NEW
HEK-Blue TLR9 Cells hkb-htlr9 hkb-mtlr9 NEW
HEK-Blue Null1 Cells hkb-nul11 -
HEK-Blue Null1-k Cells hkb-nul11k -
HEK-Blue Null1-v Cells hkb-nul11v -
HEK-Blue Null2 Cells hkb-nul12 -
HEK-Blue HEK-Blue
Null2-k Cells hkb-nul12k -
NOD1 Cells hkb-hnodl hkb-mnodl NEW
HEK-Blue HEK-Blue
NOD2 Cells hkb-hnod2 hkb-mnod2 NEW
TLR Cells
HEK-Blue NOD Cells
HEK-Blue Null Cells
Contents and Storage
HEK-Blue TLR, HEK-Blue NOD and HEK-Blue Null cells are grown in
DMEM medium, 2mM L-glutamine, 10% FBS and supplemented with
100 μg/ml Zeocin , 30 μg/ml blasticidin and/or 200 μg/ml HygroGold
(ultra-pure hygromycin) depending on the cell line. Cells are provided frozen
in a cryotube containing 5-7 x 10 6 cells and supplied with the corresponding
selective antibiotic(s), 1 ml Normocin (50 mg/ml) and 1 pouch of
QUANTI-Blue . Cells are shipped on dry ice.

Response of HEK-Blue hTLR2 cells to TLR2 agonists.
Cells were incubated in HEK-Blue Detection medium
and stimulated with 0.1 ng/ml FSL-1 (TLR2/6), 0.1 ng/ml
Pam3CSK4(TLR1/2), 10 7 cells/ml HKLM, 1 μg/ml LTA-
SA, 1 ng/ml LPS-PG or 1 μg/ml PGN-BS. After 24h
incubation, the levels of NF-kB-induced SEAP were
determined by reading the OD at 655 nm.
Response of HEK-Blue hTLR5 cells to TLR5 agonists.
Cells were incubated in HEK-Blue Detection medium
and stimulated with 100 ng/ml FLA-BS, 10 ng/ml
FLA-ST ultrapure or 10 ng/ml recFLA-ST. After 24h
incubation, the levels of NF-kB-induced SEAP were
determined by reading the OD at 655 nm.
Response of HEK-Blue hTLR9 cells to TLR9 agonists.
Cells were stimulated with 1 μg/ml ODN2006 (type B),
1 μg/ml ODN2216 (type A) or 5 μg/ml E. coli ssDNA.
After 24h incubation, the levels of NF-kB-induced SEAP
were determined using QUANTI-Blue by reading the
OD at 655 nm.
Response of HEK-Blue hTLR3 cells to TLR3 agonists.
Cells were stimulated with 100 ng/ml poly(I:C)
(HMW), 100 ng/ml poly(I:C)-LMW or 1 μg/ml
poly(A:U). After 24h incubation, the levels of NF-kBinduced
SEAP were determined using QUANTI-Blue
by reading the OD at 655 nm.
Response of HEK-Blue hTLR7 cells to TLR7/8
agonists. Cells were incubated in HEK-Blue Detection
medium and stimulated with 100 ng/ml CL264, 5 μg/ml
imiquimod, 1 μg/ml gardiquimod or 100 ng/ml R848.
After 24h incubation, the levels of NF-kB-induced SEAP
were determined by reading the OD at 655 nm.
Response of HEK-Blue hNOD1 cells to NOD1/2
agonists. Cells were incubated in HEK-Blue Detection
medium and stimulated with 1 μg/ml iE-DAP, 100 ng/ml
C12-iE-DAP, 1 μg/ml TRI-DAP or 1 μg/ml M-TriDAP.
After 24h incubation, the levels of NF-kB-induced SEAP
were determined by reading the OD at 655 nm.
www.invivogen.com/reporter-cells
Response of HEK-Blue hTLR4 cells to TLR4 agonists.
Cells were incubated in HEK-Blue Detection medium
and stimulated with 10 ng/ml LPS-EB ultrapure, 10
ng/ml LPS-EK ultrapure, 100 ng/ml MPLA or 100 ng/ml
MPLAs. After 24h incubation, the levels of NF-kBinduced
SEAP were determined by reading the OD at
655 nm.
Response of HEK-Blue hTLR8 cells to TLR7/8
agonists. Cells were incubated in HEK-Blue Detection
medium and stimulated with 5 μg/ml ssRNA40, 1 μg/ml
CL075 or1 μg/ml R848. After 24h incubation, the levels
of NF-kB-induced SEAP were determined by reading
the OD at 655 nm.
Response of HEK-Blue hNOD2 cells to NOD1/2
agonists. Cells were incubated in HEK-Blue Detection
medium and stimulated with 100 ng/ml MDP, 10 ng/ml
L18-MDP 100 ng/ml murabutide or 100 ng/ml M-TriDAP.
After 24h incubation, the levels of NF-kB-induced SEAP
were determined by reading the OD at 655 nm.
67
INNATE IMMUNITY 3

INNATE IMMUNITY 3
THP1-XBlue Cells - NF-kB/AP-1 Reporter Monocytes
THP1-XBlue are derived from THP-1, a human immune cell line that naturally expresses most TLRs. They stably express an NF-κB/AP-1-inducible reporter
(SEAP) system to facilitate the monitoring of TLR-induced NF-kB/AP-1 activation. Three cell lines are available: THP1-XBlue , THP1-XBlue -CD14, which
overexpresses the cell surface protein CD14 for enhanced sensitivity, and THP1-XBlue -defMyD characterized by a deficient MyD88 activity.
THP1-XBlue
THP1-XBlue cells were obtained by stable transfection of THP-1 cells
with a reporter construct expressing a secreted embryonic alkaline
phosphatase (SEAP) gene under the control of a promoter inducible by
the transcription factors NF-kB and AP-1. Upon TLR stimulation,
THP1-XBlue cells induce the activation of NF-kB and AP-1 and
subsequently the secretion of SEAP. The reporter protein is easily
detectable and measurable when using QUANTI-Blue , a medium that
turns purple/blue in the presence of SEAP (see page 14). THP1-XBlue
cells are resistant to the selectable marker Zeocin .
THP1-XBlue -MD2-CD14 NEW
THP1-XBlue -MD2-CD14 cells derive from the THP1-XBlue cell line by
cotranfection of the MD2 and CD14 genes. MD2 is an accessory molecule
essential for LPS-induced TLR4 response. CD14 interacts with several TLRs,
including TLR4 and TLR2. Its overexpression was found to increase the
reponse to the majority of TLR ligands (Figure 1). THP1-XBlue -CD14 cells
are resistant to the antibiotics Zeocin and G418.
THP1-XBlue -defMyD
THP1-XBlue -defMyD cells are THP1-XBlue cells deficient in MyD88
activity. They are unable to respond to the activation of receptors which
signaling is dependent on MyD88, such as TLR2, TLR4 and IL-1Rs. They
remain responsive to MyD88-independent receptors, such as NOD1 and
TNFR (Figure 2).
Quality Control
Expression of the TLR (TLR1 to TLR10), MD2 and CD14 genes was
determined by RT-PCR in THP1-XBlue , THP1-XBlue-MD2-CD14 and
THP1-XBlue-defMyD cells. All TLR mRNAs were detected but the cells
do not respond to all TLR agonists (Figure 1). Considering the
concentration of ligands used to stimulate these cells, TLR2, TLR2/1and
TLR2/6 responses are considered to be very strong. TLR4, TLR5, TLR8,
NOD1 and NOD2 responses are robust. TLR7 response is weak (a very
high concentration of its cognate ligands is needed to detect a response).
TLR3 and TLR9 responses are not detectable even when high
concentrations of the ligands are used.
MyD88 deficiency in THP1-XBlue-defMyD cells was confirmed by
qRT-PCR.
Contents
THP1-XBlue , THP1-XBlue-MD2-CD14 and THP1-XBlue-defMyD cells are grown in RPMI medium, 2mM L-glutamine, 10% FBS
supplemented with the appropriate selective antibiotic(s): 200 μg/ml
Zeocin for THP1-XBlue ; 200 μg/ml Zeocin and 250 μg/ml G418 for
THP1-XBlue-MD2-CD14; 200 μg/ml Zeocin and 100 μg/ml
HygroGold for THP1-XBlue-defMyD. Cells are provided in a vial
containing 5-7 x 10 6
cells and is supplied with 10 mg Zeocin (and 10
mg G418 or 10 mg HygroGold ) and 1 pouch of QUANTI-Blue . Cells
are shipped on dry ice. The cells are guaranteed mycoplasma-free.
68
www.invivogen.com/reporter-cells
Figure 1: TLR and NOD stimulation profile in THP1-XBlue and THP1-XBlue -
MD2-CD14. Cells were incubated with 1 ng/ml Pam3CSK4(TLR1/2), 10 ng/ml FSL-
1 (TLR2/6), 10 7 cells/ml HKLM (TLR2), 10 μg/ml poly(I:C) (TLR3), 10 ng/ml LPS-EK
(TLR4), 10 ng/ml RecFLA-ST (TLR5), 5 μg/ml imiquimod (TLR7), 5 μg/ml CL075
(TLR8), 10 μg/ml ODN2006 (TLR9), 100 ng/ml C12-iEDAP (NOD1) or 10 μg/ml
MDP (NOD2). After 24h incubation, TLR/NOD stimulation was assessed by
measuring the levels of SEAP in the supernatant by using QUANTI-Blue .
Figure 2: MyD88-dependent and -independent responses in THP1-XBlue and
THP1-XBlue -defMyD. Cells were incubated with 1 μg/ml FSL-1 (TLR2/6), 1 μg/ml
LPS-EK (TLR4), 5 μg/ml CL075 (TLR8), 10 μg/mlTri-DAP (NOD1) and 50 ng/ml
TNF-a. After 24h incubation, NF-kB activation was determined using QUANTI-Blue .
PRODUCT QUANTITY CAT. CODE
THP1-XBlue Cells 5-7 x 10 6
cells thpx-sp
THP1-XBlue -MD2-CD14 Cells 5-7 x 10 6
cells thpx-mdcdsp
THP1-XBlue -defMyD 5-7 x 10 6
cells thpx-dmyd

Ramos-Blue Cells - NF-kB/AP-1 Reporter B lymphocytes
B lymphocytes are key players in the adaptative immune system but are also prominent in the innate immune response. Consistent with their dual role, they
express Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) that allow them to discriminate among a wide spectrum of pathogen-associated
molecules (PAMPs). Upon PRR stimulation by PAMPs, various signaling pathways are induced leading to the activation of transcription factors, such as NF-kB
and AP-1, and the subsequent production of inflammatory cytokines.
Ramos-Blue Cells
Ramos-Blue is a B lymphocyte cell line that stably expresses an NF-kB/
AP-1-inducible SEAP (secreted embryonic alkaline phosphatase) reporter
gene. Ramos-Blue cells derive from a human Burkitt’s lymphoma which
is negative for Epstein Barr virus. They have the characteristics of B
lymphocytes and are routinely used as a model of B lymphocytes and for
apoptosis studies. The Ramos-Blue cell line was isolated for its ability to
respond to CpG ODNs (TLR9 ligands).
Ramos-Blue cells are responsive to NF-kB inducers, such as TNF-a and
TLR agonists. When stimulated, they produce SEAP in the supernatant
that can be readily monitored using QUANTI-Blue . QUANTI-Blue is
a SEAP detection medium that turns blue in the presence of SEAP (see
page 14). Levels of SEAP can be determined qualitatively with the naked
eye or quantitatively using a spectrophotometer at 620-655 nm.
Ramos-Blue cells are resistant to Zeocin .
Ramos-Blue -defMyD
Ramos-Blue -defMyD are MyD88-deficient cells. Due to their deficiency
in MyD88 activity, these cells are unable to respond to the activation of
receptors which signaling is dependent on MyD88, such as TLR7 and
TLR9. However, they are still responsive to TLR3 which signals through
TRIF independently of MyD88 (Figure 2).
Quality Control
Ramos-Blue cells express all TLRs and NOD1 mRNAs as detected by
RT-PCR. However, activation of NF-kB/AP-1 was only observed following
stimulation with TLR2, TLR3, TLR7, TLR9 and NOD1 agonists (Figure 1).
Considering the concentration of agonists used to stimulate these cells,
TLR3, TLR7, TLR9 and NOD1 responses are considered to be strong.
TLR2 response is detectable only at high concentrations of the ligands.
Responses to TLR4, TLR5 and NOD2 agonists are not detectable.
MyD88 deficiency in Ramos-Blue -defMyD cells was confirmed by
qRT-PCR.
Contents
Ramos-Blue cells are grown in IMDM medium, 2 mM L-glutamine, 10%
FBS supplemented with 100 μg/ml Zeocin . Each vial contains 5-7 x 10 6
cells and is supplied with 10 mg Zeocin . Cells are shipped on dry ice.
The cells are guaranteed mycoplasma-free.
Related Products
Zeocin , page 18 TLR Ligands , pages 77-80
QUANTI-Blue page 14 NOD Ligands, page 80
www.invivogen.com/reporter-cells
Figure 1: NF-kB/AP-1 activation in Ramos-Blue cells induced by various activators.
Cells were incubated with 10 μg/ml each of Pam3CSK4 (TLR1/2), FSL-1 (TLR2/6), HKLM
(TLR2, 1.10 9 cells/ml), poly(I:C) (TLR3), LPS-EK (TLR4), recFLA-ST (TLR5), imiquimod
and CL264 (TLR7), CL075 (TLR8), R848 (TLR7/8), ODN2006 and ODN2216 (TLR9),
TriDAP (NOD1), MDP (NOD2) and TNF-a.After 24h incubation, NF-kB/AP-1 activation
was assessed by measuring the levels of SEAP in the supernatant using QUANTI-Blue .
Figure 2: MyD88-dependent and -independent responses in Ramos-Blue and
Ramos-Blue -defMyD. Cells were incubated with 1 μg/ml poly(I:C) (TLR3), 10 μg/ml
CL264 (TLR7), 2 μg/ml ODN2006 (TLR9) and 100 ng/ml TNF-a. After 24h incubation,
NF-kB activation was determined using QUANTI-Blue .
PRODUCT QUANTITY CAT. CODE
Ramos-Blue Cells 5-7 x 10 6
cells rms-sp
Ramos-Blue-defMyD 5-7 x 10 6
cells rms-dmyd
69
INNATE IMMUNITY 3

INNATE IMMUNITY 3
RAW-Blue Cells
RAW-Blue Cells
TLR & NOD Reporter Macrophages
RAW-Blue Cells are derived from RAW 264.7 macrophages with
chromosomal integration of a secreted embryonic alkaline phosphatase
(SEAP) reporter construct inducible by NF-kB and AP-1. RAW-Blue
Cells are resistant to the selectable marker Zeocin .
RAW-Blue Cells express all TLRs (with the exception of TLR5) as well
as RIG-I, MDA-5, NOD1 and NOD2; expression of TLR3 and NOD1
being very low. The presence of specific agonists of these PRRs induces
signaling pathways leading to the activation of NF-kB and AP-1 and
subsequently to the secretion of SEAP, which can be easily monitored
using QUANTI-Blue .
Dectin-1 Reporter Macrophages
RAW 264.7 express low endogenous levels of Dectin-1 1 , while
RAW-Blue cells express high levels of endogenous Dectin-1. Therefore
RAW-Blue cells can be used as a Dectin-1 reporter cell line in particular
when combined with a neutralizing anti-Dectin-1 antibody. Stimulation of
RAW-Blue cells with zymosan or heat-killed preparations of yeast
induces the activation of NF-kB in a Dectin-1-dependent manner (see
graph).
1. Brown GD. et al., 2003, Dectin-1 Mediates the Biological Effects of ß-Glucans. J. Exp.
Med., 197: 1119.
Quality Control
Expression of TLRs (TLR1 to TLR9), RLRs (RIG-I and MDA-5), NODs
(NOD1 and NOD2) was determined by RT-PCR (see figure 1). Dectin-1
expression was also confirmed by RT-PCR. RT-PCR on TLR and mRNAs
were performed using InvivoGen’s Mouse TLR RT-Primer Set and RLR RT-
Primers.
The cells are guaranteed mycoplasma-free.
Contents
RAW-Blue Cells are grown in DMEM medium, 2mM L-glutamine, 10%
FBS supplemented with 200 μg/ml Zeocin . Each vial contains 5-7 x 10 6
cells and is supplied with 10 mg Zeocin . Cells are shipped on dry ice.
70
PRODUCT QUANTITY CAT. CODE
RAW-Blue Cells 5-7 x 10 6
cells raw-sp
Related Products
Blasticidin, page 17
TLR Ligands, page 77-80
NOD Ligands, see page 80
Dectin Ligands, see page 81
MAb mDectin-1, see page 72
QUANTI-Blue , see page 14
www.invivogen.com/reporter-cells
TLR1
TLR2
TLR3
TLR4
TLR5
TLR6
TLR7
TLR8
TLR9
RIG-I
MDA-5
NOD-1
NOD-2
Figure 1: Expression of TLR, RLR and NOD mRNAs in RAW-Blue cells
determined by RT-PCR. RT-PCRs were performed using InvivoGen’s Mouse TLR
RT-PCR Primer Set, NOD and RLR RT-Primer Pairs.
TLR and NOD stimulation profile in RAW-Blue Cells. RAW-Blue Cells were
incubated with TLR or NOD agonists: TLR2 (HKLM, 1.10 8 cells/ml), TLR1/2
(Pam3CSK4, 100 ng/ml), TLR2/6 (FSL-1, 100 ng/ml), TLR3 (poly(I:C), 10 μg/ml), TLR4
(LPS-EK, 1 μg/ml), TLR5 (RecFLA-ST, 1 μg/ml), TLR7 (CL075, 300 ng/ml), TLR9
(ODN1826, 10 μg/ml), NOD1 (Tri-DAP, 10 μg/ml), NOD2 (MDP, 10 μg/ml). After
24h incubation, TLR and NOD stimulation was assessed by measuring the levels of
SEAP using QUANTI-Blue .
Response of RAW-Blue cells to Dectin-1 and TLR2 agonists. RAW-Blue cells
were stimulated with FSL-1 (10 ng/ml), zymosan (10 μg/ml), depleted zymosan (100
μg/ml), HKSC (10 8 cells/ml) or HKCA (10 8 cells/ml) in the presence or absence of
10 μg/ml of an anti-mTLR2 (clone T2.5) or anti-mDectin-1 monoclonal antibody.
After 24h incubation, NF-kB activation was assessed by measuring the levels of SEAP
in the supernatant by using QUANTI-Blue .

MEF Cells
C3H/TLR4mut & C3H/WT MEFs
TLR4 Reporter Murine Embryonic Fibroblasts
C3H/TLR4mut and C3H/WT MEF cell lines were isolated from C3H/HeJ
(TLR4-deficient) and C3H/HeN (wild-type) mouse embryos respectively.
They stably express an NF-kB-inducible SEAP reporter construct that
allows to monitor in a simple and convenient manner the activation of
NF-kB.
C3H/WT MEFs express high levels of TLR2 and TLR4 and low levels of
TLR3 and TLR5. The presence of TLR2, TLR3, TLR4, or TLR5 agonists
triggers a signaling cascade in C3H/WT MEFs leading to the activation of
NF-kB and the subsequent induction of SEAP. The amount of SEAP
secreted in the supernatant can be readily detected when using
QUANTI-Blue , a SEAP detection medium. In C3H/TLR4mut MEFs, TLR4
agonists do not induce the activation of NF-kB and the production of
SEAP in contrast to TLR2, TLR3 and TLR5 ligands. Thus these two cell lines
provide a useful tool to determine whether a given compound is a specific
TLR4 agonist. They can be used to assess the purity of a given LPS by
detecting the presence of contaminants that stimulate TLR2 (see graph,
LPS-EB standard versus LPS-EB ultrapure).
Contents
C3H/TLR4mut and C3H/WT MEF cell lines are grown in DMEM medium
with 2mM L-glutamine, 10% FBS supplemented with 10 μg/ml blasticidin.
Each vial contains 5-7 x 10 6 cells and is supplied with 1 mg blasticidin.
Cells are shipped on dry ice. The cells are guaranteed mycoplasma-free.
C57/WT MEFs
RLR Reporter Murine Embryonic Fibroblasts
MEFs produce IFN-b in response to viral infection in a RLR-dependent
manner1 . Thus, these cells are commonly used to study the RLR pathway.
C57/WT MEFs were isolated from embryos under C57BL/6 background
and immortalized with the SV40 large antigen. They stably express a SEAP
reporter gene inducible by NF-kB and IRF3/7 providing a convenient
method to monitor the activation of these transcription factors upon
stimulation with a RIG-I/MDA-5 ligand.
C57/WT MEFs express both RIG-I and MDA-5. Stimulation of C57/WT
cells with poly(I:C)/LyoVec complexes induces the secretion of SEAP in
a dose-dependent manner (see figure). In contrast, stimulation with naked
poly(I:C) has no effect on SEAP secretion although the cells express also
TLR3. These data confirm that C57/WT MEFs respond to viral dsRNA
primarily through the RLR pathway1 . Both RIG-I and MDA-5 appear to
respond to transfected poly(I:C) in MEFs2 .
1. Kato H. et al., 2005. Cell type-specific involvement of RIG-I in antiviral response.
Immunity 23:19-28. 2. Venkataraman T. et al., 2007. Loss of DExD/H box RNA helicase
LGP2 manifests disparate antiviral responses. J Immunol. 178:6444-6455.
Contents
C57/WT MEFs are grown in DMEM medium with 10% FBS, 2 mM
L-glutamine and supplemented with 100 μg/ml Zeocin and 3 μg/ml
blasticidin. Each vial contains 5-7 x 10 6 cells and is supplied with 10 mg
Zeocin and 1 mg blasticidin. Cells are shipped on dry ice. The cells are
guaranteed mycoplasma-free.
www.invivogen.com/reporter-cells
TLR stimulation profile of C3H/TLR4mut and C3H/WTMEF cells. Cells were
incubated with TLR agonists: Pam3CSK4, 100 ng/ml (TLR2), poly(I:C), 10 μg/ml
(TLR3), MPLAs, 10 μg/ml (TLR4), LPS-EB standard, 10 μg/ml (TLR4, TLR2), LPS-EB
ultrapure, 10 μg/ml (TLR4), LPS-RS, 10 μg/ml (TLR2), flagellin, 1 μg/ml (TLR5). After
24h incubation, TLR stimulation was assessed by measuring the levels of SEAP in
the supernatant by using QUANTI-Blue .
PRODUCT QUANTITY CAT. CODE
C3H/TLR4mut MEFs 5-7 x 10 6
cells mef-c3h4m
C3H/WT MEFs 5-7 x 10 6
cells mef-c3hwt
RLR stimulation in C57/WT MEFs. C57/MEFs were incubated with increasing
concentrations of poly(I:C) or poly(I:C)/LyoVec complexes prepared
extemporaneously at a 1:12 ratio. After 24h incubation, RLR stimulation was assessed
by measuring the levels of SEAP secreted in the supernatant by using QUANTI-
Blue , a SEAP detection medium.
PRODUCT QUANTITY CAT. CODE
C57/WT MEFs 5-7 x 10 6
cells mef-c57wt
71
INNATE IMMUNITY 3

INNATE IMMUNITY 3
Antibodies
InvivoGen provides a collection of monoclonal and polyclonal antibodies targeting human and murine TLRs. Most of them have been
developed in house. They are listed below and classified according to their application(s):
➥ Antibodies for neutralization. Some of them can be used also for flow cytometry
➥ Antibodies for detection. The applications include Western blotting, immunoprecipitation, immunohistochemistry and flow
cytometry.
• Anti-TLR-IgA antibodies are recombinant monoclonal IgA2 antibodies against TLRs. They have been developed by InvivoGen using proprietary techniques.
They have been selected for their ability to efficiently block the biological activity of these TLRs. They can also be used for flow cytometry (FC).
• Anti-TLR IgG antibodies are mouse monoclonal antibodies against TLRs. They have been generated by InvivoGen using DNA vaccination and screened
for their ability to neutralize TLR activity.
• MAb-TLR and Mab-Dectin-1 antibodies are monoclonal mouse IgG antibodies. They can be used for various applications but have been tested in
our laboratories only for neutralization and flow cytometry. MAb-TLR antibodies are also available conjugated with FITC.
• PAb-TLR antibodies are polyclonal antibodies against human extracellular TLRs, developed by InvivoGen. These antibodies have been generated by DNA
vaccination in rats. They were obtained by purification of the IgG fraction from the sera by Protein G affinity chromatography.
Antibodies for Neutralization
72
ANTIBODY DESCRIPTION SPECIFICITY APPLICATIONS QTY CAT. CODE
Anti-CD14
Anti-hCD14-IgA Monoclonal human IgA2 Human CD14 Neutralization (TLR2, TLR4), FC 100 μg maba-hcd14
Anti-Dectin-1
MAb-mDectin-1 Monoclonal mouse IgG2b (clone 2A11) Mouse Dectin-1 Neutralization, FC 100 μg mab-mdect
Anti-TLR1
Anti-hTLR1-IgG Monoclonal mouse IgG1 (H2G2) Human TLR1 Neutralization 100 μg mabg-htlr1
PAb-hTLR1 Polyclonal rat IgG Human TLR1 Neutralization 200 μg pab-hstlr1
Anti-TLR2
Anti-hTLR2-IgA Monoclonal human IgA2 Human TLR2 Neutralization, FC 100 μg maba2-htlr2
Anti-mTLR2-IgG Monoclonal mouse IgG2a (C9A12) Mouse TLR2 Neutralization 100 μg mabg-mtlr2
MAb-mTLR2 Monoclonal mouse IgG1 (T2.5) Human/mouse TLR2 Neutralization , FC, IHC 100 μg mab-mtlr2
PAb-hTLR2 Polyclonal rat IgG Human TLR2 Neutralization 200 μg pab-hstlr2
Anti-TLR4
Anti-hTLR4-IgA Monoclonal human IgA2 Human TLR4 Neutralization 100 μg maba2-htlr4
PAb-hTLR4 Polyclonal rat IgG Human TLR4 Neutralization 200 μg pab-hstlr4
Anti-TLR5
Anti-hTLR5-IgA Monoclonal human IgA2 Human TLR5 Neutralization, FC 100 μg maba2-htlr5
Anti-mTLR5-IgG Monoclonal rat IgG2a (Q23D11) Mouse TLR5 Neutralization 100 μg mabg-mtlr5
PAb-hTLR5 Polyclonal rat IgG Human TLR5 Neutralization 200 μg pab-hstlr5
Anti-TLR6
Anti-hTLR6-IgG Monoclonal mouse IgG1 (C5C8) Human TLR6 Neutralization 100 μg mabg-htlr6
PAb-hTLR6 Polyclonal rat IgG Human TLR6 Neutralization 200 μg pab-hstlr6
* FC: flowcytometry; IHC: immunohistochemistry; WB: Western blot
www.invivogen.com/antibodies

Antibodies for Detection
ANTIBODY DESCRIPTION SPECIFICITY APPLICATIONS* QTY CAT. CODE
Anti-Flagellin
Anti-Flagellin FliC Monoclonal mouse IgG1 S. typhimurium flagellin WB 100 μg mabg-flic
Anti-TLR1
MAb-hTLR1 Monoclonal mouse IgG1 (GD2.F4) Human TLR1 FC 100 μg mab-htlr1
MAb-hTLR1-FITC Monoclonal mouse IgG1 (GD2.F4), FITC Human TLR1 FC 100 μg mab-htlr1f
Anti-TLR2
MAb-hTLR2 Monoclonal mouse IgG2a (TL2.1) Human TLR2 FC, IHC, WB 100 μg mab-htlr2
MAb-hTLR2-FITC Monoclonal mouse IgG2a (TL2.1), FITC Human TLR2 FC, IHC 100 μg mab-htlr2f
MAb-mTLR2-FITC Monoclonal mouse IgG1 (T2.5), FITC Human/mouse TLR2 FC 100 μg mab-mtlr2f
Anti-TLR3
Anti-hTLR3-IgA Monoclonal human IgA2 Human TLR3 FC 100 μg maba-htlr3
MAb-hTLR3 Monoclonal mouse IgG1 (TLR3.7) Human TLR3 FC, WB 100 μg mab-htlr3
MAb-hTLR3-FITC Monoclonal mouse IgG1 (TLR3.7), FITC Human TLR3 FC, WB 100 μg mab-htlr3f
Anti-TLR4
MAb-hTLR4 Monoclonal mouse IgG2a (HTA125) Human/monkey TLR4 FC, IHC 100 μg mab-htlr4
MAb-hTLR4-FITC Monoclonal mouse IgG2a (HTA125), FITC Human/monkey TLR4 FC 100 μg mab-htlr4f
MAb-mTLR4/MD2 Monoclonal rat IgG2a (MTS510) Mouse TLR4/MD2 FC, IHC 100 μg mab-mtlr4md2
MAb-mTLR4/MD2-FITC Monoclonal rat IgG2a (MTS510), FITC Mouse TLR4/MD2 FC 100 μg mab-mtlr4md2f
Anti-TLR9
MAb-mTLR9 Monoclonal mouse IgG2a (5G5) Human/mouse TLR9 FC, IHC, WB 100 μg mab-mtlr9
MAb-mTLR9-FITC Monoclonal mouse IgG2a (5G5), FITC Human/mouse TLR9 FC 100 μg mab-mtlr9f
* FC: flowcytometry; IHC: immunohistochemistry; WB: Western blot
Contents and Storage
MAb-TLR-IgA and MAb-TLR-IgG antibodies are provided lyophilized from a 0.2 μm filtered solution in PBS.
MAb-TLR antibodies are purified and provided as 100 μg lyophilized powder.
PAb-TLR antibodies are provided as 200 μg lyophilized sera. PAb-TLRs are sterile, azide-free (contain Pen/Strep), endotoxin-tested (

INNATE IMMUNITY 3
pNiFty - PRR Signaling Reporter Plasmids
PRR activation triggers a complex signaling cascade that leads to the activation of different transcription factors, each playing an important role in the
subsequent immune response. To monitor the induction of PRR signaling in response to ligand stimulation in a simple and efficient manner, InvivoGen
has designed pNiFty, a family of reporter plasmids expressing a reporter gene under the control of a minimal promoter inducible by these different
transcription factors, either individually or in combination. Most pNiFty plasmids are selectable with Zeocin in both E. coli and mammalian cells, and
can be used to generate stable clones
74
PLASMID NAME
Description
TRANSCRIPTION FACTOR
BINDING SITES
pNiFty plasmids are composed of three key elements: a proximal
promoter, repeated transcription factor binding sites (TFBS) and a
reporter gene. The proximal promoters are shorter than 500 bp and
contain transcription factor binding sites. Upon stimulation in 293 cells,
their expression level remains undetectable. With the addition of repeated
TFBS, the proximal promoters become inducible by the appropriate
stimulus and drive the expression of the reporter gene.
Minimal promoters
- ELAM promoter: the proximal promoter of the endothelial cellleukocyte
adhesion molecule (ELAM-1; E-selectin) gene contains three
NF-kB sites and is truly NF-kB specific, as it lacks an AP1/CREB site found
in the full-length promoter 1, 2 .
- IFN-b promoter: the mouse IFN-b minimal promoter comprises several
positive regulatory domains that bind different cooperating transcription
factors such as NF-kB, IRF3 and IRF7 3 .
- ISG-56K promoter: the minimal promoter of the human interferonstimulated
gene ISG-56K contains two interferon-stimulated regulatory
element (ISRE) sites and is fully inducible by type I IFNs and interferon
regulatory factors (IRFs) 4, 5 .
Transcription factor binding sites (TFBS)
- AP-1 binding site: Activator protein 1 (AP-1) is a transcription factor
activated by most PRRs. AP-1 is a heterodimeric complex composed of
members of Fos, Jun and, ATF protein families. AP-1 binds to the TPA
responsive element (TRE: ; TGAG/CTCA) 6 . AP-1 activation in TLR signaling
is mostly mediated by MAP kinases such as c-Jun N-terminal kinase (JNK),
p38 and extracellular signal regulated kinase (ERK).
MINIMAL
PROMOTER
www.invivogen.com/innate-immunity-pnifty
SELECTION REPORTER
CATALOG
CODE
pNiFty-SEAP NF-kB (x5) ELAM Ampicillin SEAP pnifty-seap
pNiFty-Luc NF-kB (x5) ELAM Ampicillin Luciferase pnifty-luc
pNiFty2-SEAP NF-kB (x5) ELAM Zeocin SEAP pnifty2-seap
pNiFty2-Luc NF-kB (x5) ELAM Zeocin Luciferase pnifty2-luc
pNiFty2-56K None ISG56 Zeocin SEAP pnf2-56sp
pNiFty3-SEAP NEW None IFN-b Zeocin SEAP pnf3-sp1
pNiFty3-N-SEAP NEW NF-kB (x5) IFN-b Zeocin SEAP pnf3-sp2
pNiFty3-A-SEAP NEW AP-1 (x5) IFN-b Zeocin SEAP pnf3-sp3
pNiFty3-I-SEAP NEW ISRE (x5) IFN-b Zeocin SEAP pnf3-sp4
pNiFty3-T-SEAP NEW NFAT (x5) IFN-b Zeocin SEAP pnf3-sp5
pNiFty3-AN-SEAP NEW AP-1 (x5) NF-kB (x5) IFN-b Zeocin SEAP pnf3-sp6
pNiFty3-IAN-SEAP NEW ISRE (x5) AP-1 (x5) NF-kB (x5) IFN-b Zeocin SEAP pnf3-sp7
pNiFty3-TAN-SEAP NEW NFAT (x5) AP-1 (x5) NF-kB (x5) IFN-b Zeocin SEAP pnf3-sp8
SEAP
or
Luciferase
SEAP
or
Luciferase

- NF-kB binding site: Nuclear factor (NF)-kB is a “rapid-acting” primary
transcription factor activated by a wide variety of PRRs. NF-kB is a protein
complex that belongs to the Rel-homology domain-containing protein
family. The prototypical NF-kB is composed of the p65(RelA) and p50
subunits 7 . NF-kB binds specific decameric DNA sequences
(GGGRNNYYCC, R-purine Y=pyrimidine) and activates genes involved
in the regulation of the innate and adaptative immune response.
- ISRE binding site: PRRs involved in the antiviral response induce the
activation of interferon regulatory factors (IRFs) and the production of type
I interferons (IFNs). IFNs trigger the formation of the ISGF3 complex which
contains signal transducer and activator of transcription (STAT) 1, STAT2
and IRF9. ISGF3 and IRFs bind to specific nucleotide sequences called
interferon-stimulated response elements (ISREs; AGTTTCNNTTTCC) in
the promoter of IFN-stimulated genes (ISGs) leading to their activation 8 .
- NFAT binding site: Nuclear factor of activated T-cell (NFAT) is a family
of transcription factors expressed in T cells, but also in other classes of
immune and non-immune cells 9 . NFAT is activated by stimulation of
receptors coupled to calcium mobilization, such as the PRRs Dectin-1
and Mincle 10, 11 . Calcium mobilization induces the calmodulin-dependent
phosphatase calcineurin leading to NFAT activation. NFAT binds to a 9
bp element, with the consensus sequence (A/T)GGAAA(A/N)(A/T/C)N.
Reporter Genes
- SEAP reporter gene: Secreted alkaline phosphatase (SEAP) is a
reporter widely used to study promoter activity or gene expression.
SEAP expression can be rapidly and readily measured in supernatants of
transfected cells. SEAP levels can be evaluated qualitatively with the naked
eye and quantitatively using SEAP detection media, such as HEK-Blue
Detection, or the SEAP Reporter Assay Kit (see “Related Products”).
- Luc reporter gene: The firefly luciferase gene is a highly sensitive
reporter gene and thus is ideal for detecting low-level gene expression.
Luc activity can be quantified in cell extracts by using kits commercially
available from other companies.
1. Schindler U., Baichwal VR., 1994. Three NF-kappa B binding sites in the human
E-selectin gene required for maximal tumor necrosis factor alpha-induced
expression. Mol Cell Biol. 14(9):5820-31. 2. Jensen LE. & Whitehead AS., 2003.
ELAM-1/E-selectin promoter contains an inducible AP-1/CREB site and is not NFkB-specific.
Biotechniques 35:54-58. 3. Vodjdani G. et al., 1988. Structure and
characterization of a murine chromosomal fragment containing the interferon beta
gene. J Mol Biol. 204(2):221-31. 4. Wathelet MG. et al., 1987. New inducers revealed
by the promoter sequence analysis of two interferon-activated human genes. Eur J
Biochem. 1987 Dec 1;169(2):313-21. 5. Grandvaux N, et al., 2002. Transcriptional
profiling of interferon regulatory factor 3 target genes: direct involvement in the
regulation of interferon-stimulated genes. J Virol. 2002 Jun;76(11):5532-9. 6. Hess J,
et al., 2004. AP-1 subunits: quarrel and harmony among siblings. J Cell Sci. 117(Pt
25):5965-73. 7. Kawai T. & Akira S., 2007. Signaling to NF-kappaB by Toll-like
receptors. Trends Mol Med. 13(11):460-9. 8. Wesoly J. et al., 2007. STAT activation
and differential complex formation dictate selectivity of interferon responses. Acta
Biochim Pol. 54(1):27-38. 9. Rao A. et al., 1997. Transcription factors of the NFAT
family: regulation and function. Annu Rev Immunol. 15:707-47. 10. Goodridge HS.
et al., 2007. Dectin-1 stimulation by Candida albicans yeast or zymosan triggers
NFAT activation in macrophages and dendritic cells. J Immunol. 178(5):3107-15.
11. Yamasaki S. et al., 2009. C-type lectin Mincle is an activating receptor for
pathogenic fungus, Malassezia. PNAS. 106(6):1897-902.
Contents and Storage
pNiFty plasmids are provided as lyophilized transformed E. coli strains on
paper disk with 4 pouches of Fast-Media ® (2 TB and 2 Agar), containing
the appropriate antibiotic: ampicillin for pNiFty plasmids, or Zeocin for
pNiFty2 and pNiFty3 plasmids. Products are shipped at room
temperature and should be stored at -20ºC.
Related Products
www.invivogen.com/innate-immunity-pnifty
HEK-Blue Detection, page 15
QUANTI-Blue , page 14
SEAP Reporter Assay Kit, page 13
Fast-Media ® , page 45
Zeocin , page 18
AP-1
NF-kB
ISRE
NFAT
individually
or
combined
75
INNATE IMMUNITY 3

INNATE IMMUNITY 3
Pathogen-Associated Molecular Patterns
TLRs, NODs, RLRs and Dectin-1 are pattern recognition receptors (PRRs) that recognize a wide variety of
microbial molecules, called pathogen-associated molecular patterns (PAMPs), discriminating Gram-positive and
Gram-negative bacteria from fungi and other pathogens. InvivoGen offers a comprehensive choice of high quality
PAMPs know to activate these PRRs.
All PAMPs are tested for TLR stimulation using Blue reporter cells. The endotoxin levels are determined using
a kinetic chromogenic LAL assay. EndoFit agonists contain less than 0.001 EU/μg.
TLR2 Agonists
TLR2 is involved in the recognition of a wide array of microbial molecules
representing broad groups of species such as Gram- and Gram+ bacteria,
as well as mycoplasma and yeast.
TLR3 Agonists
TLR3 recognizes double-stranded RNA (dsRNA), a molecular pattern
associated with viral infection. Polyinosine-polycytidylic acid (poly(I:C)), a
synthetic analog of dsRNA, is the ligand of choice for TLR3.
TLR4 Agonists
TLR4 is the receptor for Gram-negative lipopolysaccharide (LPS) and lipid
A, its toxic moiety. InvivoGen offers LPS from various bacteria and
monophosphoryl lipid A.
TLR5 Agonists
TLR5 recognizes flagellin, the major component of the bacterial flagellar
filament, from both Gram+ and Gram- bacteria. InvivoGen provides flagellin
purified from B. subtilis (Gram+) and S. typhimurium (Gram-) bacteria and a
recombinant form.
TLR7/8 Agonists
TLR7 and TLR8 are involved in the response to viral infection. They recognize
GU-rich short single-stranded RNA as well as small synthetic molecules
such as imidazoquinolines and nucleoside analogues.
TLR9 Agonists
TLR9 recognizes specific unmethylated CpG-ODN sequences that
distinguish microbial DNA from mammalian DNA. Three types of
stimulatory ODNs have been described: type A, B and C. InvivoGen also
provides control ODNs and inhibitory ODNs. Larger quantities are
available upon request.
NOD1/2 Agonists
NOD1 and NOD2 are intracellular pathogen-recognition molecules that
sense bacterial peptidoglycan (PGN). InvivoGen provides insoluble and
soluble PGNs from Gram- and Gram + bacteria and bioactive fragments of
PGN such as iE-DAP and MDP.
RIG-I/MDA-5 Agonist
RIG-I and MDA-5 are cytoplasmic RNA helicases that recognize intracellular
double-stranded RNA (dsRNA), a molecular pattern associated with viral
infection. InvivoGen provides poly(I:C)/LyoVec, a ligand that mimics viral
dsRNA, as well as 5’ppp-dsRNA.
76
www.invivogen.com/innate-immunity-pamps
Cytosolic DNA Sensor (CDS) Agonists
Double-stranded DNA (dsDNA) is a potent inducer of type I interferons.
Several sensors of cytosolic dsDNA have been identified, including DAI,
RIG-I and LRRFIP1. These sensors recognize AT-rich B-form dsDNA and
GC-rich Z-form dsDNA.
Dectin-1 Agonists
Dectin-1 is a specific receptor of b-glucans, which are glucose polymers
found in the cell walls of fungi, including the yeasts Saccharomyces cerevisiae
and Candida albicans.
NF-kB Activators
InvivoGen provides two TLR-independent activators of the transcription
factor NF-kB: Tumor Necrosis Factor alpha (TNF-a) and Phorbol Myristate
Acetate (PMA).
Contents and Storage
Most products are provided as a powder form and are supplied with
endotoxin-free water for their reconstitution.
Products are shipped at room temperature and should be stored at 4°C
or -20°C as mentioned in the technical datasheet.

PRODUCT ORIGIN/DESCRIPTION
TLR2 Agonists
ENDOTOXIN
LEVELS*
FSL-1 Synthetic diacylated lipoprotein - TLR2/6 EndoFit 1 - 100 ng/ml 100 μg tlrl-fsl p 82
HKAL Heat Killed Acholeplasma laidlawii EndoFit 10 6 - 10 8 cells/ml 10 9 cells tlrl-hkal p 82
HKEB NEW Heat Killed Escherichia coli 0111:B4 >1 EU/10 9 cells 10 5 - 10 7 cells/ml 10 10 cells tlrl-hkeb p 82
HKHP Heat Killed Helicobacter pylori EndoFit 10 6 - 10 8 cells/ml 10 9 cells tlrl-hkhp p 82
HKLM Heat Killed Listeria monocytogenes EndoFit 10 7 - 10 8 cells/ml 10 10 cells tlrl-hklm p 82
HKLP Heat Killed Legionella pneumophila EndoFit 10 7 - 10 8 cells/ml 10 9 cells tlrl-hklp p 82
HKLR Heat Killed Lactobacillus rhamnosus >1 EU/10 9 cells 10 8 - 10 9 cells/ml 10 10 cells tlrl-hklr p 82
HKMF NEW Heat Killed Mycoplasma fermentans EndoFit 10 6 - 10 8 cells/ml 10 9 cells tlrl-hkmf p 82
HKPA Heat Killed Pseudomonas aeruginosa >1 EU/10 8 cells 10 5 - 10 7 cells/ml 10 10 cells tlrl-hkpa p 82
HKPG Heat Killed Porphyromonas gingivalis EndoFit 10 6 - 10 8 cells/ml 10 10 cells tlrl-hkpg p 82
HKSA Heat Killed Staphylococcus aureus >1 EU/10 9 cells 10 6 - 10 8 cells/ml 10 10 cells tlrl-hksa p 82
HKSP Heat Killed Streptococcus pneumoniae EndoFit 10 7 - 10 9 cells/ml 10 10 cells tlrl-hksp p 82
LAM-MS Lipoarabinomannan from M. smegmatis EndoFit 100 ng - 10 μg/ml 500 μg tlrl-lams p 82
LM-MS Lipomannan from Mycobacterium smegmatis >5 EU/mg 1 - 10 ng/ml 250 μg tlrl-lmms2 p 82
LPS-PG Ultrapure Ultrapure LPS from P. gingivalis >10 4 EU/mg 10 ng - 10 μg/ml 1 mg tlrl-pglps p 83
LTA-BS Lipoteichoic acid from Bacillus subtilis 10 EU/mg 100 ng - 1 μg/ml 5 mg tlrl-lta p 83
LTA-SA Lipoteichoic acid from S. aureus 10 EU/mg 100 ng - 1 μg/ml 5 mg tlrl-slta p 83
LTA-SA Purified Purified lipoteichoic acid from S. aureus EndoFit 1 ng - 1 μg/ml 5 mg tlrl-pslta p 83
Pam2CSK4 Synthetic diacylated lipoprotein - TLR2(6) EndoFit 1 - 100 ng/ml 100 μg
1 mg
www.invivogen.com/innate-immunity-pamps
WORKING
CONCENTRATION QTY
CATALOG
CODE
tlrl-pam2
tlrl-pam2-1
Pam2CSK4 Biotin Biotinylated Pam2CSK4 EndoFit 1 - 100 ng/ml 50 μg tlrl-bpam2 p 83
Pam2CSK4 Rhodamine Rhodamine-labeled Pam2CSK4 EndoFit 1 - 100 ng/ml 50 μg tlrl-rpam2 p 83
Pam3CSK4 Synthetic triacylated lipoprotein - TLR1/2 EndoFit 1 - 300 ng/ml 1 mg tlrl-pms p 83
Pam3CSK4 Biotin Biotinylated Pam3CSK4 EndoFit 1 - 100 ng/ml 50 μg tlrl-bpms p 83
Pam3CSK4 Rhodamine Rhodamine-labeled Pam3CSK4 EndoFit 1 - 300 ng/ml 50 μg tlrl-rpms p 83
PGN-BS Peptidoglycan from B. subtilis EndoFit 1 - 10 μg/ml 5 mg tlrl-pgnbs p 83
PGN-EB Peptidoglycan from E. coli 0111:B4 10 2 - 10 3 EU/mg 1 - 10 μg/ml 1 mg tlrl-pgnec p 83
PGN-EK Peptidoglycan from E. coli K12 10 2 - 10 3 EU/mg 1 - 10 μg/ml 1 mg tlrl-pgnek p 83
PGN-SA Peptidoglycan from S. aureus 1 EU/mg 1 - 10 μg/ml 5 mg tlrl-pgnsa p 83
Zymosan Cell wall preparation of S. cerevisiae EndoFit TLR3 Agonists
10 μg/ml 100 mg tlrl-zyn p 83
Poly(A:U) Polyadenylic–polyuridylic acid

INNATE IMMUNITY 3
PRODUCT ORIGIN/DESCRIPTION
ENDOTOXIN
LEVELS*
WORKING
CONCENTRATION QTY
CATALOG
CODE
LPS-RS Lipopolysaccharide from Rhodobacter sphaeroides 1 x 106 TLR4 Antagonist
TLR5 Agonists
EU/mg 10 ng - 10 μg/ml 5 mg tlrl-rslps p 84
FLA-BS Standard flagellin from B. subtilis 95% pure

PRODUCT ORIGIN/DESCRIPTION
TLR9 Agonists
ODN 1668 FITC FITC-labeled CpG ODN - mouse specific, type B EndoFit 10 ng - 10 μg/ml 50 μg tlrl-1668f p 86
ODN 1826 Stimulatory CpG ODN
Type B
Mouse specific
EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
ODN 1826 control Negative control for ODN 1826 EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
www.invivogen.com/innate-immunity-pamps
tlrl-1826
tlrl-1826-1
tlrl-1826-5
tlrl-1826c
tlrl-1826c-1
tlrl-1826c-5
ODN 1826 Biotin Biotinylated CpG ODN - mouse specific, type B EndoFit 10 ng - 10 μg/ml 50 μg tlrl-1826b p 86
ODN 1826 FITC FITC-labeled CpG ODN - mouse specific, type B EndoFit 10 ng - 10 μg/ml 50 μg tlrl-1826f p 86
ODN 2006 Stimulatory CpG ODN
Type B
Human specific
EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
ODN 2006 control Negative control for ODN 2006 EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
tlrl-2006
tlrl-2006-1
tlrl-2006-5
tlrl-2006c
tlrl-2006c-1
tlrl-2006c-5
ODN 2006 Biotin Biotinylated CpG ODN - human specific, type B EndoFit 10 ng - 10 μg/ml 50 μg tlrl-2006b p 86
ODN 2006 FITC FITC-labeled CpG ODN - human specific, type B EndoFit 10 ng - 10 μg/ml 50 μg tlrl-2006f p 86
ODN 2006-G5 Stimulatory CpG ODN
Type B
Human specific
ODN 2007 Stimulatory CpG ODN
Type B
Bovine / porcine
EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
ODN 2007 control Negative control for ODN 2007 EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
ODN 2216 Stimulatory CpG ODN
Type A
Human specific
EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
ODN 2216 control Negative control for ODN 2216 EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
tlrl-2006g5
tlrl-2006g5-1
tlrl-2006g5-5
tlrl-2007
tlrl-2007-1
tlrl-2007-5
tlrl-2007c
tlrl-2007c-1
tlrl-2007c-5
tlrl-2216
tlrl-2216-1
tlrl-2216-5
tlrl-2216c
tlrl-2216c-1
tlrl-2216c-5
ODN 2216 Biotin Biotinylated CpG ODN - human specific, type A EndoFit 10 ng - 10 μg/ml 50 μg tlrl-2216b p 86
ODN 2216 FITC FITC-labeled CpG ODN - human specific, type A EndoFit 10 ng - 10 μg/ml 50 μg tlrl-2216f p 86
ODN 2336 Stimulatory CpG ODN
Type A
Human specific
EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
ODN 2336 control Negative control for ODN 2336 EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
tlrl-2336
tlrl-2336-1
tlrl-2336-5
tlrl-2336c
tlrl-2336c-1
tlrl-2336c-5
ODN 2336 FITC FITC-labeled CpG ODN - human specific, type A EndoFit 10 ng - 10 μg/ml 50 μg tlrl-2336f p 86
ODN 2395 Stimulatory CpG ODN
Type C
Human / mouse
EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
ODN 2395 control Negative control for ODN 2395 EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
tlrl-2395
tlrl-2395-1
tlrl-2395-5
tlrl-2395c
tlrl-2395c-1
tlrl-2395c-5
ODN 2395 FITC FITC-labeled CpG ODN - human specific, type C EndoFit 10 ng - 10 μg/ml 50 μg tlrl-2395f p 86
ODN M362 Stimulatory CpG ODN
Type C
Human / mouse
ENDOTOXIN
LEVELS
WORKING
CONCENTRATION QTY
EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
ODN M362 control Negative control for ODN M362 EndoFit 5 μM (10 μg/ml) 200 μg
1 mg
5 mg
CATALOG
CODE
tlrl-m362
tlrl-m362-1
tlrl-m362-5
tlrl-m362c
tlrl-m362c-1
tlrl-m362c-5
ODN M362 FITC FITC-labeled CpG ODN - human specific, type C EndoFit 10 ng - 10 μg/ml 50 μg tlrl-m362f p 86
INFO
p 86
p 86
p 86
p 86
p 86
p 86
p 86
p 86
p 86
p 86
p 86
p 86
p 86
p 86
p 86
79
INNATE IMMUNITY 3

INNATE IMMUNITY 3
ODN 2088 Inhibitory ODN, mouse preferred EndoFit 50 nM - 1 μM 200 μg
1 mg
ODN 2088 control Negative control for ODN 2088 EndoFit 50 nM - 1 μM 200 μg
1 mg
tlrl-2088
tlrl-2088-1
tlrl-2088c
tlrl-2088c-1
ODN 4084-F NEW Class B inhibitory ODN EndoFit 50 nM - 1 μM 200 μg tlrl-4084 p 87
ODN INH-1 NEW Class R inhibitory ODN EndoFit 50 nM - 1 μM 200 μg tlrl-inh1 p 87
ODN INH-47 NEW Negative control for ODN INH-1 EndoFit 50 nM - 1 μM 200 μg tlrl-inh47 p 87
ODN TTAGGG Inhibitory ODN, human preferred EndoFit 50 nM - 1 μM 200 μg tlrl-ttag p 87
1 mg tlrl-ttag-1
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PRODUCT ORIGIN/DESCRIPTION
TLR9 Agonists
ENDOTOXIN
LEVELS
ODN TTAGGG control Negative control for ODN TTAGGG EndoFit 50 nM - 1 μM 200 μg
1 mg
tlrl-ttagc
tlrl-ttagc-1
G-ODN Inhibitory guanosine-rich ODN EndoFit 50 nM - 1 μM 200 μg tlrl-godn p 87
MDP Muramyldipeptide (L-D isoform, active) EndoFit 10 ng - 10 μg/ml 5 mg tlrl-mdp p 87
MDP control Muramyldipeptide (D-D isoform, inactive) EndoFit 10 ng - 10 μg/ml 5 mg tlrl-mdpc p 88
MDP Biotin Biotinylated Muramyldipeptide EndoFit 100 ng - 10 μg/ml 500 μg tlrl-bmdp p 88
MDP FITC FITC-labeled Muramyldipeptide EndoFit 10 ng - 10 μg/ml 500 μg tlrl-fmdp p 88
MDP Rhodamine Rhodamine-labeled Muramyldipeptide EndoFit 100 ng - 10 μg/ml 500 μg tlrl-rmdp p 88
L18-MDP Muramyldipeptide with a C18 fatty acid chain EndoFit 1 - 100 ng/ml 1 mg tlrl-lmdp p 88
N-Glycolyl-MDP N-glycolylated muramyldipeptide EndoFit 100 ng - 10 μg/ml 5 mg tlrl-gmdp p 88
Murabutide Synthetic derivative of muramyldipeptide EndoFit 10 ng - 1 μg/ml 5 mg tlrl-mbt p 88
Murabutide control Murabutide analog (D isoform, inactive) EndoFit C12-iE-DAP Acylated derivative of iE-DAP EndoFit
10 ng - 1 μg/ml 5 mg tlrl-mbtc p 88
1 ng - 1 μg/ml 1 mg tlrl-c12dap p 87
iE-DAP D-g-Glu-mDAP EndoFit 1 - 100 μg/ml 5 mg tlrl-dap p 87
iE-Lys iE-DAP negative control EndoFit 1 - 100 μg/ml 5 mg tlrl-lys p 87
Tri-DAP L-Ala-g-D-Glu-mDAP EndoFit 100 ng - 10 μg/ml 1 mg tlrl-tdap p 88
Tri-Lys Tri-DAP negative control EndoFit NOD1 Agonists
NOD2 Agonists
100 ng - 10 μg/ml 1 mg tlrl-tlys p 88
www.invivogen.com/innate-immunity-pamps
WORKING
CONCENTRATION QTY
CATALOG
CODE
pCpG Giant CpG-free dcm - giant plasmid EndoFit 5 - 10 μg/ml 1 mg tlrl-cpgg p 86
Salmon sperm DNA TLR9 negative control EndoFit TLR9 Antagonists
5 - 100 μg/ml 50 mg tlrl-sdef p 86
NOD1/2 Agonists
M-TriDAP MurNAc-L-Ala-g-D-Glu-mDAP EndoFit 1 - 100 μg/ml 1 mg tlrl-mtd p 88
PGN-ECndi ultrapure Insoluble peptidoglycan from E. coli K12 EndoFit 1 - 5 μg/ml 5 mg tlrl-kipgn p 88
PGN-ECndss ultrapure Soluble sonicated peptidoglycan from E. coli K12 EndoFit 1 - 5 μg/ml 1 mg tlrl-ksspgn p 88
PGN-SAndi ultrapure Insoluble peptidoglycan from S. aureus EndoFit 1 - 5 μg/ml 5 mg tlrl-sipgn p 88
RIG-I/MDA-5 and CDS Agonists
5’ppp-dsRNA NEW 5’triphosphate blunt-end double-stranded RNA EndoFit 300 ng - 1 μg/ml 25 μg
100 μg
5’ppp-dsRNA Control NEW 5’triphosphate blunt-end double-stranded RNA EndoFit 300 ng - 1 μg/ml 25 μg
100 μg
Poly(dA:dT) Naked NEW Poly(dA-dT)•poly(dT-dA) EndoFit 1 - 5 μg/ml 200 μg
1 mg
tlrl-3prna
tlrl-3prna-100
tlrl-3prnac
tlrl-3prnac-100
tlrl-patn
tlrl-patn-1
Poly(dA:dT)/LyoVec Poly(dA-dT)•poly(dT-dA)/LyoVec complexes EndoFit 1 - 5 μg/ml 100 μg tlrl-patc p 88
Poly(dG:dC) Naked NEW Poly(dG-dC)•poly(dG-dC) EndoFit 1 - 5 μg/ml 200 μg tlrl-pgcn p 88
Poly(dG:dC)/LyoVec NEW Poly(dG-dC)•poly(dG-dC)/LyoVec complexes EndoFit 1 - 5 μg/ml 100 μg tlrl-pgcc p 88
Poly(I:C) (HMW)/LyoVec Poly(I:C) (HMW)/LyoVec complexes EndoFit 100 ng - 1 μg/ml 100 μg tlrl-piclv p 88
1 mg tlrl-piclv-10
Poly(I:C) (LMW)/LyoVec Poly(I:C) (LMW)/LyoVec complexes EndoFit 100 ng - 1 μg/ml 100 μg tlrl-picwlv p 88
1 mg tlrl-picwlv-10
INFO
p 87
p 87
p 87
p 88
p 88
p 88

PRODUCT ORIGIN/DESCRIPTION
Dectin-1 Agonists
Curdlan NEW Beta-1,3-glucan from Alcaligenes faecalis

INNATE IMMUNITY 3
TLR2 Agonists
FSL-1 - TLR2/6 Agonist
FSL-1 (Pam2CGDPKHPKSF) is a synthetic lipoprotein derived from Mycoplasma salivarium similar to MALP-2, a M. fermentans derived lipopeptide (LP) 1,2 .
Mycoplasmal LPs, such as FSL-1, contain a diacylated cysteine residue, whereas bacterial LP contain a triacylated one. FSL-1 is recognized by TLR2 and TLR6,
whereas bacterial LPs are recognized by TLR2 and TLR1 3 .
HKAL (Acholeplasma laidlawii)
Acholeplasma laidlawii, a member of the mycoplasma family, is a cell wall-less bacteria. Heat killed mycoplasma such as HKAL induce higher stimulation of
macrophage than lipoproteins from Gram- bacteria, even at low concentrations 4 . This response is mediated by TLR2 and MyD88.
HKEB (Escherichia coli) NEW
HKEB is a heat killed preparation of the gram negative bacterium, E.coli O111:B4. Cell wall components from this bacterium, such as peptidoglycan (PGN)
and lipopolysaccharide (LPS), are recognized by TLR2 and TLR4 5 . It has been demonstrated that HKEB can stimulate TLR2 and induce the production of
NF-kB and pro-inflammatory cytokines, such as IL-8 6 . HKEB is a potent stimulator of TLR2, and has a weak stimulatory effect on TLR4.
HKHP (Helicobacter pylori)
Helicobacter pylori, a Gram negative bacterium, is an important human pathogen that causes gastritis and is strongly associated with peptic ulcer, gastric
adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. Heat-killed Helicobacter pylori (HKHP) induces the production of IL-8 through the
activation of the ERK and p38 MAPK pathway. TLR2 was shown to be the sensor involved in HKHP-mediated secretion of IL-8 in monocytes 7 .
HKLM (Listeria monocytogenes)
HKLM is a freeze-dried heat-killed preparation of Listeria monocytogenes (LM), a facultative intracellular Gram-positive bacterium. Infection with LM induces
the secretion of inflammatory cytokines, such as TNF, IL-12, and several chemokines, allowing the recruitment and activation of immune cells. This response
is mediated mainly by the interaction between MyD88 and TLR2 8, 9 .
HKLP (Legionella pneumophila)
Legionella pneumophila, a Gram negative bacterium, is the causative agent of legionnaires’ disease which is characterized by severe pneumonia. Although
TLR4 is involved in host defense against gram negative bacteria infection, it is not activated or is activated only to a limited extent by L. pneumophila 10 .
L. pneumophila requires TLR2 rather than TLR4 to induce the production of cytokines 11 .
HKLR (Lactobacillus rhamnosus)
Lactobacillus rhamnosus is a nonpathogenic gram-positive inhabitant of the human microflora. It is used as a natural preservative in yogurt and other dairy products
to extend the shelf life. L. rhamnosus is known to have health beneficial effects, such as the nonspecific enhancement of the immune system. Indeed, heat-killed
L.. rhamnosus (HKLR) has been shown to be a potent inducer of TNF-a from mouse mononuclear cells. This immune response is dependent on TLR2 and CD14 12 .
HKMF (Mycoplasma fermentans) NEW
Mycoplasma fermentans, a member of the mycoplasma family, is a cell wall-less bacterium. It contains lipopeptides, in particular 2-kDa macrophage-activating
lipopetide (MALP-2), a potent stimulator of macrophages through TLR2 and TLR6 3 . Stimulation with heat killed M. fermentans (HKMF) induces rapid activation
of NF-kB and the production of pro-inflammatory cytokines.
HKPA (Pseudomonas aeruginosa)
Pseudomonas aeruginosa is a virulent gram-negative pathogen that infects patients through the respiratory tract, in particular patients with cystic fibrosis.
Heat-killed Pseudomonas aeruginosa (HKPA) initiates host inflammatory responses through TLR2 and TLR5 but not TLR4 13, 14 . The TLR5-mediated response
was shown to be induced by flagellin while LPS appears to play an important role in the TLR2-mediated response.
HKPG (Porphyromonas gingivalis)
HKPG is a freeze-dried heat-killed preparation of the periodontopathic Gram negative bacteria Porphyromonas gingivalis. In CHO cells expressing TLR2 and
CD14, exposure to HKPG induces the activation of NF-kB through TLR2. Expression of TLR4 fails to enhance the response to HKPG suggesting that either
the whole bacterial components of P. gingivalis are not recognized by TLR4 or some components of these bacteria inhibit TLR4-mediated activation 15 .
HKSA (Staphylococcus aureus)
HKSA is a lyophilized heat-killed preparation of Staphyloccocus aureus, a Gram-positive extra-cellular growing bacterium. HKSA is recognized mainly by
TLR2 16 . HKSA induces tolerance to a secondary HKSA stimulation but causes priming to LPS, suggesting a differential regulation of cytokines and chemokines
in gram-positive- and gram-negative-induced inflammatory events 17 .
HKSP (Streptococcus pneumoniae)
Streptococcus pneumoniae, a Gram positive bacterium, is the principal etiologic agent of bacterial meningitis in adults. Heat-killed Streptococcus pneumoniae
(HKSP) induce activation of NF-kB in a TLR2- and CD14-dependent manner 18 . TLR2 has been shown to play an important role in the protein- and
polysaccharide-specific type 1 IgG isotype response following immunization with HKSP 19 .
LM-MS & LAM-MS (Mycobacterium smegmatis)
Lipoarabinomannans (LAM) and lipomannans (LM) are lipoglycans found in mycobacterial cell wall. LM and LAM from the non-pathogenic Mycobacterium
smegmatis are proinflammatory molecules 20 . Both LM-MS and LAM-MS activate macrophages in a TLR2-dependent manner 21 .
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LPS-PG (Porphyromonas gingivalis)
Recognition of lipopolysaccharide from Porphyromonas gingivalis (LPS-PG), a Gram-negative bacterium, is mediated by TLR2 and CD14, and unlike enteric
LPS, is able to induce a septic shock in C3H/HeJ mice which are deficient for TLR4 and hyporesponsive to E. coli LPS. This property is attributed mainly to
the unique lipid A motif of PG-LPS 22 .
LTA-BS & LTA-SA (B. subtilis and S. aureus)
Lipoteichoic acid (LTA) is a major immunostimulatory component of Gram-positive bacteria. Like LPS, LTA is an amphiphile formed by a hydrophilic
polyphosphate polymer linked to a neutral glycolipid. LTA stimulates immune cells through TLR2 to produce TNF-a and other inflammatory cytokines 23 .
Recognition of LTA involves also LPS-binding protein (LBP) and CD14 24 .
InvivoGen provides LTA from B. subtilis (LTA-BS) and S. aureus (LTA-SA) and a purified form of LTA-SA, which is EndoFit (

INNATE IMMUNITY 3
TLR3 Agonists
Poly(I:C) & Poly(I:C)-LMW
Polyinosine-polycytidylic acid (poly(I:C)) is a synthetic analog of double-stranded RNA (dsRNA), a molecular pattern associated with viral infection. Poly(I:C)
is recognized by TLR3 inducing the activation of NF-kB and the production of cytokines through distinct mechanisms that are MyD88-dependent or MyD88independent
1,2 . InvivoGen provides poly(I:C) with a high molecular weight (HMW) or a low molecular weight (LMW) that might activate the immune system
differently:
• Poly(I:C) (HMW) has an average size of 1.5-8 kb.
• Poly(I:C)-LMW has an average size of 0.2-1 kb.
Poly(A:U)
Polyadenylic–polyuridylic acid (poly(A:U)) is a synthetic double stranded RNA molecule that signals only through TLR3. Recognition of poly(A:U) by TLR3
induces the activation of dendritic cells and T lymphocytes. When combined with an antigen in mice, poly(A:U) has been shown to promote antigen-specific
Th1-immune responses and boost antibody production 3 . The potent adjuvant activity of poly(A:U) has been exploited in the treatment of breast cancers
that express TLR3 4 .
1. Yamamoto M. et al., 2003. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science, 301(5633):640-3. 2. Alexopoulou L. et al., 2001. Recognition of
double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature, 413(6857):732-8. 3. Wang L. et al., 2002. Noncoding RNA danger motifs bridge innate and adaptive
immunity and are potent adjuvants for vaccination. J Clin Invest 110:1175–84. 4. Conforti R. et al., 2010. Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically
uncoupled to optimize the anticancer efficacy of TLR3 ligands. Cancer Res. 70(2):490-500.
TLR4 Agonists
Bacterial lipopolysaccharide (LPS) is the major structural component of the outer wall of all Gram-negative bacteria and a potent activator of the immune
system. LPS is recognized by Toll-like receptor 4 (TLR4) which interacts with three different extracellular proteins: LPS binding protein (LBP), CD14 and,
myeloid differentiation protein 2 (MD-2), to induce a signaling cascade leading to the activation of NF-kB and the production of proinflammatory cytokines.
LPS consists of a polysaccharide region that is anchored in the outer bacterial membrane by a specific carbohydrate lipid moiety termed lipid A. Lipid A, also
known as endotoxin, is responsible for the immunostimulatory activity of LPS. The most active form of lipid A contains six fatty acyl groups and is found in
pathogenic bacteria such as Escherichia coli and Salmonella species 1 . Underacylated lipid A structures, containing four or five fatty acids, induce markedly less
host defense responses and can inhibit in a dose-dependent manner the strong endotoxic response triggered by hexa-acylated LPS 2 .
LPS-EB & LPS-EK standard (E. coli 0111:B4) - TLR4 (TLR2) Agonists
LPS-EB and LPS-EK are standard preparations of lipopolysaccharide. They are extracted by a phenol-water mixture. LPS-EB and LPS-EK contain other
bacterial components, such as lipopeptides, and therefore stimulate both TLR4 and TLR2.
LPS-EB, LPS-EK & LPS-SM Ultrapure (E. coli 0111:B4, E. coli K12 and S. minnesota)
Ultrapure LPS-EB (E. coli 0111:B4), LPS-EK (E. coli K12) and LPS-SM (S. minnesota Re type) are extracted by successive enzymatic hydrolysis steps and purified
by the phenol-TEA-DOC extraction protocol described by Hirschfeld M. et al. 3
LPS-RS (Rhodobacter sphaeroides) - TLR4 Antagonist
LPS from the photosynthetic bacterium Rhodobacter sphaeroides (LPS-RS) is a potent antagonist of LPS from pathogenic bacteria 1 . Complete competitive
inhibition of LPS activity is possible at a 100 fold excess of the antagonist. LPS-RS does not induce TLR4 signaling but is detected by the LAL assay, the
standard endotoxin detection assay.
MPLA & MPLAs (synthetic)
MPLA (monophosphoryl lipid A) is a derivative of lipid A from Salmonella minnesota R595 lipopolysaccharide (LPS or endotoxin). While LPS is a complex
heterogeneous molecule, its lipid A portion is relatively similar across a wide variety of pathogenic strains of bacteria 4 . MPLA, used extensively as a vaccine
adjuvant, has been shown to activate TLR4.
MPLAs is a synthetic monophosphoryl lipid A from E. coli with 6 fatty acyl groups. It is structurally very similar to natural MPLA except that natural MPLA
contains a mixture of 5, 6, and 7 acyl Lipid A. This E. coli synthetic MPLA activates TLR4 but does not activate TLR2 even at high concentrations reflecting its
high purity.
1. Coats SR. et al., 2005. MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex.
J Immunol.;175(7):4490-8. 2. Teghanemt A. et al., 2005. Molecular basis of reduced potency of underacylated endotoxins. J Immunol. 175(7):4669-76. 3. Hirschfeld M. et al., 2000. Cutting edge:
repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2. J Immunol. ;165(2):618-22. 4. Martin M. et al., 2003. Role of innate immune
factors in the adjuvant activity of monophosphoryl lipid A. Infect Immun. 71(5):2498-507.
TLR5 Agonists
FLA-BS, FLA-ST, FLA-ST Ultrapure & RecFLA-ST (B. subtilis and S. typhimurium)
Flagellin is the major component of the bacterial flagellar filament, which confers motility on a wide range of bacterial species. Flagellin is recognized by TLR5 1
and induces the activation of NF-kB and the production of cytokines and nitric oxide depending on the nature of the TLR5 signaling complex 2 .
• FLA-BS and FLA-ST are flagellins isolated from the Gram-positive bacteria B. subtilis and from the Gram-negative bacteria S. typhimurium, respectively. They
are purified by acid hydrolysis, heating and ultrafiltration according to Ibrahim GF. et al. 3 . The purity of FLA-ST is estimated at 10%.
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• FLA-ST ultrapure was purified by monoclonal anti-FliC affinity chromatography. The purity of FLA-ST ultrapure is >95%.
• RecFLA-ST is a recombinant flagellin purified from mammalian cells transfected with the FliC gene which encodes flagellin in S. typhimurium. RecFLA-ST is
endotoxin-free according to the gel clot LAL Assay. It activates TLR5 but does not activate TLR2 nor TLR4.
1. Hayashi F. et al., 2001.The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature. 410(6832):1099-103. 2. Mizel SB. et al., 2003. Induction of macrophage
nitric oxide production by Gram-negative flagellin involves signaling via heteromeric Toll-like receptor 5/Toll-like receptor 4 complexes. J Immunol. 170(12):6217-23. 3. Ibrahim GF. et al., 1985.
Method for the isolation of highly purified Salmonella flagellins. J. Clin. Microbiol. 22(6):1040-1044.
TLR7/8 Agonists
CL075 - TLR7/8 Agonist
CL075 (3M002) is a thiazoloquinolone derivative that stimulates TLR8 in human PBMC. It activates NF-kB and triggers preferentially the production of
TNF-a and IL-12 1 . CL075 seems also to induce the secretion of IFN-a through TLR7 but to a lesser extent. It induces the activation of NF-kB at 0.4 μM (0.1
μg/ml) in TLR8-transfected HEK293 cells, and ~ 10 times more CL075 is required to activate NF-kB in TLR7-transfected HEK293 cells.
CL097 - TLR7/8 Agonist
CL097 is a highly water-soluble derivative of the imidazoquinoline compound R848 (>= 20 mg/ml). Similarly to R848, CL097 is a TLR7 and TLR8 ligand 2, 3 .
It induces the activation of NF-kB at 0.4 μM (0.1 μg/ml) in TLR7-transfected HEK293 cells and at 4 μM (1 μg/ml) in TLR8-transfected HEK293 cells.
CL264 - TLR7 Agonist
CL264 is a novel 9-substituted-8 hydroxyadenine derivative. Similarly to SM360320, CL264 induces the activation of NF-kB and the secretion of IFN-a in
TLR7-expressing cells 4 . CL264 is a TLR7-specific ligand, it does not stimulate TLR8 even at high concentrations (> 10 μg/ml). In TLR7-transfected HEK293
cells, CL264 triggers NF-kB activation at a concentration of 0.1 μM which is 5-10 times less than imiquimod.
Gardiquimod - TLR7 Agonist
Gardiquimod is a new imidazoquinoline compound developed and manufactured by InvivoGen. Similarly to Imiquimod, Gardiquimod induces the activation
of NF-kB in HEK293 cells expressing human or mouse TLR7. However Gardiquimod is 10 times more active as a concentration of 0.1 μg/ml is sufficient to
detect NF-kB activation whereas Imiquimod requires a concentration of 1 μg/ml. Gardiquimod shares the same actions as R848 7 .
Imiquimod - TLR7 Agonist
Imiquimod (R837), an imidazoquinoline amine analogue to guanosine, is an immune response modifier with potent indirect antiviral activity. This low molecular
weight synthetic molecule induces the production of cytokines such as IFN-a through the activation of TLR7 8 . This activation is MyD88-dependent and leads
to the induction of the transcription factor NF-kB 9 .
Loxoribine - TLR7 Agonist
Loxoribine is a guanosine analog derivatized at position N7 and C8. This L-nucleoside is a strong stimulator of the immune system 10 . It signals through TLR7
leading to the activation of NF-kB 8, 11 . Similarly to the imidazoquinoline compound imiquimod, loxoribine recognition is restricted to TLR7.
Poly(dT) - TLR7/8 Modulator
Poly(dT), a thymidine homopolymer phosphorothioate ODN, is a modulator of human TLR7 and TLR8. In combination with an imidazoquinoline, such as
R848 and CL075, it increases TLR8-mediated signaling but abolishes TLR7-mediated signaling 12, 13 . Alone poly(dT) has no significant effect on either of these
TLRs. Furthermore, co-incubation of poly(dT) and an imidazoquinoline was shown to induce NF-kB activation in HEK293 cells transfected with murine
TLR8- and primary TLR8-expressing mouse cells, demonstrating that murine TLR8 is functional 14 .
R848 - TLR7/8 agonist
R848 is an imidazoquinoline compound with potent anti-viral activity. This low molecular weight synthetic molecule activates immune cells via the TLR7/TLR8
MyD88-dependent signaling pathway 9, 12 . Recently, R848 was shown to trigger NF-kB activation in cells expressing murine TLR8 when combined with
poly(dT) 14 . Unlike other commercially available R848 preparations, InvivoGen’s R848 is water soluble (~5 mg/ml).
Single-stranded RNAs: ssPolyU, ssRNA40, ssRNA-DR & ORN02/06 - TLR8 agonists
Single-stranded RNA (ssRNA) has been identified as the natural ligand of TLR7 and TLR8 15, 16 . ssRNA derived from HIV-1 or the influenza virus were shown
to induce the production of proinflammatory cytokines in pDC. This induction was reproduced using polyU or GU-rich (ssRNA40) ODNs complexed with
cationic lipids to protect them from degradation. Upon stimulation with ssRNA, murine TLR7 and human TLR8 induced the activation of NF-kB, whereas
human TLR7 and murine TLR8 failed, implying a species specificity difference in ssRNA recognition.
• ssPolyU & ssRNA40: ssPolyU is a single-stranded poly-uridine (polyU) oligonucleotide while ssRNA40 is a 20-mer phosphorothioate protected singlestranded
RNA oligonucleotide containing a GU-rich sequence. Both single-stranded RNAs are complexed with the cationic lipid LyoVec , to protect them
from degradation and facilitate their uptake. They are provided as lyophilized powder.
• ssRNA41 is a 20-mer phosphorothioate protected single-stranded RNA oligonucleotide. It derives from ssRNA40 by replacement of all U nucleotides with
adenosine 2 . ssRNA41 is complexed with the cationic lipid LyoVec , to protect it from degradation and facilitate its uptake, and lyophilized to generate
ssRNA41/LyoVec. Unlike ssRNA40, ssRNA41 is unable to induce the production of type IFNs, and therefore can be used as a negative control for ssRNA40 16, 17 .
• ssRNA-DR is a short single stranded RNA (

INNATE IMMUNITY 3
1. Gorden KB. et al., 2005. Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J Immunol. 174(3):1259-68. 2. Salio M. et al., 2007. Modulation of human
natural killer T cell ligands on TLR-mediated antigen-presenting cell activation. PNAS 104: 20490 - 20495. 3. Butchi NJ. et al., 2008., Analysis of the Neuroinflammatory Response to TLR7
Stimulation in the Brain: Comparison of Multiple TLR7 and/or TLR8 Agonists. J Immunol 180: 7604-7612. 4. Lee J. et al., 2006. Activation of anti-hepatitis C virus responses via Toll-like receptor
7. Proc Natl Acad Sci U S A. 103(6):1828-33. 5 Koski GK. et al., 2004. Cutting edge: innate immune system discriminates between RNA containing bacterial versus eukaryotic structural features
that prime for high-level IL-12 secretion by dendritic cells.J Immunol. 172(7):3989-93. 6. Kariko K. et al., 2005. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside
modification and the evolutionary origin of RNA. Immunity. 23(2):165-75. 7. Morris GE., et al., 2006. Cooperative molecular and cellular networks regulate Toll-like receptor-dependent
inflammatory responses. FASEB J. 20: 2153 - 2155. 8. Lee J et al., 2003. Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: Activation of Toll-like receptor 7.
Proc Natl Acad Sci U S A. 100(11):6646-6651. 9. Hemmi H. et al. 2002. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol,
3(2):196-200. 10. Pope BL. et al., 1995. The immunostimulatory compound 7-Allyl-8-oxoguanosine (Loxoribine) induces a distinct subset of murine cytokines. Cell Immunol, 162:333-339.
11. Heil F. et al., 2003. The Toll-like receptor 7 (TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily. Eur J Immunol. 33(11):2987-97. 12. Jurk
M. et al. 2002. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R848. Nat Immunol, 3(6):499. 13. Gorden KKB. et al., 2006. Oligodeoxynucleotides
Differentially Modulate Activation of TLR7 and TLR8 by Imidazoquinolines. J. Immunol. 177: 8164 - 8170. 14. Gorden KKB. et al., 2006. Cutting Edge: Activation of Murine TLR8 by a Combination
of Imidazoquinoline Immune Response Modifiers and PolyT OligodeoxynucleotidesJ. Immunol., 177: 6584 - 6587. 15. Diebold SS. et al., 2004. Innate antiviral responses by means of TLR7mediated
recognition of single-stranded RNA. Science. 5;303(5663):1529-31 16. Heil F. et al., 2004. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science.
5;303(5663):1526-9. 17. Alter G. et al., 2007. Single-Stranded RNA Derived from HIV-1 Serves as a Potent Activator of NK Cells. J Immunol. 178:7658-7666. 18. Hornung V. et al., 2005. Sequencespecific
potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7.Nat Med. 11(3):263-70. 19. Forsbach A. et al., 2008. Identification of RNA Sequence
Motifs Stimulating Sequence-Specific TLR8-Dependent Immune Responses. J. Immunol., 180: 3729-38.
TLR9 Agonists
E. coli DNA ef (endotoxin-free)
Bacterial DNA contains 20-fold more unmethylated CpG motifs than mammalian DNA and thus activates TLR9 1 . E. coli DNA ef is an ultrapure, endotoxinfree
(ef) preparation of bacterial double-stranded DNA devoid of TLR2 and TLR4 activities.
E. coli ssDNA
E. coli ssDNA is an ultrapure, endotoxin-free preparation of bacterial single-stranded DNA (ssDNA). It is a better TLR9 ligand than E. coli DNA ef (dsDNA
endotoxin-free) as TLR9 binds directly and sequence-specifically to single-stranded unmethylated CpG-DNA 2 . E. coli ssDNA is complexed with the cationic
lipid LyoVec to allow a better internalization of the immunostimulatory DNA to the acidic compartment where TLR9 is expressed.
Stimulatory CpG ODNs & Control CpG ODNs
Toll-Like Receptor 9 (TLR9) detects unmethylated CpG dinucleotides in bacterial or viral DNA inducing strong immunostimulatory effects. TLR9 activation
can be mimicked by synthetic phosphorothioate-stabilized oligodeoxynucleotides (ODN) containing immune stimulatory “CpG motifs”. Three types of
stimulatory CpG ODNs have been identified, types A (or D), B (or K) and C, which differ in their immune-stimulatory activities:
- Type A CpG ODNs are characterized by a phosphodiester central CpG-containing palindromic motif and a phosphorothioate 3’ poly-G string. They induce
high IFN-a production from plasmacytoid dendritic cells (pDC) but are weak stimulators of TLR9-dependent NF-kB signaling.
- Type B CpG ODNs contain a full phosphorothioate backbone with one or more CpG dinucleotides. They strongly activate B cells but weakly stimulate
IFN-a secretion.
- Type C CpG ODNs combine features of both types A and B. They contain a complete phosphorothioate backbone and a CpG-containing palindromic
motif. Type C CpG ODNs induce strong IFN-a production from pDC and B cell stimulation.
These stimulatory CpG ODNs induce differentially the stimulation of human and murine immune cells in vitro. This species-specificity is also observed with
nonresponsive cells such as HEK293 cells transfected with human or mouse TLR9. InvivoGen offers a comprehensive collection of stimulatory CpG ODNs
and control CpG ODNs that provide useful tools for studying TLR9-mediated activation. InvivoGen’s CpG ODNs are endotoxin-free and tested for activity
in various cell lines expressing human or mouse TLR9.
Control CpG ODNs that do not stimulate TLR9 have been designed for each stimulatory CpG ODN. They feature the same sequence as their stimulatory
counterparts but contain GpC dinucleotides in place of CpG dinucleotides.
ODN 1585 3 (Type A, mouse specific) 5’-ggGGTCAACGTTGAgggggg-3’ ODN 1585 control 5’-ggGGTCAAGCTTGAgggggg-3’
ODN 1668 4 (Type B, mouse specific) 5’-tccatgacgttcctgatgct-3’ ODN 1668 control 5’-tccatgagcttcctgatgct-3’
ODN 1826 5 (Type B, mouse specific) 5’-tccatgacgttcctgacgtt-3’ ODN 1826 control 5’-tccatgagcttcctgagctt-3’
ODN 2006 6, 10 (Type B, human specific) 5’-tcgtcgttttgtcgttttgtcgtt-3’ ODN 2006 control 5’-tgctgcttttgtgcttttgtgctt-3’
ODN 2007 7, 8 (Type B, bovine/porcine) 5’-tcgtcgttgtcgttttgtcgtt-3’ ODN 2007 control 5’-tgctgcttgtgcttttgtgctt-3’
ODN 2216 9 (Type A, human specific) 5’-ggGGGACGA:TCGTCgggggg-3’ ODN 2216 control 5’-ggGGGAGCA:TGCTGgggggc-3’
ODN 2336 10 (Type A, human specific) 5’-gggGACGAC:GTCGTGgggggg-3’ ODN 2336 control 5’-gggGAGCAG:CTGCTGgggggg-3’
ODN 2395 6, 10 (Type C, human/mouse) 5’-tcgtcgttttcggcgc:gcgccg-3’ ODN 2395 control 5'-tgctgcttttggggggcccccc-3'
ODN D19 11, 12 (Type A, human/porcine) 5’-ggTGCATC:GATGCAGggggg-3’ ODN D19 control 5’-ggTGCATG:CATGCAGggggg-3’
ODN M362 13 (Type C, human/mouse) 5’-tcgtcgtcgttc:gaacgacgttgat-3’ ODN M362 control 5’-tgctgctgcttg:caagcagcttgat-3’
Bases in capital letters are phosphodiester, bases in lower case are phosphorothioate. Palindrome is underlined.
pCpG Giant - TLR9 Control
pCpG giant is a high molecular weight plasmid entirely devoid of CpG dinucleotides. This DNA also features no detectable amounts of endotoxin, as determined
using a kinetic chromogenic LAL assay and the HEK -Blue -4 cell line-based assay (LPS Detection Kit, see page 90) and no detectable TLR2 activity. In addition,
this plasmid DNA displays no Dcm methylation and a reduced level of Dam methylation. pCpG Giant can be used as a control in studies on CpG methylations.
Salmon Sperm DNA - TLR9 Control
Salmon sperm DNA does not activate any TLR and can be used as a negative control for TLR induction experiments in particular in TLR9 studies. Salmon
sperm DNA has been tested for endotoxin and has been found to be EndoFit (

1. Zhao H. et al., 2004. Contribution of toll-like receptor 9 signaling to the acute inflammatory response to nonviral vectors. Mol. Ther. 9(2)241-248. 2. Rutz M. et al., 2004. Toll-like receptor
9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur. J. Immunol. 34:1-10. 3. Ballas ZK. et al., 2001. Divergent therapeutic and immunologic effects of
oligodeoxynucleotides with distinct CpG motifs. J Immunol. 167(9):4878-86. 4. Heit A. et al., 2004. CpG-DNA aided cross-priming by cross-presenting B cells. J Immunol. 172(3):1501-7.
5. Li J. et al., 2007. Lymphoma immunotherapy with CpG oligodeoxynucleotides requires TLR9 either in the host or in the tumor itself. J Immunol. 179: 2493-2500. 6. Moseman EA. et al.,
2004. Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4+CD25+ regulatory T cells. J Immunol. 173(7):4433-42. 7. Mena A. et al.,
2003. Bovine and ovine blood mononuclear leukocytes differ markedly in innate immune responses induced by Class A and Class B CpG-oligodeoxynucleotide. Oligonucleotides.
2003;13(4):245-59. 8. Kringel H. et al., 2004. CpG-oligodeoxynucleotides enhance porcine immunity to Toxoplasma gondii. Vet Parasitol. 123(1-2):55-66. 9. Tomescu C. et al., 2007. NK cell
lysis of HIV-1-infected autologous CD4 primary T cells: requirement for IFN-mediated NK activation by plasmacytoid dendritic cells. J Immunol. 179(4):2097-104. 10. Roda JM. et al., 2005.
CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells. J Immunol. 175(3):1619-27. 11. Verthelyi D. et al., 2001.
Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs. J Immunol. 166(4):2372-7. 12. Guzylack-Piriou L. et al., 2004. Type-A CpG oligonucleotides
activate exclusively porcine natural interferon-producing cells to secrete interferon-alpha, tumour necrosis factor-alpha and interleukin-12. Immunology. 112(1):28-37. 13. Marshall JD. et al.,
2005. Superior activity of the type C class of ISS in vitro and in vivo across multiple species. DNA Cell Biol. 24(2):63-72.
TLR9 Antagonists
Inhibitory ODNs
• ODN 2088, ODN TTAGGG and G-ODN
Recent studies suggest the existence of DNA sequences that can neutralize the stimulatory effect of CpG ODNs. The most potent inhibitory sequences
are (TTAGGG)4 found in mammalian telomeres 1 and ODN 2088 which derives from a murine stimulatory CpG ODN by replacement of 3 bases 2 . Recently,
another inhibitory guanosine-rich ODN, named G-ODN, was described 3 . G-ODN was suppressive in murine DC and macrophages as well as in human
plasmacytoid DC. Inhibitory ODNs seem to act by disrupting the colocalization of CpG ODNs with TLR9 in endosomal vesicles without affecting cellular
binding and uptake. Inhibitory ODNs are often utilized to demonstrate a TLR9 dependence in murine systems.
ODN 2088 1 (Mouse preferred) 5’-tcctggcggggaagt-3’ ODN 2088 control 5’-tcctgagcttgaagt-3’
ODN TTAGGG 2 (Human preferred) 5’-tttagggttagggttagggttaggg-3’ ODN TTAGGG control 5’-gctagatgttagcgt-3’
G-ODN 5’-ctcctattgggggtttcctat-3’
Bases are phosphorothioate.
• ODN 4084-F and ODN INH-1
ODN 4084-F and ODN INH-1 belong to a new class of inhibitory ODNs 4 . They contain an inhibitory DNA motif consisting of two nucleotide triplets, a
proximal CCT and a more distal GGG, spaced from each other by four nucleotides. ODN 4084-F is the shortest active inhibitory ODN. ODN INH-1
derives from ODN 4084-F by addition of a complementary strand of nucleotides forming a complete palindrome. ODN INH-47 is a palindromic variant
of ODN INH-1 in which the CCT and GGG have been replaced by random nucleotide triplets. ODN 4084-F is linear and a class B (‘broadly-active)
inhibitory ODN, while ODN INH-1 is palindromic and a class R (‘restricted’) inhibitory ODN. ODN 4084-F and ODN INH-1 are potent inhibitors of
TLR9-induced B cells and macrophages, whereas ODN INH-47 has no effect 5 .
ODN 4084-F NEW 5’-cctggatgggaa-3’’
ODN INH-1 NEW 5’-cctggatgggaa:ttcccatccagg-3’
ODN INH-47 (control) NEW 5’-tatggattttaa:ttaaaatccata-3’
Bases are phosphorothioate.
1. Stunz LL. et al., 2002. Inhibitory oligonucleotides specifically block effects of stimulatory CpG oligonucleotides in B cells. Eur J Immunol. 32(5):1212-22. 2. Gursel L. et al., 2003. Repetitive
elements in mammalian telomeres suppress bacterial DNA-induced immune activation. J Immunol. 171(3):1393-400. 3. Peter M. et al., 2008. Characterization of suppressive
oligodeoxynucleotides that inhibit Toll-like receptor-9-mediated activation of innate immunity. Immunology. 123(1):118-28. 4. Lenert P, et al., 2003. Structural characterization of the inhibitory
DNA motif for the type A (D)-CpG-induced cytokine secretion and NK-cell lytic activity in mouse spleen cells. DNA Cell Biol. 22(10):621-31 .5. Lenert P, et al., 2009. DNA-like class R
inhibitory oligonucleotides (INH-ODNs) preferentially block autoantigen-induced B-cell and dendritic cell activation in vitro and autoantibody production in lupus-prone MRL-Fas(lpr/lpr)
mice in vivo. Arthritis Res Ther. 11(3):R79.
NOD1/2 Agonists
iE-DAP & iE-Lys - NOD1 Agonists
• iE-DAP (D-g-Glu-mDAP) is a dipeptide present in the PGN of a subset of bacteria that include Gram-negative bacilli and particular Gram-positive bacteria
such as Bacillus subtilis and Listeria monocytogenes 1 . iE-DAP is the minimal motif recognized by NOD1.
• iE-Lys is a peptide found in the PGN of Gram-positive bacteria. It is not recognized by NOD1 and thus can be used as negative control for iE-DAP.
C12-iE-DAP - NOD1 Agonist
C12-iE-DAP is an acylated derivative of iE-DAP. It was generated by addition of a lauroyl (C12) group to the glutamic residue of iE-DAP. C12-iE-DAP
stimulates specifically NOD1 at concentrations 100- to 1000-fold lower than the original iE-DAP.
MDP, MDP control, L18-MDP & N-glycolyl-MDP - NOD2 Agonists
• MDP (MurNAc-L-Ala-D-isoGln, also known as muramyl dipeptide), is the minimal bioactive peptidoglycan motif common to all bacteria and the essential
structure required for adjuvant activity in vaccines. MDP has been shown to be recognized by NOD2, but not TLR2, nor TLR2/1 or TLR2/6 associations 2, 3 .
This recognition is highly stereospecific of the L-D isomer, excluding any reaction to the D-D or L-L analogs 3, 4 . NOD2 mutants associated with susceptibility
to Crohn‘s disease have been found to be deficient in their recognition of MDP 2, 3 . The potent adjuvant activity of MDP may also be linked to an activation
of the CIAS1/NALP3/Cryopyrin inflammasome 5 .
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INNATE IMMUNITY 3

INNATE IMMUNITY 3
• MDP control (MurNAc-D-Ala-D-isoGln), the D-D isomer of muramyl dipeptide, is unable to induce NOD2 signaling.
• L18-MDP, a synthetic derivative of MDP, has been shown to display a higher adjuvant activity than MDP 6 .
• N-glycolyl-MDP - The cell wall of mycobacteria is known to be extremely immunogenic. This potent activity is attributed to their MDP which is Nglycolylated
in contrast to the MDP of most bacteria which is N-acetylated. N-glycolyl-MDP has been reported to display a stronger NOD2-mediated
activity than N-acetyl-MDP and thus to be a more potent vaccine adjuvant than N-acetyl-MDP 7 .
Murabutide & Murabutide control - NOD2 Ligands
Murabutide (MurNAc-L-Ala-D-GlnOBu) is a safe synthetic immunomodulator derived from muramyl dipeptide (MDP). In contrast to MDP, murabutide is
devoid of pyrogenic activity 8 and lacks somnogenic activity 9 . Murabutide is recognized by the intracellular receptor NOD2 inducing the activation of NF-kB.
Murabutide control contains D-alanine instead of L-alanine and is inactive on NOD2.
M-Tri DAP - NOD1/ NOD2 Agonist
MurNAc-L-Ala-D-g-Glu-mDAP (M-TriDAP), also called DAP-containing muramyl tripeptide is a peptidoglycan (PGN) degradation product found mostly in
Gram-negative bacteria. M-TriDAP is recognized by NOD1 and to a lesser extent NOD2. M-TriDAP induces the activation of NF-kB at similar levels to Tri-
DAP 10 .
PGN-Ecndi & PGN-SAndi insoluble, ultrapure (E. coli and S. aureus) - NOD1/2 Agonists
PGN-ECndi from E. coli K12 and PGN-SAndi from S. aureus are insoluble preparations of PGNs purified by detergent lysis and hydrolysis under basic
conditions to eliminate lipophilic constituents. These PGN preparations have lost their ability to activate TLR2-transfected HEK293 cells but still activate
NOD2-transfected cells. PGN-ECndi activates also NOD1-transfected cells.
PGN-Ecndss soluble, sonicated, ultrapure (E. coli) - NOD1/2 Agonists
PGN-ECndss from E. coli K12 is obtained after sonication of insoluble PGNs. At the working concentrations, it activates HEK293 cells transfected with either
NOD1 or NOD2 but not cells transfected with TLR2 or TLR4.
Tri-DAP & Tri-Lys - NOD1 Agonists
Tri-DAP (L-Ala-g-D-Glu-mDAP) comprises the iE-DAP dipeptide and an L-Ala residue. Similarly to iE-DAP, this tripeptide is specifically recognized by Nod1
but exhibits a ~3-fold higher ability to activate NF-kB than iE-DAP 10 .
Tri-Lys is a peptide found in the PGN of Gram-positive bacteria. It is not recognized by NOD1 and thus can be used as negative control for Tri-DAP.
1. Chamaillard M. et al., 2003. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat. Immunol.4(7):702-7. 2. Girardin SE. et al., 2003.
Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 278(11):8869-72. 3. Inohara N. et al., 2003. Host recognition of bacterial muramyl
dipeptide mediated through NOD2. Implications for Crohn’s disease. J Biol Chem. 278(8):5509-12. 4. Traub S. et al., 2004. Structural requirements of synthetic muropeptides to synergize
with lipopolysaccharide in cytokine induction. J Biol Chem. 279(10):8694-700. 5. Martinon F. et al., 2004. Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin
inflammasome. Curr Biol. 14(21):1929-34. 6. Ishihara C. et al. 1985. Effect of muramyl dipeptide and its stearoyl derivatives on resistance to Sendai virus infection in mice. Vaccine. 3(5):370-4.
7. Coulombe F,. et al., 2009. Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide. J Exp Med. 206(8):1709-16. 8. Chedid LA. et al., 1982. Biological activity of a new
synthetic muramyl peptide adjuvant devoid of pyrogenicity. Infect Immun. 35(2):417-24. 9. Krueger JM. et al., 1984. Muramyl peptides. Variation of somnogenic activity with structure. J Exp
Med. 159(1):68-76. 10. Girardin SE. et al., 2003. Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2. J Biol Chem. 278(43):41702-8.
RIG-I/MDA5 and CDS Agonists
5’ppp-dsRNA - RIG-I Agonist NEW
5’ppp-dsRNA is a short (19 mer) blunt-end double-stranded RNA with a 5’ triphosphate. Transfected 5’ppp-dsRNA is a ligand for RIG-I 1 . This dsRNA sensor
is specifically activated by the uncapped 5’ triphosphate moiety on viral RNA. This triphosphate occurs during viral replication and is absent from most
cytosolic self-RNA. A synthetic approach to the exact structure requirement to RIG-I recognition demonstrated that a short blunt double-stranded
conformation containing a triphosphate at the 5’ end is required 2 . 5’ppp-dsRNA Control is a 19 mer blunt-end dsRNA without a 5’triphosphate.
5’ppp-dsRNA 5’- pppGCAUGCGACCUCUGUUUGA -3’ 5’ppp-dsRNA Control 5’- pppGCAUGCGACCUCUGUUUGA -3’
3’- CGUACGCUGGAGACAAACU -5’ 3’- CGUACGCUGGAGACAAACU -5’
Poly(dA:dT) - CDS Agonist NEW
Poly(dA:dT) is a repetitive synthetic double-stranded DNA sequence of poly(dA-dT)•poly(dT-dA) and a synthetic analog of B-DNA. Poly(dA:dT) is
recognized by several sensors, including DAI, LRRFIP1 and AIM2 3-5 . It has also been shown to be transcribed by RNA polymerase III into dsRNA with a 5’triphosphate
moiety which is a ligand for RIG-I 6 . Poly(dA:dT) is available naked or complexed with the cationic lipid LyoVec to facilitate their uptake.
Poly(dG:dC) - CDS Agonist NEW
Poly(dG:dC) is a repetitive synthetic double-stranded DNA sequence of poly(dG-dC)•poly(dC-dG). Poly(dG:dC) is a synthetic analog of the Z-DNA form.
It has been reported to be recognized by LRRFIP1 4 . Poly(dG:dC) is available naked or complexed with the cationic lipid LyoVec to facilitate their uptake.
Poly(I:C)/LyoVec & Poly(I:C)-LMW/LyoVec Complexes
Unlike naked poly(I:C) which is recognized by TLR3, transfected poly(I:C) is sensed by RIG-I/MDA-5 in a cell-type-specific manner 7, 8 . Poly(I:C)/LyoVec and
poly(I:C)-LMW/LyoVec are preformed complexes between poly(I:C) or poly(I:C)-LMW and the transfection reagent LyoVec . These complexes induce the
activation of the RIG-I/MDA-5 signaling pathway at concentrations ranging from 100 ng to 1 μg/ml in C57/WT murine embryonic fibroblasts (MEFs),
InvivoGen’s RLR reporter cell line.
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1. Hornung V. et al., 2006. 5'-Triphosphate RNA is the ligand for RIG-I. Science. 314(5801):994-7. 2. Schlee m. et al., 2009. Recognition of 5' triphosphate by RIG-I helicase requires short blunt
double-stranded RNA as contained in panhandle of negative-strand virus. Immunity. 17;31(1):25-34. 3. Takaoka A. et al., 2007. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator
of innate immune response. Nature. 448(7152):501-5. 4. Yang P. et al., 2010. The cytosolic nucleic acid sensor LRRFIP1 mediates the production of type I interferon via a beta-catenindependent
pathway. Nat Immunol. 11(6):487-94. 5. Jones JW. et al., 2010. Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis. PNAS, 107(21):9771-6.
6. Ablasser A. et al., 2009. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat Immunol. 10(10):1065-72. 7. Gitlin
L. et al., 2006. Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephelamyocarditis picornavirus. PNAS 103(22):8459-8464. 8. Kato H. et al.,
2005. Cell type-specific involvement of RIG-I in antiviral response. Immunity. 23(1):19-28.
Dectin-1 Agonists
Curdlan NEW
Curdlan is a high molecular weight linear polymer consisting of ß-(1 -> 3)-linked glucose residues. Curdlan is produced as a water-insoluble polysaccharide
by the soil bacterium, Alcaligenes faecalis. Curdlan is recognized by the membrane bound Dectin-1 receptor leading to the CARD9-dependent activation of
NF-kB and MAP kinases 1 . Furthermore, Dectin-1 signaling activates the NFAT transcription factor. Recent data suggest that Curdlan is also recognized by
the cytosolic NLRP3 inflammasome complex which cooperates with Dectin-1 resulting in in robust activation of IL-1b–mediated inflammatory response 2 .
HKCA (Candida albicans)
HKCA is a heat-killed preparation of Candida albicans. C. albicans is an opportunistic yeast that causes serious infections in immunocompromised patients.
Beta-glucans represent 40% of the cell wall of C. albicans. Heat killing of this yeast result in the exposure of the beta-glucans on the surface of the cell wall
and their subsequent recognition by the beta-glucan receptor, dectin-1 3 . HKCA derives from the strain ATCC 10231.
HKSC (Saccharomyces cerevisiae)
HKSC is a heat-killed preparation of the yeast Saccharomyces cerevisiae. The cell wall of S. cerevisiae consists mainly of equal amounts of a-mannans and
b-glucans 4 . Early studies have suggested that the phagocytosis of unopsonized HKSC is mediated by both mannose and b-glucans receptors. However, recent
data show that the b-glucan receptor, dectin-1, is the predominant receptor involved in this process 5 .
Zymosan - TLR2 & Dectin-1 Agonist
Zymosan, an insoluble preparation of yeast cell, activates macrophages via TLR2. TLR2 cooperates with TLR6 and CD14 in response to zymosan 6 . Zymosan
is also recognized by Dectin-1, a phagocytic receptor expressed on macrophages and dendritic cells, which collaborates with TLR2 and TLR6 enhancing the
immune responses triggered by the recognition of Zymosan by each receptor 7 .
Zymosan Depleted
Zymosan depleted is a S. cerevisiae cell wall preparation treated with hot alkali to remove all its TLR-stimulating properties 8 . Zymosan depleted activates
Dectin-1 but not TLR2.
1. Goodridge HS, et al., 2009. Beta-glucan recognition by the innate immune system. Immunol Rev. 230(1):38-50. 2. Kankkunen P., 2010. (1,3)-beta-glucans activate both dectin-1 and NLRP3
inflammasome in human macrophages. J Immunol. 184(11):6335-42. 3. Gow NA. et al., 2007. Immune recognition of Candida albicans beta-glucan by dectin-1.J Infect Dis. 196(10):1565-71.
4. Giaimis J. et al., 1993. Both mannose and b-glucan receptors are involved in phagocytosis of unopsonized, heat-killed Saccharomyces cerevisiae by murine macrophages. J. Leukoc. Biol., 54:
564-571. 5. Brown GD. et al., 2002. Dectin-1 Is A Major ß-Glucan Receptor On Macrophages. J. Exp. Med., 196: 407-412. 6. Ozinsky A. et al., 2000. The repertoire for pattern recognition
of pathogens by the innate immune system is defined by cooperation between toll-like receptors. PNAS. 97(25):13766-71. 7. Gantner BN. et al., 2003. Collaborative induction of inflammatory
responses by dectin-1 and Toll-like receptor 2. J Exp Med. 197(9):1107-17. 8. Gantner BN. et al., 2003. Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor
2. J Exp Med. 197(9):1107-17.
NF-kB Activators
PMA
Phorbol 12-myristate 13-acetate (PMA), also known as 12-O-tetradecanoylphorbol 13-acetate (TPA) is a specific activator of Protein Kinase C (PKC) and
hence of NF-kB. PMA is the most commonly used phorbol ester. It is active at nM concentrations. PMA causes an extremely wide range of effects in cells
and tissues, and is a very potent mouse skin tumor promoter 1 .
TNF-a
Tumor necrosis factor-alpha (TNF-a) is a cytokine that plays a role in a variety of biological processes including cell proliferation, differentiation and apoptosis 2 .
It acts by activating transcription factors such as NF-kB and AP-1. InvivoGen provides a recombinant human TNF-a produced in CHO cells and purified by
affinity chromatography.
1. Chang MS. et al., 2005. Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-kappaB, but not p38
MAPK, pathways. Cell Signal. 17(3):299-310. 2. Leong KG & Karsan A. 2000. Signaling pathways mediated by tumor necrosis factor alpha. Histol Histopathol. 15(4):1303-25. Review.
www.invivogen.com/innate-immunity-pamps
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INNATE IMMUNITY 3

INNATE IMMUNITY 3
90
HEK-Blue LPS Detection Kit
Lipopolysaccharide (LPS), also known as endotoxin, the major cell wall component of Gram-negative bacteria, induces the activation of NF-kB
and the production of proinflammatory cytokines. In vivo, this response can cause fever, septic shock and eventually death of the animal. In vitro,
it can introduce a bias in experiments involving cells sensitive to low levels of LPS such as monocytes. In addition, repeated passages of cell
lines in a medium containing LPS might render these cells unresponsive to further stimulation by LPS. This desensitization, termed LPS tolerance,
does not only affect inflammatory responses but also other essential functions including antigen presentation by monocytes. Thus, monitoring
the presence of LPS in biological reagents is crucial. InvivoGen provides the HEK-Blue LPS Detection Kit, a simple, rapid and reliable system
to detect the presence of endotoxins in your samples.
➥ Detection range: 1.5 EU/ml in the sample
Description
The HEK-Blue LPS Detection Kit is based on the ability of TLR4 to
recognize structurally different LPS from gram-negative bacteria and in
particular lipid A, their toxic moiety. Proprietary cells engineered to
become extremely sensitive to LPS, called HEK-Blue -4 cells, are the main
feature of this endotoxin detection kit. The presence of very low
concentrations of LPS, starting as low as 0.03 ng/ml, are detected by the
HEK-Blue -4 cells leading to the activation of NF-kB. Using HEK-Blue
Detection, a specific detection medium, NF-kB activation can be observed
with the naked eye or quantified by reading the OD at 650 nm.
Key Features
HEK-Blue -4 cells, the endotoxin sensor cells, are engineered HEK293
cells. These cells stably express TLR4, MD2, CD14 and multiple genes from
the TLR4 pathway. They coexpress an optimized SEAP reporter gene,
placed under the control of a promoter inducible by the transcription
factors NF-kB and AP-1.
HEK-Blue Selection is a solution that combines several selective
antibiotics. These antibiotics guarantee the persistent expression of the
various transgenes introduced in HEK-Blue -4 cells. Furthermore,
Normocin is included in the kit to protect HEK-Blue -4 cells from any
potential microbial contamination, whether caused by mycoplasmas,
bacteria or fungi (see page 11).
HEK-Blue Detection is a medium specifically designed for the detection
of SEAP. It contains a color substrate that produces a purple/blue color
following its hydrolysis by SEAP (see page 15).
Contents and Storage
The HEK-Blue LPS Detection Kit is composed of the following
components:
- 1 vial of HEK-Blue -4 cells (3-5x 10 6 cells)
- 4 tubes of 250X HEK-Blue Selection (2 ml)
- 4 tubes of 500X Normocin (1 ml)
- 2 pouches of HEK-Blue Detection (50 ml each)
- 1 tube of E. coli K12 LPS, LPS-EK, (100 μg) as a positive control
- 1 tube of endotoxin-free water (1.5 ml) as a negative control
Buy the HEK-Blue LPS Detection Kit once then reorder only the
reagents to perform further assays.
www.invivogen.com/innate-immunity-pamps
HEK-Blue LPS Detection Kit Procedure
PRODUCT QTY CAT. CODE
HEK-Blue LPS Detection Kit 1 kit rep-lps
HEK-Blue Selection 5 x 2 ml hb-sel
Normocin 10 x 1 ml ant-nr-1
HEK-Blue Detection 2 pouches hb-det1
LPS-EK (control) 5 mg tlrl-eklps

TLR/NOD Ligand Screening
InvivoGen has developed a high-throughput method to determine whether a compound is recognized by Toll-Like Receptors or NOD1/NOD2
and acts either as an agonist or antagonist. This method is based on TLR- or NOD-induced activation of various transcription factors including
NF-kB, AP1 and IRF7 in HEK293 clones.
Key Service Features
Short turnaround time - Screening turnaround: ONLY 2 weeks
Screening flexibility - Screening parameters can be selected and/or modified
based on customer requirements. Level 1 and level 2 can be carried out on
additional TLRs, including mouse TLRs. More compound concentrations can be
tested.
Cost effective - A set-up charge applies for the first compound. Subsequent
compounds are heavily discounted.
Description
TLR Ligand Screening
Two choices of services are offered, level 1 and level 2, that can be performed
sequentially or individually.
Level 1: Single dose testing on a panel of human TLRs
Testing is carried out on seven TLRs.
- TLR2: wide array of microbial molecules
- TLR3: dsRNA
- TLR4: Gram-negative lipopolysaccharide
- TLR5: flagellin
- TLR7: single-stranded RNA and small antiviral molecules
- TLR8: single-stranded RNA and small antiviral molecules
- TLR9: specific unmethylated CpG oligonucleotide sequences
Screening is performed at a single concentration, typically a 1/10 dilution of the
original compound solution provided, or customer specified. Duplicate tests (plus
controls, see list).
Level 2: Dose response on one or two TLRs
Three concentrations of the compound(s), typically 1/10, 1/100 and 1/1000
dilutions of the original compound solution, are tested on the TLR(s) recognizing
the compound(s) as determined in level 1 or specified by the customer. Triplicate
tests (plus controls).
TLR ligands are typically recognized by a single TLR, potentially two (TLR7 and
TLR8). Recognition by TLR4 usually reflects the presence of endotoxins in the
sample.
NOD Ligand Screening
Two cytoplasmic proteins involved in pathogen recognition, NOD1 and
NOD2, can be added to this screening service. Their activation by a given
compound is detected using the same method as the one described above.
Compounds can be supplied as stock solutions or as solid materials for watersoluble
compounds.
A detailed report will be prepared and provided to the customer electronically
and in hard copy. All procedures are performed accordingly to strict guidelines.
Confidentiality is guaranteed.
TLR Ligand Concentration
TLR2
TLR3
TLR4
TLR5
TLR7
TLR8
TLR9
NOD1
NOD2
www.invivogen.com/innate-immunity-pamps
HKLM
Poly(I:C)
E. coli K12 LPS
S. typhimurium flagellin
Imiquimod
ssPolyU/LyoVec
CpG ODN 2006
Tri-DAP
Muramyl dipeptide
108 cells/ml
1 μg/ml
100 ng/ml
100 ng/ml
3 μg/ml
10 μg/ml
10 μg/ml
1 μg/ml
100 ng/ml
PRODUCT CAT. CODE
TLR / NOD Ligand Screening tlrl-test
Contact us for more information.
info@invivogen.com
91
INNATE IMMUNITY 3

INNATE IMMUNITY 3
Ready-Made psiRNA - shRNAs targeting PRR & Related Genes
RNA interference using small interfering RNA (siRNA) or short-hairpin RNA (shRNA) has become a common technique for gene silencing studies.
InvivoGen provides an extensive list of plasmid-based shRNAs that target genes involved in the innate immunity. These plasmid-based shRNAs, called
ready-made psiRNA, are useful to study the role of these target genes in innate immunity.
Description
Ready-made psiRNAs are psiRNA-h7SKGFPzeo-derived plasmids that express high amounts
of shRNAs through the human 7SK RNA Pol III promoter. They feature a GFP::Zeo fusion
gene which confers both reporter and antibiotic resistance activities allowing simple
monitoring of transfection efficiency and selection in both E. coli and mammalian cells.
Contents and Storage
Ready-made psiRNA plasmids are available alone or in a kit.
Each ready-made psiRNA plasmids is provided as 20 μg of lyophilized DNA.
Each ready-made psiRNA kit contains the following components:
- 20 μg of the ready-made psiRNA plasmid of your choice
- 20 μg of a control psiRNA plasmid targeting Luciferase GL3
- 1 vial of LyoComp GT116
- 4 pouches of Fast-Media ® Zeo
Products are shipped at room temperature. Store at -20°C.
GENE NAME SPECIES CAT.
CODE
Toll-Like Receptors (TLRs)
92
TLR1 h, m p 149
TLR2 h, m p 149
TLR3 h, m p 149
TLR4 h, m p 149
TLR5 h, m p 149
TLR6 h, m p 149
TLR7 h, m p 149
TLR8 h, m p 149
TLR9 h, m p 149
TLR10 h p 149
NOD-Like Receptors (NLRs)
IPAF / CARD12 h p 149
NAIP5 / BIRC1E m p 149
NALP1 / CARD7 h p 149
NALP2 h p 149
NALP3 / NLRP3 h, m p 149
NALP12 / Monarch1 h p 149
NOD1 / CARD4 h, m p 149
NOD2 / CARD15 h, m p 149
NOD9 / NLRX1 h, m p 149
RIG-I-Like Receptors (RLRs)
LGP2 / DHX58 h, m p 149
MDA5 / IFIH1 h, m p 149
RIG-I / DDX58 h, m p 149
Other Pathogen Sensors
AIM2 / IFI210 h, m p 148
CLEC9A h, m p 148
DAI / ZBP1 h p 148
DC-SIGN / CD209 h, m p 148
Dectin-1 h, m p 148
IFI16 NEW h, m p 148
GENE NAME SPECIES
Other Pathogen Sensors
MBL2 h p 149
SIGNR1 m p 149
SIGNR2 m p 149
SIGNR3 m p 149
CD14 h, m p 148
CD36 h, m p 148
MD2 NEW h p 149
BCL10 / CLAP h, m p 148
CARD9 h, m p 148
CD44 h, m p 148
DDX3 / DDX3X h p 148
FADD / MORT1 h, m p 148
IKKε / IKBKE / IKK-i h, m p 149
IRAK-1 h, m p 149
IRAK-4 h, m p 149
LRRFIP2 h p 149
NAP1 / AZI2 h, m p 149
Pellino1 / PELI1 h p 149
Pellino2 / PELI2 h p 149
CAT.
CODE
Adaptors
ASC / CARD5 h, m p 148
Cardinal / CARD8 h p 148
IPS-1 / MAVS / VISA h, m p 149
MyD88 h, m p 149
RAC1 h, m p 149
SARM1 h p 149
TICAM1 / TRIF h, m p 149
TICAM2 / TRAM h, m p 149
TIRAP / Mal
Co-receptors
h, m p 149
Signaling Effectors
www.invivogen.com/readymade-psirna
GENE NAME
Signaling Effectors
SPECIES
Pellino3 / PELI3 h, p 149
PKD1 / PKRD1 h p 149
PKR / EIF2AK2 h, m p 149
PRKRA / PACT h, m p 149
RIP1 / RIPK1 h, m p 149
RIP2 / RIPK2 h, m p 149
STING NEW h, m p 149
SUGT1 NEW h p 149
TAK1 / MAP3K7 h, m p 149
TANK h, m p 149
TBK1 / NAK h, m p 149
TRADD h, m p 149
TRAF3 / CAP-1 h, m p 149
TRAF6 / RNF85
Signaling Inhibitors
h, m p 149
A20 / TNFAIP3 h, m p 148
ATF3 h p 148
Bcl-3 h, m p 148
DAK h, m p 148
DUBA m p 148
FLII / Fliih h p 148
IRAK-M h p 149
MULAN / Dublin h, m p 149
PIN1 / DOB h, m p 149
RNF125 / TRAC-1 h, m p 149
RP105 / CD180 h p 149
SIGIRR / TIR8 m p 149
SIKE h, m p 149
ST2 / IL1RL1 / T1 h p 149
Tollip / IL-1RAcPIP h p 149
TRAFD1 / FLN29 h p 149
Triad3A h p 149
CAT.
CODE

Signal Transduction Inhibitors
InvivoGen offers a selection of known inhibitors of the signaling pathways initiated by PRRs. These inhibitory molecules intervene in the different
steps of PRR activation and signaling cascade, including ligand binding, interaction with adapters and activation of MAP kinases and NF-kB.
PRODUCT DESCRIPTION
ENDOTOXIN
LEVELS
WORKING
CONCENTRATION
QUANTITY CATALOG
CODE
2-Aminopurine PKR inhibitor

INNATE IMMUNITY 3
AG490 - JAK2 Inhibitor
AG490 is a specific and potent inhibitor of the Janus kinase 2 protein (JAK2) 1 . JAK2 regulates the phosphorylation of JNK, primarily through PI3K. It has been
established that JAK2 plays an important role in TLR-mediated biological responses, blocking TLR4-mediated responses to LPS 2 and TLR5-mediated responses
to flagellin 3 .
2-Aminopurine - PKR Inhibitor
2-aminopurine (2-AP) is a potent inhibitor of double-stranded RNA (dsRNA)-activated protein kinase (PKR), a critical mediator of apoptosis. PKR is
phosphorylated and activated by dsRNA and poly(I:C) and contributes to the induction of type I interferons, such as IFN-b, which can further increase its
expression 4 . PKR plays also a role in TLR-induced antiviral activities as an intermediary in TLR3, TLR4 and TLR9 signaling 5 . .
Bay 11-7082 - IkB-a Inhibitor
Bay 11-7082 is an irreversible inhibitor of TNF-a-induced IkB-a phosphorylation resulting in the inactivation of NF-kB 6 . TNF-a-dependent effects of NF-kB
are important for TLR expression and cytokine production 7 .
BX795 - TBK/IKKε Inhibitor
BX795, an aminopyrimidine compound, was developed as an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1) 8 . It was recently shown to be a
potent inhibitor of the IKK-related kinases, TANK-binding kinase 1 (TBK1) and IKKε, and hence of IRF3 activation and IFN-β production 9 . BX795 inhibits the
catalytic activity of TBK1/IKKε by blocking their phosphorylation.
Celastrol - NF-kB Inhibitor
Celastrol is a triterpenoid compound isolated from the medicinal plant Tripterygium wilfordii known for its anti-inflammatory properties. Its mode of action
and spectrum of cellular targets are still poorly understood. Celastrol was recently shown to act as an effective inhibitor of the transcription factor NF-kB
resulting in the attenuation of nitric oxide and proinflammatory cytokine production 10 .
Chloroquine - Inhibitor of Endosomal Acidification
Chloroquine is a lysosomotropic agent that prevents endosomal acidification 11 . It accumulates inside the acidic parts of the cell, including endosomes and
lysosomes. This accumulation leads to inhibition of lysosomal enzymes that require an acidic pH, and prevents fusion of endosomes and lysosomes. Chloroquine
is commonly used to study the role of endosomal acidification in cellular processes, such as the signaling of intracellular TLRs 12 .
CLI095 - TLR4 Signaling Inhibitor
CLI095, also known as TAK-242, is a novel cyclohexene derivative that suppresses specifically TLR4 signaling, inhibiting the production of NO and
pro-inflammatory cytokines 13 . It acts by blocking the signaling mediated by the intracellular domain of TLR4, but not the extracellular domain. It potently
suppresses both ligand-dependent and -independent signaling of TLR4 14 .
Cyclosporin A - Calcineurin inhibitor NEW
Cyclopsporin A, a calcineurin inhibitor, exerts its immunosuppressive effects through the down-regulation of NFAT (nuclear factor of activated T cells),
thus preventing the transcription of T cell effector cytokines. NFAT has been implicated in the downstream signaling of Dectin-1 15 . Conversely, it has been
demonstrated that the inhibition of calcineurin in macrophages can trigger TLR signaling and enhance NF-κB activation 16 .
Gefitinib - RIP2 Tyrosine Kinase inhibitor NEW
Gefitinib (also known as IRESSA) is a selective inhibitor of epidermal growth factor (EGFR), a growth factor that plays a pivotal role in the control of cell growth,
apoptosis, and angiogenesis. Recent studies demonstrated that Gefitinib can inhibit NOD2-induced cytokine release and NF-kB activation by inhibiting RIP2
(receptor-interacting protein 2) tyrosine phosphylation which is critical for activation of NOD2 downsream signaling pathways 17 .
H-89 - PKA Inhibitor
H-89 is a selective, potent cell permeable inhibitor of cAMP-dependent protein kinase (PKA) 18 . It can be used to determine the role of PKA in TLR and
other PRR mediated signaling. PKA has be shown to participate in the TLR-mediated TREM-1 expression on macrophages following LPS stimulation 19 .
Leptomycin B - Nuclear export inhibitor NEW
Leptomycin B, an inhibitor of nuclear export, can be used to study nucleo-cytoplasmic translocation. It has been demonstrated that Leptomycin B can provoke the
nuclear accumulation of proteins that shuttle between the cytosol and nucleus such as MAPK, MAPKAP kinase 2, IRAK-1 and NLRC5 20 . The cellular target of
Leptomycin B has been identified as CRM1 (exportin 1), an evolutionarily conserved receptor for the nuclear export signal of proteins.
LY294002 - PI3K Inhibitor
LY294002 is a potent, cell permeable inhibitor of phosphatidylinositol 3-kinase (PI3K) that acts on the ATP binding site of the enzyme 21 . The PI3K pathway
is extensively studied for its property in inhibiting apoptosis. PI3K is also known to regulate TLR-mediated inflammatory responses 22 .
OxPAPC - TLR2 and TLR4 Inhibitor
OxPAPC (1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine), is an oxidized phospholipid that has been shown to inhibit the signaling induced by
bacterial lipopeptide and lipopolysaccharide (LPS). It acts by competing with CD14, LBP and MD2, the accessory proteins that interact with bacterial lipids,
thus blocking the signaling of TLR2 and TLR4 23 .
PD98059 - MAP Kinase Kinase Inhibitor
PD98059 is a potent and selective inhibitor of MAP kinase kinase (also known as MAPK/ERK kinase or MEK kinase). It mediates its inhibitory properties by
binding to the ERK-specific MAP kinase MEK, therefore preventing phosphorylation of ERK1/2 (p44/p42 MAPK) by MEK1/2. MAPK ERK1/2 is involved in
TLR-induced production of cytokines 24 .
94
www.invivogen.com/inhibitors

Pepinh-MYD - MyD88 inhibitory peptide
Pepinh-MYD is a 26 aa peptide that blocks MyD88 signaling by inhibiting its homodimerization through binding. Pepinh-MYD contains a sequence from the
MyD88 TIR homodimerization domain (RDVLPGT) 25 preceeded by a protein transduction sequence (RQIKIWFQNRMKWKK) derived from antennapedia
which enables the peptide to translocate through the cell membrane 26 . Pepinh-MYD is provided with a control peptide.
Pepinh-TRIF - TRIF inhibitory peptide
Pepinh-TRIF is a 30 aa peptide that blocks TRIF signaling by interfering with TLR-TRIF interaction. Pepinh-TRIF contains the 14 aa that correspond to the
sequence of the BB loop of TRIF (FCEEFQVPGRGELH) 27 linked to the cell-penetrating segment of the antennapedia homoedomain
(RQIKIWFQNRMKWKK) 26 . Pepinh-TRIF is provided with a control peptide.
Polymyxin B - Inhibitor of LPS-induced TLR4 activation
Polymyxin B (PMB) is a cyclic cationic polypeptide antibiotic produced by the soil bacterium Paenibacillus polymixa. PMB blocks the biological effects of Gram
negative LPS (endotoxin) through binding to lipid A, the toxic component of LPS, which is negatively charged. The neutralizing effect of PMB on LPS is doserelated
and specific for LPS 28 . PMB is widely used to eliminate the effects of endotoxin contamination, both in vitro and in vivo.
Rapamycin - mTOR Inhibitor
Rapamycin is an inhibitor of the Ser/Thr protein kinase named "mammalian target of rapamycin" (mTOR) that regulates cell growth and metabolism in
response to environmental cues. mTOR is a downstream target of PI3K, an important actor in TLR signaling. Rapamycin was shown to block TLR2- and
TLR4-mediated expression of TNF-a and IL-6 in neutrophils stimulated with Pam3CSK4 or LPS 29 .
SB203580 - p38/RK MAP Kinase Inhibitor
SB203580 is a pyridinyl imidazole inhibitor widely used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase 30 . SB203580 inhibits also the phosphorylation
and activation of protein kinase B (PKB, also known as Akt) 31 . Both kinases are involved in a wide array of signaling pathways, including the TLR signaling pathway.
SP600125 - JNK Inhibitor
SP600125 is a potent, cell-permeable, selective and reversible inhibitor of c-Jun N-terminal kinase (JNK) 32 . It inhibits in a dose-dependent manner the phosphorylation
of JNK. JNK is a member of the mitogen-activated protein kinase (MAPK) family and plays an essential role in TLR-mediated inflammatory responses.
U0126 - MEK1 and MEK2 Inhibitor
U0126 is a selective inhibitor of the MAP kinase kinases, MEK1 and MEK2. It acts by inhibiting the kinase activity of MEK1/2 thus preventing the activation of
MAP kinases p42 and p44 which are encoded by the erk2 and erk1 genes respectively 33 . MAPK p42/p44 are involved in the signaling cascade triggered by
LPS and other ligands through stimulation of the TLRs.
Wortmannin - PI3K Inhibitor
Wortmannin is a cell-permeable, fungal metabolite that acts as a potent, selective and irrevesible inhibitor of phosphatidylinositol 3-kinase (PI3K). There is
increasing evidence of the involvement of PI3K in TLR signaling. Inhibition of PI3K by wortmannin was shown to greatly enhance TLR-mediated inducible
nitric-oxide synthase (iNOS) expression and cytokine production in the mouse macrophage cell line RAW264.7. The effect of wortmannin was common
to TLR2, -3, -4, and -9 and was accompanied by activation of NF-kB and up-regulation of cytokine mRNA production 34 .
1. Levitzki A., 1990. Tyrphostins- potential antiprolierative agents and novel molecular tools.
Biochem. Pharmacol. 40:913-918. 2. Kimura A et al., 2005. Suppressor of cytokine signaling-1
selectively inhibits LPS-induced IL-6 production by regulating JAK–STAT. PNAS 102:17089-
17094. 3. Ha H et al., 2008. Stimulation by TLR5 Modulates Osteoclast Differentiation through
STAT1/IFN-b1. J. Immuno. 180:1382-1389.4. Samuel CE., 2001. Antiviral actions of interferons.
Clin Microbiol Rev. 14(4):778-809. 5. García MA. et al., 2006. Impact of Protein Kinase PKR in
Cell Biology: from Antiviral to Antiproliferative Action. Microbiol. Mol. Biol. Rev. 70: 1032-1060.
6. Pierce JW. et al., 1997. Novel Inhibitors of Cytokine-induced Ikappa Balpha Phosphorylation
and Endothelial Cell Adhesion Molecule Expression Show Anti-inflammatory Effects in Vivo. J.
Biol. Chem. 272: 21096. 7. Phulwani NK. et al., 2008.TLR2 Expression in Astrocytes Is Induced
by TNF-{alpha}- and NF-{kappa}B-Dependent Pathways. J. Immunol. 181: 3841-3849.
8. Feldman RI. et al., 2005. Novel Small Molecule Inhibitors of 3-Phosphoinositide-dependent
Kinase-1. J. Biol. Chem. 280: 19867-19874. 9. Clark K. et al., 2009. Use of the Pharmacological
Inhibitor BX795 to Study the Regulation and Physiological Roles of TBK1 and I{kappa}B Kinase
{epsilon}: a distinct upstream kinase mediates Ser-172 phosphorylation and activation. J. Biol.
Chem. 284: 14136-14146. 10. Sethi G. et al., 2007. Celastrol, a novel triterpene, potentiates
TNF-induced apoptosis and suppresses invasion of tumor cells by inhibiting NF-kB–regulated
gene products and TAK1-mediated NF-kB activation. Blood 109: 2727-2735. 11. Steinman RM.
et al., 1983. Endocytosis and the recycling of plasma membrane. J. Cell. Biol. 96:1-27. 12. Hart
OM. et al., 2005. TLR7/8-Mediated Activation of Human NK Cells Results in Accessory Cell-
Dependent IFN-{gamma} Production. J. Immunol. 175: 1636-1642. 13. Li M. et al., 2006. A
Novel Cyclohexene Derivative, Ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl] cyclohex-
1-ene-1-carboxylate (TAK-242), Selectively Inhibits Toll-Like Receptor 4-Mediated Cytokine
Production through Suppression of Intracellular Signaling. Mol. Pharmacol. 69: 1288-1295.
14. Kawamoto T. et al., 2008. TAK-242 selectively suppresses Toll-like receptor 4-signaling
mediated by the intracellular domain. Eur J Pharmacol. 584(1):40-8. 15. Goodridge et al. 2007.
Dectin-1stimulation by Candida albicans yeast or zymosan triggers NFAT activation in
macrophages and dendritic cells. J Immunol. 178( 5): 3107-15. 16. Kang Y. et al., 2007. Calcineurin
negatively regulates TLR-mediated activation pathways. J Immunol 179(7):4598–607.
17. Tigno-Aranjuez J. et al., 2010. Inhibition of RIP2’s tyrosine kinase activity limits NOD2-driven
cytokine responses. Genes Dev. 24(23):2666-77. 18. Chijiwa, T., et al.,1990. Inhibition of
forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized
selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)
ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J. Biol. Chem.
www.invivogen.com/inhibitors
265: 5267-5272. 19. Murakami Y. et al., 2007. Lipopolysaccharide-Induced Up-Regulation of
Triggering Receptor Expressed on Myeloid Cells-1 Expression on Macrophages Is Regulated
by Endogenous Prostaglandin E2. J. Immunol. 178: 44. 20. Benko S. et al., 2010. NLRC5 limits
the activation of inflammatory pathways. J Immunol. 185(3):1681-91. 21. Vlahos CJ. et al., 1994.
A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one
(LY294002). J. Biol. Chem 69(7):5241-8.. 22. Guiducci C. et al., 2008. PI3K
is critical for the nuclear translocation of IRF-7 and type I IFN production by human plasmacytoid
predendritic cells in response to TLR activation. J. Exp. Med. 205: 315-322. 23. Erridge C. et al.,
2008. Oxidized Phospholipid Inhibition of Toll-like Receptor (TLR) Signaling Is Restricted to
TLR2 and TLR4: roles for CD14, LPS-binding protein, and MD2 as targets for specificity of
inhibition. J. Biol. Chem. 283: 24748-24759. 24. Banerjee A. et al., 2006. Diverse Toll-like
receptors utilize Tpl2 to activate extracellular signal-regulated kinase (ERK) in hemopoietic cells.
PNAS. 103: 3274-3279. 25. Loiarro M. et al., 2005. Peptide-mediated Interference of TIR
Domain Dimerization in MyD88 Inhibits Interleukin-1-dependent Activation of NF-{kappa}B. J.
Biol. Chem. 280: 15809-14. 26. Derossi D. et al., 1994. The third helix of the Antennapedia
homeodomain translocates through biological membranes. J. Biol. Chem., 269: 10444-50.
27. Toshchakov VU. et al., 2005. Differential Involvement of BB Loops of Toll-IL-1 Resistance
(TIR) Domain-Containing Adapter Proteins in TLR4- versus TLR2-Mediated Signal Transduction.
J. Immunol. 175: 494 - 500. 28. Duff GW. & Atkins E. 1982. The inhibitory effect of polymyxin
B on endotoxin-induced endogenous pyrogen production. J Immunol Methods. 52(3):333-40.
29. Lorne E. et al., 2009. Participation of Mammalian Target of Rapamycin Complex 1 in Toll-
Like Receptor 2– and 4–Induced Neutrophil Activation and Acute Lung Injury. Am. J. Respir.
Cell Mol. Biol. 41: 237 - 245. 30. Cuenda A. et al., 1995. SB203580 is a specific inhibitor of a
MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett.
364:229-233. 31. Lali FV. et al., 2000. The pyridinyl imidazole inhibitor SB203580 blocks
phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and
retinoblastoma hyperphosphorylation in interleukin-2- stimulated T cells independently of p38
mitogen-activated protein kinase. J Biol Chem. 275(10):7395-402. 32. Bennett BL. et al., 2001.
SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. PNAS. 98:13681-13686.
33. Favata MF. et al., 1998. Identification of a novel inhibitor of mitogen-activated protein kinase.J.
Biol. Chem. 273(29):18623-32. 34. Hazeki K. et al, 2006. Opposite Effects of Wortmannin and
2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one Hydrochloride on Toll-Like Receptor-
Mediated Nitric Oxide Production: Negative Regulation of Nuclear Factor-{kappa}B by
Phosphoinositide 3-Kinase. Mol. Pharmacol. 69: 1717 - 1724.
95
INNATE IMMUNITY 3

INNATE IMMUNITY 3
Inflammasomes
The nucleotide-binding oligomerization domain-like receptor (NLR) family
of proteins is involved in the regulation of innate immunity responses.
Certain members of the NLR family sense pathogen-associated molecular
patterns (PAMPs) in the cytosol and induce the assembly of large
caspase-1-activating complexes called inflammasomes 1 . Activation of
caspase-1 through autoproteolytic maturation leads to the processing and
secretion of the pro-inflammatory cytokines interleukin-1b (IL-1b) and
IL-18. Several inflammasomes have been identified and defined by the NLR
protein that they contain. IPAF (ICE protease-activating factor)
inflammasome is triggered by bacterial flagellin 2 , whereas NALP1b (NACHT
domain-, leucine-rich repeat-, and PYD-containing protein 1b)
inflammasome is induced by anthrax lethal toxin 3 . NALP3/cryopyrin/NLRP3
inflammasome assembles in response to a variety of PAMPs and ‘danger’
signals, such as uric acid crystals. A new inflammasome has just been
identified composed of AIM2 (absent in melanoma 2) which recognizes
cytoplasmic double-stranded DNA.
IL-1b and IL-18 are related cytokines that cause a wide variety of biological
efffects associated with infection, inflammation and autoimmune processes.
IL-1b participates in the generation of systemic and local responses to
infection and injury by generating fever, activating lymphocytes and by
promoting leukocyte infiltration at sites of infection or injury. IL-1b signaling
initiates signaling cascades leading to the activation of NF-kB and MAP
kinases which trigger the secretion of inflammatory cytokines. IL-18 induces
IFN-g production and contributes to T-helper 1 (Th1) cell polarization.
IL-18 signaling pathways are similar to those initiated by IL-1b. Both
cytokines share a common maturation mechanism that requires
caspase-1. Caspase-1 itself is synthesized as an inactive 45 kDa zymogen
(pro-caspase-1) that undergoes autocatalytic processing following an
appropriate stimulus. The active form of the enzyme comprises the subunits
p20 and p10 which assemble into a heterotetramer 4 . Caspase-1 is activated
within the inflammasome through interaction with ASC (apoptosisassociated
speck-like protein containing a carboxy-terminal CARD), a
bipartite adapter protein that bridges NLRs and caspase-1 5 .
It is now generally accepted that activation and release of IL-1b requires
two distinct signals. The nature of these signals in vivo during infection or
inflammation is not completely defined. However, in vitro studies indicate
that the first signal can be triggered by various PAMPs following Toll-like
receptor (TLR) activation which induces the synthesis of pro-IL-1b. The
second signal is provided by the activation of the inflammasome and
caspase-1 leading to IL-1b processing. The requirement for a second signal
for IL-1b maturation might constitute a fail-safe mechanism to ensure that
induction of potent inflammatory responses occurs only in the presence
of a bona fide stimulus, such as pathogen infection and/or tissue injury.
NALP3 Inflammasome
Among the inflammasomes, NALP3 inflammasome is the most studied. Its
activation in macrophages can be achieved with PAMPs, such as LPS,
peptidoglycan, and bacterial nucleic acids, provided the cells are exposed
to ATP. Indeed, in the absence of ATP, macrophages stimulated with LPS
produce large quantities of pro-IL-1b, but release little mature cytokine to
the medium. ATP and certain bacterial toxins, such as nigericin and
maitotoxin, cause a change in the intracellular ion composition leading to
the activation of the NALP3 inflammasome. The effect of ATP is mediated
by the purinergic P2X7 receptor, which causes a rapid potassium efflux
from the cytosol upon activation. ATP induces rapid opening of the nonselective
cation channel of P2X7, followed by the gradual opening of a larger
96
www.invivogen.com/inflammasome
pore. The larger pore is mediated by the hemichannel pannexin-1 which is
recruited upon activation of the P2X7 receptor. Recent studies have
demonstrated that pannexin is required for caspase-1 activation in response
to ATP, nigericin and maitotoxin 6 . However, the trigger between pannexin-1
and the NALP3 inflammasome remains unclear. Potassium (K+) efflux is
thought to be an essential trigger of NALP3-induced caspase-1 activation.
Macrophages derived from mice lacking the P2X7 receptor failed to activate
caspase-1 7 . However, K+ efflux is not sufficient for activation of the NALP3
inflammasome, since activation of the P2X7 receptor does not induce
caspase-1 maturation in macrophages that have not been exposed to LPS.
It has been suggested that pannexin-1 may mediate the delivery of PAMPs
into the cytosol which might explain the lack of requirement for TLR signaling
in caspase-1 activation induced via the pannexin-1/NALP3 pathway 8 .
A recent study demonstrated that monosodium urate (MSU) and calcium
phosphate dihydrate (CPPD) crystals activate caspase-1 in a NALP3dependent
manner 9 . Deposition of MSU and CPPD crystals in joints is
responsible for the inflammatory conditions gout and pseudogout,
respectively. Thus, the NALP3 inflammasome was suggested to participate
in the etiology of these auto-inflammatory diseases. In addition to its role
in gout, uric acid is a major component released into the extracellular milieu
by necrotic cells, suggesting an important role of NALP3 in the detection
of endogenous ‘danger’ signal. Crystalline silica and asbestos were shown
to induce the activation of the NALP3 inflammasome, suggesting that the
NALP3 inflammasome participates in the pathogenesis of silicosis and
asbestosis 10-12 . Aluminium salt (alum) crystals also activate the
inflammasome formed by NALP3 and seem to require the presence of
PAMPs. Indeed, several groups have reported that alum activates the
NALP3 inflammasome only if the cells are pretreated with LPS 12-14 . NALP3
activation by crystals has been shown to require phagocytosis, causing
lysosomal swelling and damage, and to involve cathepsin B, a lysosomal
cysteine protease. As crystal-independent lysosomal damage was sufficient
to induce NALP activation, it was suggested that rather than the crystal
structure itself, the NALP3 inflammasome senses lysosomal pertubation as
a ‘danger’ signal 12 .
AIM2 Inflammasome
Recently, several groups have identified AIM2 (absent in melanoma 2) as a
new receptor for cytoplasmic DNA, which forms an inflammasome with
the ligand and ASC to activate caspase-1 15-17 . AIM2 is an interferon-inducible
HIN-200 family member that contains an amino-terminal pyrin domain and
a carboxy-terminal oligonucleotide/oligosaccharide-binding domain. AIM2
senses cytoplasmic double-stranded DNA through its oligonucleotide/
oligosaccharide-binding domain and interacts with ASC via its pyrin domain
to activate caspase-1. The interaction of AIM2 with ASC also leads to the
formation of the ASC pyroptosome, which induces pyroptotic cell death
in cells containing caspase-1. Knockdown of AIM2 expression by RNAmediated
interference was shown to reduce DNA-induced maturation of
IL-1b in macrophages whereas stable expression of AIM2 in the human
embryonic kidney 293T cell line conferred responsiveness to cytoplasmic
DNA. These data suggest that AIM2 is both required and sufficient for
inflammasome activation in reponse to cytoplasmic DNA.
Clearly, inflammasomes fulfill a central role in innate immunity. They detect
and respond to bacterial components, ‘danger signals’ and potentially
dangerous cytoplasmic DNA. Further understanding on how they are
activated should provide new insights into the mechanism of host defense
and the pathogenesis of autoimmune diseases.

1. Martinon F,. et al., 2002. The inflammasome: a molecular platform triggering activation of
inflammatory caspases and processing of proIL-beta. Mol Cell. 2002 10(2):417-26. 2. Miao
EA. et al., 2006. Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1beta
via Ipaf. Nat Immunol. 7(6):569-75. 3. Boyden ED & Dietrich WF., 2006. Nalp1b controls
mouse macrophage susceptibility to anthrax lethal toxin. Nat Genet. 38(2):240-4. 4.
Mariathasan S & Monack DM., 2007. Inflammasome adaptors and sensors: intracellular
regulators of infection and inflammation. Nat Rev Immunol. 7(1):31-40. 5. Martinon F, &
Tschopp J., 2004. Inflammatory caspases: linking an intracellular innate immune system to
autoinflammatory diseases. Cell. 117(5):561-74. 6. Pelegrin P, & Surprenant A., 2007.
Pannexin-1 couples to maitotoxin- and nigericin-induced interleukin-1beta release through
a dye uptake-independent pathway. J Biol Chem. 282(4):2386-94. 7. Solle M. et al., 2001.
Altered cytokine production in mice lacking P2X(7) receptors. J Biol Chem. 276(1):125-32.
8. Kanneganti TD, et al., 2007. Pannexin-1-mediated recognition of bacterial molecules
activates the cryopyrin inflammasome independent of Toll-like receptor signaling. Immunity.
26(4):433-43. 9. Martinon F. et al., 2006. Gout-associated uric acid crystals activate the
www.invivogen.com/inflammasome
NALP3 inflammasome. Nature. 440(7081):237-41. 10. Dostert C. et al., 2008. Innate
immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science.
320(5876):674-7. 11. Cassel SL. et al., 2008. The Nalp3 inflammasome is essential for the
development of silicosis. Proc Natl Acad Sci U S A. 105(26):9035-40. 12. Hornung V. et al.,
2008. Silica crystals and aluminum salts activate the NALP3 inflammasome through
phagosomal destabilization. Nat Immunol. 9(8):847-56. 13. Eisenbarth SC. et al., 2008. Crucial
role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium
adjuvants. Nature. 453(7198):1122-6. 14. Li H. et al., 2008. Cutting edge: inflammasome
activation by alum and alum's adjuvant effect are mediated by NLRP3. J Immunol. 181(1):17-
21. 15. Hornung V. et al., 2009. AIM2 recognizes cytosolic dsDNA and forms a caspase-1activating
inflammasome with ASC. Nature. 458(7237):514-8. 16. Fernandes-Alnemri T. et
al., 2009. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA.
Nature. 458(7237):509-13. 17. Bürckstümmer T. et al., 2009. An orthogonal proteomicgenomic
screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat
Immunol. 10(3):266-72.
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Inflammasome Inducers & Inhibitors
The inflammasome is a multi-protein complex involved in the production of mature IL-1b. It is activated by a large variety of microbial molecules,
danger signals and crystalline substances. InvivoGen provides a selection of these molecules known to induce the assembly of the NLRP3 or
AIM2 inflammasomes. We also offer some inhibitors to confirm the ability of a given molecule to activate the inflammasome or to test the
importance of phagocytosis or potassium efflux in this process. These inflammasome inducers and inhibitors have been validated using the
THP-1/HEK-Blue -IL-1b Assay (see page 100).
98
PRODUCT DESCRIPTION
Inflammasome Inducers
Alum Crystals Aluminium potassium sulfate 10 - 200 μg/ml 1 g tlrl-alk
ATP Adenosine 5′-triphosphate disodium salt 5 mM 1 g tlrl-atp
CPPD Crystals Calcium pyrophosphate dihydrate 50 - 200 μg/ml 5 mg tlrl-cppd
Hemozoin NEW Synthetic heme crystal 50 - 400 μg/ml 5 mg tlrl-hz
MSU Crystals Monosodium urate (uric acid) 50 - 200 μg/ml 5 mg tlrl-msu
Nigericin Nigericin, sodium salt 1 μM 10 mg tlrl-nig
Poly(dA:dT), double-stranded B DNA Poly(dA-dT)•poly(dT-dA)/LyoVec complex 1 - 5 μg/ml 100 μg tlrl-pat
Inflammasome Inhibitors
Inflammasome Inducers
Alum Crystals - NLRP3 Inflammasome Inducer
Aluminum hydroxide and potassium salts (alum) are commonly used vaccine adjuvants. Adjuvants are vaccine additives that stimulate the immune system
without having any specific antigenic effect. Alum has been demonstrated to activate caspase-1 and triggers IL-1b and IL-18 secretion 1 . All alum preparations
contain crystals. The alum-induced release of IL-1b in macrophages is dependent on NLRP3 and ASC, indicating that alum triggers inflammation through
activation of the NLRP3 inflammasome 2 . Alum has been shown to trigger NLRP3 activation through lysosomal destabilization 2 .
ATP - NLRP3 Inflammasome Inducer
Adenosine triphosphate (ATP), a potassium efflux agent, can trigger the activation of NLRP3 inflammasome in response to PAMPs, such as LPS and
peptidoglycan. It stimulates the caspase-1-dependent cleavage and secretion of IL-1b from LPS-stimulated cells 3 . ATP triggers the opening of the nonselective
cation channel of the purinergic P2X7 receptor, followed by the gradual opening of a larger pore. The larger pore is attributed to pannexin-1, which
is recruited upon P2X7 receptor activation 4 . Activation of the P2X7 receptor results in potassium efflux which is necessary for activation of the posttranslational
maturation of IL-1b 5 .
Hemozoin - NLRP3 Inflammasome Inducer NEW
Hemozoin is a dark-brown heme crystal produced by the intraerythrocytic parasite Plasmodium, the causative agent of malaria. Hemozoin is taken up by
macrophages initiating signals that lead to the production of IL-1b. Hemozoin-induced IL-1b production is dependent on the activation of the NLRP3
inflammasome 6, 7 . Synthetic hemozoin has been shown to possess adjuvant properties that differ depending on the method of synthesis 8 . InvivoGen provides
a chemically synthesized hemozoin using an acidic method.
MSU & CPPD Crystals - NLRP3 Inflammasome Inducers
Crystals of monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) are the aetiological agents of the inflammatory joint diseases gout
and pseudo-gout, respectively. Both pathogenic crystals have been recently shown to be potent activators of caspase-1 through the NLRP3 inflammasome 9 .
Involvement of the inflammasome is suggested by the finding that macrophages from mice deficient in various components of the inflammasome are
defective in crystal-induced IL-1b induction. NLRP3 activation requires the phagocytosis of crystals that leads to lysosomal damage which appears to be
the signal recognized by the inflammasome resulting in its activation 2 .
www.invivogen.com/inflammasome
WORKING
CONCENTRATION
QUANTITY CATALOG
CODE
Glybenclamide Proton pump inhibitor 25 μg/ml 1 g tlrl-gly
Z-VAD-FMK Pan-caspase Inhibitor 10 μg/ml (20 μM) 1 mg tlrl-vad

Nigericin - NLRP3 Inflammasome Inducer
Nigericin is a microbial toxin derived from Streptomyces hygroscopicus. Nigericin acts as a potassium ionophore. The release of IL-1b in response to nigericin
has been demonstrated to be NLRP3-dependent 3 . Similar to ATP, nigericin induces a net decrease in intracellular levels of potassium which is crucial for the
activation of caspase-1 5 . Nigericin requires signaling through pannexin-1 to induce caspase-1 maturation and IL-1b processing and release 10 .
Poly(dA:dT) - AIM2 Inflammasome Inducer
Poly(dA:dT) is a repetitive synthetic double-stranded DNA sequence of poly(dA-dT)•poly(dT-dA). Poly(dA:dT) is complexed with the cationic lipid
LyoVec to facilitate its uptake. Transfection of macrophages with poly(dA:dT) leads to the production of IL-1b 11 . This response to transfected poly(dA:dT)
is ASC-dependent, but NLRP3 independent. AIM2 was recently shown to sense poly(dA:dT), form an inflammasome with ASC and trigger caspase-1
activation 12-14 . Poly(dA:dT) binds to AIM2 and induces its oligomerization, which is the first demonstration of an inflammasome bound to its ligand 12 .
Inflammasome Inhibitors
Glybenclamide (glyburide) - Proton Pump Inhibitor
Glybenclamide, also known as glyburide, blocks the maturation of caspase-1 and pro-IL-1b by inhibiting the K+ efflux 15 . Glybenclamide was shown to potently
block the activation of the NLRP3 inflammasome induced by PAMPs, DAMPs and crystalline substances 16, 17 . Recent data suggest that glybenclamide works
downstream of the P2X 7 receptor but upstream of NLRP3 16 .
Z-VAD-FMK - Caspase Inhibitor
Z-VAD-FMK is a cell-permeable pan-caspase inhibitor that irreversibly binds to the catalytic site of caspase proteases 18 . The peptide is O-methylated in the
P1 position on aspartic acid, providing enhanced stability and increased cell permeability. Z-VAD-FMK is used in apoptosis studies and also in inflammasome
studies. It is a potent inhibitor of caspase-1 activation in NLRP3-induced cells 17 .
1. Li H. et al., 2008. Cutting Edge: Inflammasome activation by Alum and Alum’s adjuvant effect are mediated by NLRP3. J Immunol. 181:17-21. 2. Hornung V. et al., 2008. Silica crystals and
aluminium salts activate the NALP3 inflammasome through phagosomal destabilization. Nature Immunol. 9:847-856. 3. Mariathasan S. et al., 2006. Cryopyrin activates the inflammasome and
ATP. Nature 440;228-32. 4. Locovei S. et al., 2007. Pannexin1 is part of the pore forming unit of the P2X(7) receptor death complex. FEBS Lett. 581(3):483-8. 5. Perregaux D. & Gabel CA.,
1994. Interleukin-1b maturation and release in response to ATP and nigericin. J Biol. Chem. 269:15195-15203. 6. Shio MT. et al., 2009. Malarial hemozoin activates the NLRP3 inflammasome
through Lyn and Syk kinases. PLoS Pathog. 5(8):e1000559. 7. Dostert C. et al., 2009. Malarial hemozoin is a Nalp3 inflammasome activating danger signal. PLoS One. 4(8):e6510. 8. Coban
C. et al., 2010. The malarial metabolite hemozoin and its potential use as a vaccine adjuvant. Allergol Int. 59(2):115-24. 9. Martinon F. et al., 2006. Gout-associated uric acid crystals activate
the NALP3 inflammasome. Nature.440(7081):237-41. 10. Pelegrin P, & Surprenant A., 2007. Pannexin-1 couples to maitotoxin- and nigericin-induced interleukin-1beta release through a dye
uptake-independent pathway. J Biol Chem. 282(4):2386-94. 11. Muruve DA. et al., 2008. The inflammasome recognizes cytosolic microbial and host DNA and triggers an innate immune
response. Nature. 452(7183):103-7. 12. Hornung V. et al., 2009. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature. 458(7237):514-8.
13. Fernandes-Alnemri T. et al., 2009. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature. 458(7237):509-13. 14. Bürckstümmer T. et al., 2008. An
orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat Immunol. 10(3):266-72. 15. Laliberte RE. et al., 1999. ATP treatment of
human monocytes promotes caspase-1 maturation and externalization. J Biol Chem. 274(52):36944-51. 16. Lamkanfi M. et al., 2009. Glyburide inhibits the Cryopyrin/Nalp3 inflammasome.
J. Cell Biol., 187: 61 - 70. 17. Dostert C. et al., 2009. Malarial hemozoin is a Nalp3 inflammasome activating danger signal. PLoS One. 4(8):e6510. 18. Slee EA. et al., 1996. Benzyloxycarbonyl-
Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32. Biochem J. 315 ( Pt 1):21-4.
Contents and Storage
Each product is provided as a solid and shipped at room temperature. Store at room temperature, 4°C or -20°C according to the product label.
www.invivogen.com/inflammasome
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HEK-Blue IL-1b Cells - IL-1b Sensor Cells for Inflammasome Studies
Many studies on the inflammasome use the human monocytic THP-1 cell line and Western blot or ELISA for the detection of mature IL-1b. InvivoGen
has developed a new method to detect bioactive IL-1b that is simple, rapid and cost-effective. This method is based on HEK293 cells specifically engineered to
selectively respond to IL-1b, named HEK-Blue IL-1b.
Description
HEK-Blue IL-1b cells provide a convenient read-out to determine the
amount of IL-1b secreted by THP-1 cells following stimulation by NLRP3
inflammasome inducers.
HEK-Blue IL-1b cells feature the SEAP (secreted embryonic alkaline
phosphotase) reporter gene under the control of an NF-kB-inducible
promoter. They naturally express the IL-1b receptor (IL-1R), and all the proteins
involved in the MyD88-dependent IL-1R signaling pathway that leads to
NF-kB activation. Thus upon IL-1b binding to IL-1R, a signaling cascade is
initiated triggering NF-kB activation and the subsequent production of SEAP.
Detection of SEAP in the supernatant of HEK-Blue IL-1b cells can be readily
assessed using QUANTI-Blue , a SEAP detection medium. QUANTI-Blue
turns blue in the presence of SEAP which can be easily quantified using a
spectrophotometer.
The specificity of the HEK-Blue IL-1b cells for the detection of IL-1b can be
confirmed using a neutralizing antibody against IL-1b, such as anti-hIL-1b-IgA.
HEK-Blue IL-1b cells are resistant to the selective antibiotics Zeocin and
hygromycin B.
Contents and Storage
HEK-Blue IL-1b cells are grown in DMEM medium with 10% FBS, 2mM
L-glutamine, 100 μg/ml Zeocin and 200 μg/ml HygroGold (ultrapure
Hygromycin). Each vial contains 5-7 x 10 6
cells and is supplied with 10 mg
Zeocin , 10 mg HygroGold and 1 pouch of QUANTI-Blue Cells are
shipped on dry ice.
THP1 cells pretreated with PMA and primed with LPS (1 μg/ml) were stimulated with
ATP (5 mM), nigericin (1 μM), alum (200 μg/ml), MSU (200 μg/ml) or CPPD (200 μg/ml).
After 24h incubation, THP-1 supernatants or recombinant IL-1b (0.1 ng/ml) were added
to HEK-Blue IL-1b cells. IL-1b-induced NF-kB activation was assessed by measuring the
levels of SEAP in the supernatant of HEK-Blue IL-1b cells using the QUANTI-Blue assay.
100
Related Products
Zeocin , page 18 QUANTI-Blue , see page 14
Anti-hIL-1b-IgA, page 104
www.invivogen.com/inflammasome
THP-1/HEK-Blue IL-1b Assay
1- Production of IL-1b by THP-1 cells: Typically, THP-1 cells are pretreated with
phorbol 12-myristate acetate (PMA) to become more susceptible to
inflammasome activators, then are primed with lipopolysaccharide (LPS). These
treatments induce the production of pro-IL-1b, the immature form of IL-1b.
Subsequent stimulation with inflammasome inducers, such as crystals or ATP, leads
to NRLP3 and caspase-1 activation resulting in IL-1b maturation and secretion.
2- Detection of IL-1b by HEK-Blue IL-1b cells: IL-1b-containing THP-1
supernatants are added to HEK-Blue IL-1b cells leading to NF-kB activation and
the subsequent production of SEAP. The presence of SEAP in HEK-Blue IL-1b
supernatants is assessed using QUANTI-Blue , a SEAP detection medium.
➥ Detection range for human IL-1b: 100 pg - 100 ng/ml
PRODUCT QUANTITY CAT. CODE
HEK-Blue IL-1b cells 5-7 x 10 6 cells hkb-il1b

Autophagy is a pathway by which cytoplasmic constituents, including
organelles and intracellular pathogens, are sequestred in a doublemembrane-bound
autophagosome and delivered to the lysosome for
degradation. The role of autophagy is to eliminate unwanted constituents
from the cell and recycle cytoplasmic material allowing the cell to maintain
macromolecular synthesis and energy homeostatis during starvation and
other stressful conditions. The autophagy pathway proceeds through a
series of stages. It starts with the nucleation of the autophagic vesicle
followed by the elongation and closure of the autophagosome membrane
to envelop cytoplasmic constituents. Then the autophagosome docks with
the lysosome leading to the breakdown of the inner membrane and the
subsequent exposure of the sequestred cytoplasmic material to lysosomal
hydrolases1, 2.
The autophagy pathway requires the concerted action of evolutionarily
conserved genes. Vesicle nucleation depends on a class III
phosphatidylinositol-3-OH kinase (PI(3)K) complex formed by Beclin 1,
Vps34 and other proteins. Atg7 participates in two ubiquitin-like conjugation
pathways, conjugation of Atg5 to Atg12 and conversion of LC3 to its
phosphatidylethanolamine (PE)-conjugated LC3-II form. The Atg5-Atg12
conjugate forms a large complex with Atg16L1. Both conjugation systems
are required for the generation of the autophagosome.
Autophagy plays a key role in the prevention of aging, cancer and
neurodegeneration but also in the innate immune system against intracellular
Autophagy Genes
Description
Autophagy genes are provided as open reading frames (coding sequences)
from the Start codon to the Stop codon. They are cloned in the pUNO
plasmid (see page 58) downstream of the EF-1a/HTLV promoter. pUNO
plasmids are selectable in E. coli and mammalian cells with blasticidin.
Contents and Storage
Each pUNO plasmid is provided as a lyophilized transformed E. coli strain
on a paper disk. Transformed strains are shipped at room temperature and
should be stored at -20°C. They are provided with 4 pouches of E. coli
Fast-Media ® Blas (2 TB and 2 Agar). For more information about
Fast-Media ® , see pages 44-45.
Autophagy and TLRs
pathogens. Autophagy has been shown to interact with pattern recognition
receptors (PRRs), such as the Toll-like receptors (TLRs). It was recently
reported that the autophagic machinery can deliver pathogen-associated
molecular patterns (PAMPs) to endosomal TLRs 3 , suggesting that autophagy
enhances TLR recognition of PAMPs. Conversely, TLRs have been shown to
promote autophagy. Several groups have reported the induction of
autophagy by signaling through TLR4, TLR7, TLR3, TLR2 and TLR5 4-6 . TLRinduced
autophagy appears to depend on MyD88 and TRIF. Both adapters
trigger autophagy through a direct interaction with Beclin 1 (Shi). Autophagy
seems also to participate in the regulation of inflammation. Macrophages
from mice deficient for Atg16L1 produce more of the proinflammatory
cytokines IL-1b and IL-18 after endotoxin stimulation of TLR4 7 . These data
demonstrate that induction of autophagy and TLR signaling are connected.
1. Levine B. & Kroemer G., 2008. Autophagy in the pathogenesis of disease. Cell.
132(1):27-42. Review. 2. Mizushima N. et al., 2008. Autophagy fights disease through
cellular self-digestion. Nature. 451(7182):1069-75. Review. 3. Lee HK. et al., 2007.
Autophagy-dependent viral recognition by plasmacytoid dendritic cells. Science.
315(5817):1398-401. 4. Xu Y. et al., 2007. Toll-like receptor 4 is a sensor for autophagy
associated with innate immunity. Immunity. 27(1):135-44. 5. Delgado MA,. et al., 2008.
Toll-like receptors control autophagy. EMBO J. 27(7):1110-21. 6. Shi CS. &Kehrl JH.,
2008. MyD88 and Trif target Beclin 1 to trigger autophagy in macrophages. J Biol Chem.
283(48):33175-82. 7. Saitoh T. et al., 2008. Loss of the autophagy protein Atg16L1
enhances endotoxin-induced IL-1beta production. Nature. 2456(7219):264-8.
PRODUCT QTY
www.invivogen.com/autophagy
CAT. CODE
(Human)
CAT. CODE
(Mouse)
pUNO1-ATG3 E. coli disk puno1-hatg3 puno1-matg3
pUNO1-ATG4A E. coli disk puno1-hatg4a puno1-matg4a
pUNO1-ATG4B E. coli disk puno1-hatg4b puno1-matg4b
pUNO1-ATG5 E. coli disk puno1-hatg5 puno1-matg5
pUNO1-ATG7 E. coli disk puno1-hatg7 puno1-matg7
pUNO1-ATG10 E. coli disk puno1-hatg10 puno1-matg10
pUNO1-ATG16L1 E. coli disk puno1-hatg16l1 puno1-matg16l1
pUNO1-BECLIN1 E. coli disk puno1-hbecn1 puno1-mbecn1
pUNO1-LC3A E. coli disk puno1-hlc3a puno1-mlc3a
pUNO1-LC3B E. coli disk puno1-hlc3b puno1-mlc3b
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pSELECT-GFP-LC3 - GFP-LC3 Expression Plasmid
Description
pSELECT-GFP-LC3 is a mammalian expression vector containing the
human LC3B gene fused at its 5’ end to the green fluorescent protein
(GFP) gene. The same plasmid is available with the GFP gene alone as a
control. This control plasmid is called pSELECT-NGFP-zeo. Both plasmids
are selectable in bacteria and mammalian cells with Zeocin .
Application
Expression of the GFP-LC3 fusion gene allows to visualize autophagosome
formation in real time in live cells. During autophagosome formation, GFP-
LC3 is processed and recruited to the autophagosome membrane, where
it can be imaged as cytoplasmic puncta by fluorescence microscopy (see
picture). The percentage of GFP-LC3 positive cells can be determined and
is indicative of autophagosome formation.
Contents and Storage
pSELECT-GFP-LC3 and pSELECT-NGFP-zeo are provided as 20 μg of
lyophilized DNA. The plasmids can be purchased individually or together.
They are shipped at room temperature and should be stored at -20°C.
Both plasmids are provided with 4 pouches of E. coli Fast-Media ® Zeo (2
TB and 2 Agar). For more information about Fast-Media ® , see pages 44-45.
Autophagy Inducers & Inhibitors
Green puncta representing
autophagosome formation in cells
expressing a GFP-LC3 fusion.
PRODUCT QUANTITY CAT. CODE
pSELECT-GFP-LC3 20 μg psetz-gfplc3
pSELECT-NGFP-zeo 20 μg psetz-ngfp
Autophagy is regulated at different levels. Two connected signaling pathways encompassing class III phosphatidylinositol 3-kinase (PI3K) and
mammalian target of rapamycin (mTOR) play a central role in this regulation. Autophagy requires PI3K autophagy but is negatively regulated
by mTOR. Thus inhibitors of PI3K act as autophagy inhibitors while inhibitors of mTOR act as autophagy inducers.
PRODUCT DESCRIPTION
Autophagy Inducers
Rapamycin (mTOR inhibitor)
Tamoxifen
Autophagy Inhibitors
mimics cellular starvation by blocking signals required for
cell growth and proliferation
stimulates autophagy by increasing the intracellular level
of ceramide and abolishing the inhibitory effect of PI3K
1. Jung CH. et al., 2010. mTOR regulation of autophagy. FEBS Lett. 584(7):1287-95.
2. Scarlatti F. et al., 2004. Ceramide-mediated macroautophagy involves inhibition of
protein kinase B and up-regulation of beclin 1. J Biol Chem. 279(18):18384-91. 3.
Blommaart EF. et al., 1997.The phosphatidylinositol 3-kinase inhibitors wortmannin
and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur. J. Biochem. 243: 240–
246. 4. Yamamoto A. et al., 1998. Bafilomycin A1 prevents maturation of autophagic
vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat
hepatoma cell line, H-4-II-E cells. Cell Struct Funct. 23(1):33-42.
WORKING
CONCENTRATION QTY
CATALOG
CODE
10 - 500 nM 5 mg tlrl-rap 1
10 - 100 μM 200 mg tlrl-txf 2
3-Methyladenine (3-MA, PI3K inhibitor) inhibits the formation of autophagosome 5 mM 50 mg tlrl-3ma 3
Bafilomycin A1 (V-ATPase inhibitor) prevents maturation of autophagic vacuoles 0.1 - 1 μM 10 μg tlrl-baf 4
LY294002 (PI3K inhibitor) inhibits the formation of autophagosome 10 - 100 μM 5 mg tlrl-ly29 3
Wortmannin (PI3K inhibitor) inhibits the formation of autophagosome 0.1 - 10 μM 5 mg tlrl-wtm 3
Contents and Storage
102 www.invivogen.com/autophagy
REF.
Each product is provided as a solid and shipped at room temperature.
Store at room temperature, 4°C or -20°C according to the product label.

4
CYTOKINE SIGNALING
Cytokine Genes 104
Cytokine Antibodies 104
Cytokine Reporter Cells 105-116

CYTOKINE SIGNALING 4
Cytokine and Related Genes
Cytokines are key modulators of the immune system. They are produced mainly by T lymphocytes in response to infectious agents. Some
cytokines induce the inflammatory response or stimulate B cells to produce antibodies while others act to offset these effects by
downregulation. InvivoGen offers a comprehensive list of cytokine genes classified in different families including interleukins and chemokines.
Description
Cytokine and related genes are provided either in a pORF or a pUNO
plasmid (see page 37), both containing the complete coding sequence from
the ATG to the Stop codon. Each gene is fully sequenced.
Gene families in pORF plasmids:
- Chemokine genes - Immune receptor genes
- Chemokine receptor genes - Interferon genes
- Cytokine genes - Interleukin genes
- Cytokine suppressor genes - Interleukin receptor genes
Gene family in pUNO plasmids:
- Interferon signaling genes
Contents and Storage
Each pORF and pUNO plasmid is provided as a lyophilized transformed
E. coli strain on a paper disk. Transformed strains are shipped at room
temperature and should be stored at -20°C.
Each pORF is provided with 4 pouches of E. coli Fast-Media ® Amp (2 TB
and 2 Agar) and each pUNO plasmid with 4 pouches of E. coli
Fast-Media ® Blas (see pages 44-45).
PRODUCT QUANTITY CAT. CODE*
pORF- E. coli disk porf-
pUNO- E. coli disk puno-
* Catalog codes are available on our website
Anti-Cytokine Neutralizing IgA Antibodies
Description
Neutralizing IgA antibodies are chimeric monoclonal antibodies in which
the constant domains of the human IgA molecule were combined with
murine variable regions. They have been selected for their ability to
efficiently neutralize the biological activity of selected cytokines. The
neutralizing activity of these IgA antibodies was determined using
InvivoGen’s HEK-Blue Cytokine Cells.
Contents and Storage
Each neutralizing IgA antibody is provided lyophilized from a 0.2 μm
filtered solution in PBS. Product should be reconstituted in sterile water.
Lyophilized antibodies are stable greater than six months when stored
at -20ºC. Reconstituted IgAs are stable 1 month when stored at 4ºC and
6 months when aliquoted and stored at -20ºC.
PRODUCT ANTIGEN REACTIVITY QUANTITY
CATALOG
CODE
Anti-hCD40L-IgA2 Human CD40 ligand Human 100 μg maba-h40l
Anti-hIFNa-IgA2 Human Interferon a Human 100 μg maba-hifna
Anti-hIFNg-IgA2 Human Interferon g Human 100 μg maba-hifng
Anti-hIL-1b-IgA2 Human Interleukin 1b Human 100 μg maba-hil1b
Anti-hIL-4-IgA2 Human Interleukin 4 Human 100 μg maba-hil4
Anti-hIL-6-IgA2 Human Interleukin 6 Human 100 μg maba-hil6
Anti-hIL-13-IgA2 Human Interleukin 13 Human 100 μg maba-hil13
Anti-hIL-18-IgA2 Human Interleukin 18 Human 100 μg maba-hil18
Anti-hTGFb-IgA2 Human Tumor Growth Factor b Human 100 μg maba-htgfb
Anti-hTNFa-IgA2 Human Tumor Necrosis Factor a Human 100 μg htnfa-mab7
104 www.invivogen.com/cytokine-signaling

Blue Cytokine Reporter Cells
Blue Cytokine Reporter Cells is an expanding family of engineered cell lines designed to provide a simple, rapid
and reliable method to monitor the activation of signaling pathways induced by key cytokines. They allow to detect
these biologically active cytokines and can also be used to screen for compounds exhibiting agonist and antagonist
activities. Blue Cytokine Reporter Cells include B16-Blue and HEK-Blue Cells. B16-Blue and HEK-Blue Cells
are engineered murine melanoma and HEK293 cells, respectively. They express an inducible secreted embryonic
alkaline phosphatase (SEAP) reporter gene that can be quantitatively detected using QUANTI-Blue , a SEAP
colorimetric detection medium. Blue Cytokine Reporter Cells express this SEAP reporter gene under the control
of specific promoters that are selectively activated by the cytokines or other immune modulators known to induce
the pathway of interest. Therefore, in the presence of the cytokine, agonist or antagonist compound, the pathway
is activated or inhibited modulating the SEAP activity. The amount of SEAP secreted in the cell supernatant can
be measured spectrophotometrically with QUANTI-Blue .
JAK/STAT Pathway Smad Pathway
• B16-Blue IFN-a/b Cells • HEK-Blue TGF-b Cells
• HEK-Blue IFN-a/b Cells
• HEK-Blue IL-4/IL-13 Cells
• HEK-Blue IL-6 Cells
NF-kB Pathway CD40 Pathway
• HEK-Blue TNF-a/IL-1b Cells • HEK-Blue CD40L Cells
• HEK-Blue IL-1b Cells
• HEK-Blue IL-18/IL-1b Cells
• HEK-Blue IL-33/IL-1b Cells NEW
CYTOKINE SENSOR CELLS IFNa IFNb IFNg IL-1b IL-4 IL-6 IL-13 IL-18 IL-33 TGF-b TNF-a CD40L INFO
B16-Blue IFN-a/b cells +++ +++ - - - - - - - - - - p 106
HEK-Blue CD40L cells - - - +++ - - - - - - +++ ++++ p 115
HEK-Blue IFN-a/b cells +++ +++ - - - - - - - - - - p 107
HEK-Blue IL-1b cells - - - ++++ - - - - - - - - p 111
HEK-Blue IL-4/IL-13 cells +/- - - - ++ - ++ - - - - - p 108
HEK-Blue IL-6 cells - - - - - ++ - - - - - - p 109
HEK-Blue IL-18/IL-1b cells - - - +++ - - - ++++ - - - - p 112
HEK-Blue IL-33/IL-1b cells - - - +++ - - - - ++++ - - - p 113
HEK-Blue TGF-b cells - - - - - - - - - +++ - - p 114
HEK-Blue TNF-a/IL-1b cells - - - +++ - - - - - - ++++ - p 110
www.invivogen.com/cytokine-sensor-cells 105
CYTOKINE SIGNALING 4

CYTOKINE SIGNALING 4
B16-Blue IFN-a/b - Murine Type I IFNs Reporter Cells
➥ Monitor the JAK/STAT pathway
➥ Monitor the RIG-I/MDA-5 pathway
➥ Detection of murine IFN-a and IFN-b
Background
Interferon-alpha (IFN-a) and interferon beta (IFN-b), play an important
role in viral infections. They bind to an IFN receptor complex consisting of
two alpha chains (IFNAR1 and IFNAR2) and recruit JAK1 and TyK2. These
kinases phosphorylate STAT1 and STAT2 leading to the formation of the
ISGF3 complex. ISGF3 binds to IFN-stimulated response elements (ISRE)
in the promoters of IFN-stimulated genes (ISG) to regulate their
expression (figure 1). IFN-a and IFN-b are produced in response to viral
pathogen associated molecular patterns (PAMPs), such as viral RNA and
DNA. These PAMPs are recognized by cytoplasmic pattern recognition
receptors including the RNA helicases RIG-I or MDA5. Stimulation of
B16-Blue IFN-a/b cells with RIG-I/MDA-5 agonists, such as transfected
poly(I:C), triggers the secretion of IFN-a and IFN-b.
Description
B16-Blue IFN-a/b cells allow the detection of bioactive murine type I IFNs
by monitoring the activation of the JAK/STAT/ISGF3 pathway. They derive
from the murine B16 melanoma cell line of C57B1/6 origin after stable
transfection with a SEAP reporter gene under the control of the IFN-a/binducible
ISG54 promoter.
Stimulation of B16-Blue IFN-a/b cells with murine IFN-a or IFN-b, or
type I IFN inducers, such as tranfected poly(I:C) or poly(dA:dT), activates
the JAK/STAT/ISGF3 pathway and triggers the subsequent production of
SEAP (figures 2&3). Levels of SEAP in the supernatant can be easily
determined with QUANTI-Blue , a medium that turns purple/blue in the
presence of SEAP and by reading the OD at 655 nm.
• Detection range for mIFN-a: 10 2 - 10 4 IU/ml
• Detection range for mIFN-b: 10 2 - 10 4 IU/ml
B16-Blue IFN-a/b cells are resistant to Zeocin
Contents and Storage
B16-Blue IFN-a/b cells are grown in RPMI medium, 2 mM L-glutamine,
10% FBS supplemented with 100 μg/ml Zeocin . Each vial contains
5-7x 10 6
cells and is supplied with 10 mg Zeocin , 50 mg Normocin and
1 pouch of QUANTI-Blue . Cells are shipped on dry ice.
PRODUCT QUANTITY CAT. CODE
B16-Blue IFN-a/b cells 5-7 x 10 6 cells bb-ifnab
Related Products
Blasticidin, page 17 Anti-hIFN-a-IgA , page 104
Zeocin , page 18 QUANTI-Blue page 14
Poly(I:C) (HMW), page 77 Poly(I:C) (HMW)/LyoVec , page 80
Poly(dA:dT) Naked, page 80 Poly(dA:dT)/LyoVec, page 80
106 www.invivogen.com/cytokine-sensor-cells
Figure 1: JAK/STAT and RIG-I/MDA-5 signaling pathways
Figure 2: Stimulation of B16-Blue IFN-a/b cells by recombinant human and
murine IFN-a and IFN-b was assessed by measuring the levels of SEAP using
QUANTI-Blue .
Figure 3: Response of B16-Blue IFN-a/b cells following their stimulation with
naked poly(I:C) or poly(:C)/LyoVec. Induction of type I IFNs was assessed by
determining the levels of SEAP using QUANTI-Blue .

HEK-Blue IFN-a/b Cells - Human Type I IFN Reporter Cells
➥ Monitor the JAK/STAT/ISGF3 pathway
➥ Detection of human IFN-a and IFN-b
Background
Type I interferons, in particular interferon alpha (IFN-a) and interferon beta
(IFN-b), play a vital role in host resistance to viral infections. They signal
mainly through the JAK-STAT pathway. Following their production, IFN-a
and IFN-b bind to a common receptor (IFNAR) and recruit the Janus
kinases (JAK1 and TyK2). JAKs phosphorylate STAT1 and STAT2, which
then dimerize and interact with IFN regulatory factor 9 (IRF9), forming a
complex named ISGF3. ISGF3 binds to IFN-stimulated response elements
(ISRE) in the promoters of IFN-stimulated genes (ISG) to regulate their
expression (figure 1).
Description
HEK-Blue IFN-a/b cells allow the detection of bioactive human type I
IFNs by monitoring the activation of the ISGF3 pathway. These cells were
generated by stable transfection of HEK293 cells with the human STAT2
and IRF9 genes to obtain a fully active type I IFN signaling pathway. The
other genes of the pathway (IFNAR1, IFNAR2, JAK1, TyK2 and STAT1) are
naturally expressed in sufficient amounts. The cells were further transfected
with a SEAP reporter gene under the control of the IFN-a/b-inducible
ISG54 promoter.
Stimulation of HEK-Blue IFN-a/b cells with human IFN-a or IFN-b
activates the JAK/STAT/ISGF3 pathway and subsequently induces the
production of SEAP. Levels of SEAP in the supernatant can be easily
determined with QUANTI-Blue , a medium that turns purple/blue in the
presence of SEAP and by reading the OD at 655 nm (figure 2).
Stimulation of HEK-Blue IFN-a/b cells with recombinant human IFN-a
can be blocked by anti-hIFN-a-IgA, a neutralizing monoclonal antibody of
the IgA isotype (see page 104).
• Detection range for hIFN-a: 5 - 10 4 IU/ml
• Detection range for hIFN-b: 20 - 10 4 IU/ml
HEK-Blue IFN-a/b cells are resistant to blasticidin and Zeocin .
Contents and Storage
HEK-Blue IFN-a/b cells are grown in standard DMEM medium, 2mM
L-glutamine,10% FBS supplemented with blasticidin (30 μg/ml) and
Zeocin (100 μg/ml). Cells are provided frozen in a cryotube containing
5-7 x 10 6 cells and supplied with 100 μl of blasticidin at 10 mg/ml, 100 μl
of Zeocin at 100 mg/ml, 1 ml Normocin at 50 mg/ml, and 1 pouch of
QUANTI-Blue . Cells are shipped on dry ice.
PRODUCT QUANTITY CAT. CODE
HEK-Blue IFN-a/b cells 5-7x 10 6 cells hkb-ifnab
Figure 1: JAK-STAT pathway induced by type I IFNs.
Figure 2: Stimulation of HEK-Blue IFN-a/b cells by human IFN-a2b, IFN-b1a and
IFN-g was assessed by measuring the levels of SEAP using QUANTI-Blue .
Related Products
Blasticidin, page 17 Anti-hIFN-a-IgA , page 104
Zeocin , page 18 QUANTI-Blue page 14
www.invivogen.com/cytokine-sensor-cells 107
CYTOKINE SIGNALING 4

CYTOKINE SIGNALING 4
108
HEK-Blue IL-4/IL-13 Cells - IL-4 and IL-13 Reporter Cells
➥ Monitor the JAK/STAT-6 pathway
➥ Detection of human IL-4 and human/murine IL-13
Background
The transcription factor STAT6 is activated primarily by two cytokines
with overlapping biologic functions, IL-4 and IL-13. It can also be activated
by IFN-a in a cell-specific manner. In non-hematopoietic cells, IL-4 and
IL-13 bind a receptor complex composed of the IL-4Ralpha and
IL-13Ralpha1. Upon ligand binding, the receptor complex activates the
receptor-associated Janus kinases (JAK1 and Tyk2) leading to the
recruitment of STAT6 and its phosphorylation. Activated STAT6 forms
homodimers that translocate to the nucleus where they bind the
promoter of responsive genes inducing gene transcription.
Description
HEK-Blue IL-4/IL-13 cells allow the detection of bioactive IL-4 and
IL-13 by monitoring the activation of the STAT-6 pathway. These cells
were generated by stable transfection of HEK293 cells with the human
STAT6 gene to obtain a fully active STAT6 pathway. The other genes of
the pathway are naturally expressed in sufficient amounts. The cells were
further transfected with a SEAP reporter gene under the control of the
IFNb minimal promoter fused to four STAT6 binding sites. HEK-Blue
IL-4/IL-13 cells produce SEAP in response to IL-4 or IL-13 stimulation
and to a lower extent IFN-a. The levels of SEAP secreted in the
supernatant can be easily determined with QUANTI-Blue , a SEAP
detection medium (Figure 2).
Stimulation of HEK-Blue IL-4/IL-13 cells with IL-4 or IL-13 can be blocked
by the neutralizing monoclonal anti-hIL-4-IgA and anti-hIL-13-IgA
antibodies, respectively (see page 104).
• Detection range for IL-4: 0.5 - 100 ng/ml
• Detection range for IL-13: 5 - 1000 ng/ml
HEK-Blue IL-4/IL-13 cells are resistant to blasticidin and Zeocin .
Contents and Storage
HEK-Blue IL-4/IL-13 cells are grown in standard DMEM medium, 2 mM
L-glutamine with 10% FBS supplemented with blasticidin (10 μg/ml) and
Zeocin (100 μg/ml). Each vial contains 5-7 x 10 6 cells and is supplied
with 100 μl of blasticidin at 10 mg/ml, 100 μl of Zeocin at 100 mg/ml,
1 ml Normocin at 50 mg/ml and 1 pouch of QUANTI-Blue . Cells are
shipped on dry ice.
HEK-Blue IL-4/IL-13 cells are also available in a kit. See below:
HEK-Blue IL-4/IL-13 Kit
• HEK-Blue IL-4/IL-13 Cells 5-7 x 10 6 cells
• Anti-hIL-4-IgA antibody 100 μg
• Anti-hIL-13-IgA antibody 100 μg
• QUANTI-Blue 1 pouch (100 ml)
• Normocin 1 ml (50 mg/ml)
• Blasticidin 100 μl (10 mg/ml)
• Zeocin 100 μl (100 mg/ml)
www.invivogen.com/cytokine-sensor-cells
Figure 1: JAK/STAT6 signaling pathway induced by IL-4 and
IL-13.
Figure 2: Stimulation of HEK-Blue IL-4/IL-13 cells by recombinant human IL-4 and
IL-13 was assessed by measuring the levels of SEAP secreted in the supernatant using
QUANTI-Blue .
PRODUCT QUANTITY CAT. CODE
HEK-Blue IL-4/IL-13 Cells 5-7 x 10 6 cells hkb-stat6
HEK-Blue IL-4/IL-13 Kit 1 kit hkb-stat6-kit

HEK-Blue IL-6 Cells - IL6 Reporter Cells
➥ Monitor the JAK/STAT-3 pathway
➥ Detection of human IL-6
Background
The proinflammatory cytokine IL-6 is one of the most important
mediators of fever and of the acute phase response. IL-6 exerts its
action by first binding to the IL-6R. The complex of IL-6 and IL-6R
associates with the signal-transducing membrane protein gp130, thereby
inducing its dimerization. This leads to the activation by phosphorylation
of the tyrosine kinases of the Janus family (JAK1, JAK2, and Tyk2).
Activated JAKs induce the dimerization and translocation to the nucleus
of STAT3 where it binds enhancer elements of IL-6-inducible genes.
Description
HEK-Blue IL-6 cells allow the detection of bioactive IL-6 by
monitoring the activation of the STAT-3 pathway. These cells were
generated by stable transfection of HEK293 cells with the human
IL-6R gene. They were further transfected with a SEAP reporter gene
under the control of the IFN-b minimal promoter fused to four STAT3
binding sites. Upon IL-6 stimulation, HEK-Blue IL-6 cells trigger the
activation of STAT3 and the subsequent secretion of SEAP. SEAP can
be readily monitored when using the SEAP detection medium
QUANTI-Blue .
Stimulation of HEK-Blue IL-6 cells with recombinant human IL-6 can be
blocked by anti-hIL-6-IgA, a neutralizing monoclonal antibody of the IgA
isotype (see page 104).
• Detection range for IL-6: 0.5 - 50 ng/ml
HEK-Blue /IL-6 cells are resistant to both hygromycin and Zeocin .
Contents and Storage
HEK-Blue IL-6 cells are grown in standard DMEM medium, 2 mM
L-glutamine, 10% FBS supplemented with 200 µg/ml HygroGold
(ultrapure hygromycin) and 100 µg/ml Zeocin . Cells are provided frozen
in a cryotube containing 5-7 x 10 6 cells and supplied with 100 µl of
HygroGold at 100 mg/ml, 100 µl of Zeocin at 100 mg/ml, 1 ml
Normocin at 50 mg/ml and 1 pouch of QUANTI-Blue . Cells are
shipped on dry ice.
HEK-Blue IL-6 cells are also available in a kit. See below:
HEK-Blue IL-6 Kit
• HEK-Blue IL-6 Cells 5-7 x 10 6 cells
• Anti-hIL-6-IgA antibody 100 μg
• QUANTI-Blue 1 pouch (100 ml)
• Normocin 1 ml (50 mg/ml)
• HygroGold 100 μl (100 mg/ml)
• Zeocin 100 μl (100 mg/ml)
www.invivogen.com/cytokine-sensor-cells
Figure 1: JAK/STAT3 signaling pathway.
Figure 2: Stimulation of HEK-Blue IL-6 cells by recombinant human IL-6 was assessed
by measuring the levels of SEAP using QUANTI-Blue .
PRODUCT QUANTITY CAT. CODE
HEK-Blue IL-6 Cells 5-7 x 106 cells hkb-il6
HEK-Blue IL-6 Kit 1 kit hkb-il6-kit
109
CYTOKINE SIGNALING 4

CYTOKINE SIGNALING 4
HEK-Blue TNF-a/IL-1b Cells - TNF-a and IL-1b Reporter Cells
➥ Monitor the TNF-a- and IL-1b-induced NF-kB and AP-1 pathways
➥ Detection of human and murine TNF-a and IL-1b(a)
Background
Tumor necrosis factor alpha (TNF-a) and interleukin 1 beta (IL-1b) are
proinflammatory cytokines produced in response to microbial infection.
The common inflammatory responses of TNF-a and IL-1b are mediated
by interactions of these cytokines with distinct receptors: TNF-a binds
TNFR1 and TNFR2, IL-1b binds IL-1RI. Binding of these cytokines to their
receptors induces intracellular signaling. TNF-a signaling involves TRADD,
TRAF2 and RIP, while IL-1b signaling is mediated by MyD88, IRAK-1,
TRAF-6, and Rac1. Both signalings lead to the activation of the
transcription factors NF-kB and AP-1 (figure 1).
Description
HEK-Blue TNF-a/IL-1b cells are designed to detect bioactive TNF-a and
IL-1b (and IL-1a) by monitoring the activation of the NF-kB pathway.
HEK-Blue TNF-a/IL-1b cells derive from HEK293 cells. They
endogenously express the receptors for TNF-a and IL-1b and stably
express a SEAP reporter gene under the control of the IFN-b minimal
promoter fused to five NF-kB and five AP-1 binding sites.
HEK-Blue TNF-a/IL-1b cells secrete SEAP upon stimulation by human
TNF-a and IL-1b (figure 2). SEAP production is also detected in the
presence of murine TNF-a and IL-1b, and human or murine IL-1a. SEAP
levels can be readily determined using a SEAP detection medium, such
as QUANTI-Blue or HEK-Blue Detection medium.
Stimulation of HEK-Blue TNF-a/IL-1b cells with recombinant human
TNF-a or IL-1b can be blocked by the neutralizing monoclonal antihTNF-a-IgA
or anti-hIL-1b-IgA antibody, respectively (see page 104).
• Detection range for TNF-a: 0.5 ng - 1 μg/ml
• Detection range for IL-1b: 0.2 - 100 ng/ml
HEK-Blue TNF-a/IL-1b cells are resistant to Zeocin .
Contents and Storage
HEK-Blue TNF-a/IL-1b cells are grown in DMEM medium, 2 mM
L-glutamine, 10% FBS supplemented with Zeocin (100 μg/ml). Each vial
contains 5-7 x 10 6 cells and supplied with 100 μl of Zeocin at 100
mg/ml, 1 ml Normocin at 50 mg/ml and 1 pouch of QUANTI-Blue .
Cells are shipped on dry ice.
HEK-Blue TNF-a/IL-1b cells are also available in a kit. See below:
110
HEK-Blue TNF-a/IL-1b Kit
• HEK-Blue TNF-a/IL-1b Cells 5-7 x 10 6 cells
• Anti-hTNF-a-IgA antibody 100 μg
• Anti-hIL-1b-IgA antibody 100 μg
• QUANTI-Blue 1 pouch (100 ml)
• Normocin 1 ml (50 mg/ml)
• Zeocin 100 μl (100 mg/ml)
www.invivogen.com/cytokine-sensor-cells
Figure 1: NF-kB/AP-1 signaling pathways induced by TNF-a
and IL-1b.
Figure 2: Stimulation of HEK-Blue TNF-a/IL-1b cells by recombinant human
TNF-a and IL-1b was assessed by measuring the levels of SEAP secreted in the
supernatant using QUANTI-Blue .
PRODUCT QUANTITY CAT. CODE
HEK-Blue TNFa/IL-1b Cells 5-7 x 10 6 cells hkb-tnfil1
HEK-Blue TNF-a/IL-1b Kit 1 kit hkb-tnfil1-kit

HEK-Blue IL-1b Cells - IL-1b Reporter Cells
➥ Monitor the IL-1b-induced NF-kB and AP-1 pathways
➥ Detection of human and murine IL-1b(a)
Background
Interleukin-1b (IL-1b) is an important mediator of the inflammatory response
to infection and injury. IL-1b binds to the type 1 IL-1 receptor (IL-1R) which
requires the IL-1 receptor accessory protein (IL-1RAcP) to transduce a
signal. IL-1b signaling is mediated by MyD88, IRAK-1/2 and TRAF-6 leading
to the activation of NF-kB and AP-1 (figure 1).
Description
HEK-Blue IL-1b cells allow to detect bioactive IL-1b by monitoring the
activation of the NF-kB and AP-1 pathways. They derive from HEK-Blue
TNF-a/IL-1b cells in which the TNF-a response has been blocked.
Therefore, HEK-Blue IL-1b cells respond specifically to IL-1b. They express
a SEAP reporter gene under the control of the IFN-b minimal promoter
fused to five NF-kB and five AP-1 binding sites.
Binding of IL-1b to its receptor on the surface of HEK-Blue IL-1b cells
triggers the IL1-R signaling pathway leading to the activation of NF-kB/
AP-1 and the subsequent production of SEAP. The levels of SEAP in the
supernatant can be easily monitored using QUANTI-Blue .
HEK-Blue IL-1b cells respond to IL-1b and also IL-1a (data not shown)
but do not respond to TNF-a (figure 2).
Stimulation of HEK-Blue IL-1b cells with recombinant human IL-1b can
be blocked by the neutralizing monoclonal anti-hIL-1b-IgA antibody (see
page 104),
• Detection range for human IL-1b: 100 pg - 100 ng/ml
• Detection range for murine IL-1b: 10 ng - 1 μg/ml
HEK-Blue IL-1b cells are resistant to Zeocin and hygromycin B.
Contents and Storage
HEK-Blue IL-1b cells are grown in DMEM medium, 2 mM L-glutamine, 10%
FBS supplemented with 100 μg/ml Zeocin and 200 μg/ml HygroGold (ultrapure Hygromycin). Each vial contains 5-7 x 10 6
cells and is supplied
with 10 mg Zeocin , 10 mg HygroGold , 50 mg Normocin and 1 pouch
of QUANTI-Blue Cells are shipped on dry ice.
HEK-Blue IL-1b cells are also available in a kit. See below:
HEK-Blue IL-1b Kit
• HEK-Blue IL-1b Cells 5-7 x 10 6 cells
• Anti-hIL-1b-IgA antibody 100 μg
• QUANTI-Blue 1 pouch (100 ml)
• Normocin 1 ml (50 mg/ml)
• HygroGold 100 μl (100 mg/ml)
• Zeocin 100 μl (100 mg/ml)
www.invivogen.com/cytokine-sensor-cells
Figure 1: IL-1b-induced NF-kB signaling pathway.
Figure 2: Stimulation of HEK-Blue IL-1b cells by human and murine IL-1b and
human TNF-a was assessed by measuring the levels of SEAP using QUANTI-Blue .
PRODUCT QUANTITY CAT. CODE
HEK-Blue IL-1b Cells 5-7 x 10 6 cells hkb-il1b
HEK-Blue IL-1b Kit 1 kit hkb-il1b-kit
111
CYTOKINE SIGNALING 4

CYTOKINE SIGNALING 4
HEK-Blue IL-18/IL-1b Cells - IL-18/IL-1b Reporter Cells
➥ Monitor the IL-18/IL-1b-induced NF-kB and AP-1 pathways
➥ Detection of IL-18 and IL-1b(a)
Background
Interleukin-1b (IL-1b) and IL-18 are related pro-inflammatory cytokines
that cause a wide variety of biological efffects associated with infection,
inflammation and autoimmune processes. IL-1b binds to the type 1 IL-1
receptor which requires the IL-1 receptor accessory protein (IL-1RAcP)
to transduce a signal. IL-18 binds to an heterodimeric receptor consisting
of IL-18R and IL-18 receptor accessory protein (IL18RAP). IL-1b and IL-
18 share common signaling pathways that involve MyD88, and TRAF-6
leading to the activation of NF-kB and AP-1 (figure 1).
Description
HEK-Blue IL-18/IL-1b cells are designed to detect bioactive IL-18 and
IL-1b (and IL-1a) by monitoring the activation of the NF-kB and AP-1
pathways. They were generated by stable transfection of HEK-Blue
IL-1b cells with the gene encoding IL-18RAP. They express a SEAP
reporter gene under the control of the IFN-b minimal promoter fused
to five NF-kB and five AP-1 binding sites.
Stimulation of HEK-Blue IL-18/IL-1b cells with human IL-18 or IL-1b,
but not with human TNF-a, activates the NF-kB and AP-1 pathways
triggering the production of SEAP (figure 2). Levels of SEAP in the
supernatant can be easily determined with QUANTI-Blue .
Stimulation of HEK-Blue IL-18/IL-1b cells with recombinant human
IL-18 or IL-1b can be blocked by the neutralizing antibodies
anti-hIL-18-IgA and anti-hIL-1b-IgA, respectively (see page 104).
• Detection range for human IL-18: 0.5 - 100 ng/ml
• Detection range for human IL-1b: 0.5 - 100 ng/ml
HEK-Blue IL-18/IL-1b cells are resistant to blasticidin, Zeocin and
hygromycin B.
Contents and Storage
HEK-Blue IL-18/IL-1b cells are grown in DMEM medium, 2mM
L-glutamine, 10% FBS supplemented with 30 μg/ml blasticidin, 100 μg/ml
Zeocin and 200 μg/ml HygroGold (ultrapure Hygromycin). Each vial
contains 5-7 x 10 6
cells and is supplied with 1 mg blasticidin, 10 mg Zeocin ,
10 mg HygroGold , 50 mg Normocin and 1 pouch of QUANTI-Blue Cells are shipped on dry ice.
HEK-Blue IL-18/IL-1b cells are also available in a kit, see below:
HEK-Blue IL-18/IL-1b Kit
• HEK-Blue IL-18/IL-1b Cells 5-7 x 10 6 cells
• Anti-hIL-18-IgA antibody 100 μg
• Anti-hIL-1b-IgA antibody 100 μg
• QUANTI-Blue 1 pouch (100 ml)
• Normocin 1 ml (50 mg/ml)
• Blasticidin 100 μl (10 mg/ml)
• HygroGold 100 μl (100 mg/ml)
• Zeocin 100 μl (100 mg/ml)
112 www.invivogen.com/cytokine-sensor-cells
Figure 1: IL-18- and IL-1b--induced signaling pathways.
Figure 2: Stimulation of HEK-Blue IL-18/IL-1b cells by human IL-18, IL-1b and
TNF-a was assessed by measuring the levels of SEAP using QUANTI-Blue .
PRODUCT QUANTITY CAT. CODE
HEK-Blue IL-18/IL-1b Cells 5-7 x 10 6 cells hkb-il18
HEK-Blue IL-18/IL-1b Kit 1 kit hkb-il18-kit

HEK-Blue IL-33/IL-1b Cells - IL-33/IL-1b Reporter Cells NEW
➥ Monitor the IL-33/IL-1b-induced NF-kB and AP-1 pathways
➥ Detection of IL-33 and IL-1b(a)
Background
Interleukin-33 (IL-33) is a member of the IL-1 family, a group of cytokines
that play important roles in host defense, immune regulation and
inflammation. IL-33 mediates its biological effects through ST2 (also known
as IL1RL1), a receptor expressed on Th2 and mast cells. IL-33 and ST2 form
a complex with IL-1R accessory protein (IL-1RAcP), a signaling receptor
subunit that is also a member of the IL-1R complex. IL-33 signaling leads to
the activation of NF-kB and MAP kinases, and the production of Th2associated
cytokines.
Description
HEK-Blue IL-33/IL-1b cells are designed to detect bioactive IL-33 and
IL-1b (and IL-1a) by monitoring the activation of the NF-kB and AP-1
pathways. They were generated by stable transfection of HEK-Blue
IL-1b cells with the IL1RL1 gene. They express a SEAP reporter gene
under the control of the IFN-b minimal promoter fused to five NF-kB
and five AP-1 binding sites.
Stimulation of HEK-Blue IL-33/IL-1b cells with human IL-33 or IL-1b,
but not with IL-18 nor TNF-a, activates the NF-kB and AP-1 pathways
triggering the production of SEAP (figure 2). Levels of SEAP in the
supernatant can be easily determined with QUANTI-Blue .
Stimulation of HEK-Blue IL-33/IL-1b cells with recombinant human
IL-1b can be blocked by using the neutralizing antibody anti-hIL-1b-IgA.
• Detection range for human IL-33: 0.5 - 100 ng/ml
• Detection range for human IL-1b: 0.5 - 100 ng/ml
HEK-Blue IL-33/IL-1b cells are resistant to blasticidin, Zeocin and
hygromycin B.
Contents and Storage
HEK-Blue IL-33/IL-1b cells are grown in DMEM medium, 2mM
L-glutamine, 10% FBS supplemented with 30 μg/ml blasticidin, 100 μg/ml
Zeocin and 200 μg/ml HygroGold (ultrapure Hygromycin). Each vial
contains 5-7 x 10 6
cells and is supplied with 1 mg blasticidin, 10 mg Zeocin ,
10 mg HygroGold , 50 mg Normocin and 1 pouch of QUANTI-Blue Cells are shipped on dry ice.
Related Products
Figure 1: IL-33- and IL-1b--induced signaling pathways.
Figure 2: Stimulation of HEK-Blue IL-33/IL-1b cells by human IL-33, IL-1b, IL-18
and TNF-a was assessed by measuring the levels of SEAP using QUANTI-Blue .
Anti-hIL-1b-IgA , page 104 Blasticidin, page 17
Normocin , page 11 HygroGold , page 18
QUANTI-Blue , page 14 Zeocin , page 18 PRODUCT QUANTITY CAT. CODE
HEK-Blue IL-33/IL-1b Cells 5-7 x 10 6 cells hkb-il33
www.invivogen.com/cytokine-sensor-cells 113
CYTOKINE SIGNALING 4

CYTOKINE SIGNALING 4
HEK-Blue TGF-b Cells - TGF-b Reporter Cells
➥ Monitor the TGF-b/Smad pathway
➥ Detection of human TGF-b
Background
Tumor growth factor-beta (TGF-b) belongs to a family of structurally
related cytokines that regulate a plethora of cellular functions, such as
proliferation, apoptosis, differentiation and migration. TGF-b binds to a
type II receptor which recruits and activates a type I receptor. The type I
receptor then phosphorylates receptor-regulated Smads (R-Smads), such
as Smad2 and Smad3, which associate with Smad4. R-Smad/Smad4
complexes accumulate in the nucleus where they regulate the
transcription of target genes.
Description
HEK-Blue TGF-b cells allow the detection of bioactive TGF-b by
monitoring the activation of the TGF-b/Smad pathway. They were
generated by stable transfection of HEK293 cells with the human TGFBRI,
Smad3 and Smad4 genes. They further express a SEAP reporter gene
under the control of the b-globin minimal promoter fused to three
Smad3/4-binding elements (SBE).
Stimulation of HEK-Blue TGF-b cells with TGF-b induces the activation
of the TGF-b/Smad signaling pathway leading to the formation of a
Smad3/Smad4 complex. The heterocomplex enters the nucleus and binds
SBE sites inducing the production of SEAP (figure 1). The quantity of SEAP
secreted in the supernatant can be easily assessed using QUANTI-Blue
(figure 2). TGF-b-mediated SEAP production can be blocked using a
neutralizing antibody, such as anti-hTGF-b-IgA (see page 104).
• Detection range for TGF-b: 0.1 - 10 ng/ml
HEK-Blue TGF-b cells are resistant to blasticidin, hygromycin and
Zeocin .
Contents and Storage
HEK-Blue TGF-b cells are grown in DMEM medium, 2 mM L-glutamine,
10% FBS supplemented with 30 μg/ml blasticidin, 200 µg/ml HygroGold
(ultrapure hygromycin) and 100 µg/ml Zeocin . Cells are provided frozen
in a cryotube containing 5-7 x 10 6 cells and supplied with 100 µl of
blasticidin at 10 mg/ml, 100 µl of HygroGold at 100 mg/ml, 100 µl of
Zeocin at 100 mg/ml and 1 pouch of QUANTI-Blue . Cells are shipped
on dry ice.
HEK-Blue TGF-b cells are also available in a kit, see below:
HEK-Blue CD40L Kit
• HEK-Blue TGF-b Cells 5-7 x 10 6 cells
• Anti-hTGF-b-IgA antibody 100 μg
• QUANTI-Blue 1 pouch (100 ml)
• Normocin 1 ml (50 mg/ml)
• Blasticidin 100 μl (10 mg/ml)
• HygroGold 100 μl (100 mg/ml)
• Zeocin 100 μl (100 mg/ml)
114 www.invivogen.com/cytokine-sensor-cells
Figure 1: TGF-b-induced Smad signaling pathway
Figure 2: Stimulation of HEK-Blue TGF-b cells by human TGF-b was assessed by
measuring the levels of SEAP using QUANTI-Blue .
PRODUCT QUANTITY CAT. CODE
HEK-Blue TGF-b Cells 5-7 x 10 6 cells hkb-tgfb
HEK-Blue TGF-b Kit 1 kit hkb-tgfb-kit

HEK-Blue CD40L Cells - CD40L Reporter Cells
➥ Study CD40L-CD40 interactions
➥ Screen for molecules that interfere with CD40L-CD40 cross-talk
Background
CD40 ligand (CD40L) is a member of the tumor necrosis factor (TNF)
family of cell surface interaction molecules. It is mainly expressed in
CD4+-T cells and interacts with CD40 on antigen-presenting cells to
regulate both humoral and cellular immune responses. CD40L-CD40
interactions are thought to play an important role in the pathogenesis of
certain diseases, including AIDS, Alzheimer’s disease and rheumatoid
arthritis.
Description
HEK-Blue CD40L cells can serve to measure the bioactivity of CD40L
through the secretion of embryonic alkaline phosphatase (SEAP) upon
NF-kB activation. These cells were generated by stable transfection of
HEK293 cells with the human CD40 gene and an NF-kB-inducible SEAP
construct. The SEAP construct consists of the SEAP reporter gene under
the control of the IFN-b minimal promoter fused to five NF-kB binding
sites. Binding of CD40L to its receptor CD40 triggers a signaling cascade
leading to the activation of NF-kB and the subsequent production of
SEAP (figure 1). CD40L-CD40 interaction can be monitored by assessing
the levels of SEAP using a SEAP detection assay, such as QUANTI-Blue
(figure 2). HEK293 cells express endogenously the receptors for the
cytokines IL-1b and TNF-a which share a common signaling pathway with
CD40L. Consequently, HEK-Blue CD40L cells also respond to IL-1b and
TNF-a. IL-1b- and TNF-a-mediated SEAP production can be blocked
using neutralizing antibodies, such as anti-hIL-1b-IgA and anti-hTNF-a-IgA
antibodies respectively (see page 104).
• Detection range for CD40L: 5 ng - 1 μg/ml
HEK-Blue CD40L cells are resistant to the selective antibiotics blasticidin
and Zeocin .
Contents and Storage
HEK-Blue CD40L cells are grown in DMEM medium, 2mM L-glutamine,
10% FBS supplemented with 100 μg/ml Zeocin and 30 μg/ml blasticidin.
Each vial contains 5-7 x 10 6
cells and is supplied with 10 mg Zeocin ,
1 mg blasticidin, 50 mg Normocin and 1 pouch of QUANTI-Blue . Cells
are shipped on dry ice.
HEK-Blue CD40L cells are also available in a kit, see below: Figure 2: Stimulation of HEK-Blue CD40L cells by human CD40L was assessed by
measuring the levels of SEAP using QUANTI-Blue .
HEK-Blue CD40L Kit
• HEK-Blue CD40L Cells 5-7 x 10 6 cells
• Anti-hCD40L-IgA antibody 100 μg
• QUANTI-Blue 1 pouch (100 ml)
• Normocin 1 ml (50 mg/ml)
• Blasticidin 100 μl (10 mg/ml)
• Zeocin 100 μl (100 mg/ml)
Figure 1: CD40L-induced NF-kB signaling pathway.
PRODUCT QUANTITY CAT. CODE
HEK-Blue CD40L Cells 5-7 x 10 6 cells hkb-cd40
HEK-Blue CD40L Kit 1 kit hkb-cd40-kit
www.invivogen.com/cytokine-sensor-cells 115
CYTOKINE SIGNALING 4

CYTOKINE SIGNALING 4
HEK-Blue Cytokine Kits
PRODUCT CONTENTS CAT. CODE
HEK-Blue IL-4/IL-13 Kit • HEK-Blue IL-4/IL-13 Cells (5-7 x 10 6 cells) • Normocin (1 ml @ 50 mg/ml)
• Anti-hIL-4-IgA neutralizing antibody (100 μg) • Blasticidin (100 μl @ 10 mg/ml)
• Anti-hIL-13-IgA neutralizing antibody (100 μg) • Zeocin (100 μl @ 100 mg/ml)
• QUANTI-Blue (1 pouch, 100 ml)
HEK-Blue IL-6 Kit • HEK-Blue IL-6 Cells (5-7 x 10 6 cells) • Normocin (1 ml @ 50 mg/ml)
• Anti-hIL-6-IgA neutralizing antibody (100 μg) • HygroGold (100 μl @ 100 mg/ml)
• QUANTI-Blue (1 pouch, 100 ml) • Zeocin (100 μl @ 100 mg/ml)
HEK-Blue TNF-a/IL-1b Kit • HEK-Blue TNF-a/IL-1b Cells (5-7 x 10 6 cells) • QUANTI-Blue (1 pouch, 100 ml)
• Anti-hTNF-a-IgA neutralizing antibody (100 μg) • Normocin (1 ml @ 50 mg/ml)
• Anti-hIL-1b-IgA neutralizing antibody (100 μg) • Zeocin (100 μl @ 100 mg/ml)
HEK-Blue IL-1b Kit • HEK-Blue IL-1b Cells (5-7 x 10 6 cells) • Normocin (1 ml @ 50 mg/ml)
• Anti-hIL-1b-IgA neutralizing antibody (100 μg) • HygroGold (100 μl @ 100 mg/ml)
• QUANTI-Blue (1 pouch, 100 ml) • Zeocin (100 μl @ 100 mg/ml)
HEK-Blue IL-18/IL-1b Kit • HEK-Blue IL-18/IL-1b Cells (5-7 x 10 6 cells) • Normocin (1 ml @ 50 mg/ml)
• Anti-hIL-18-IgA neutralizing antibody (100 μg) • Blasticidin (100 μl @ 10 mg/ml)
• Anti-hIL-1b-IgA neutralizing antibody (100 μg) • HygroGold (100 μl @ 100 mg/ml)
• QUANTI-Blue (1 pouch, 100 ml) • Zeocin (100 μl @ 100 mg/ml)
HEK-Blue TGF-b Kit • HEK-Blue TGF-b Cells (5-7 x 10 6 cells) • Blasticidin (100 μl @ 10 mg/ml)
• Anti-hTGF-b-IgA neutralizing antibody (100 μg) • HygroGold (100 μl @ 100 mg/ml)
• QUANTI-Blue (1 pouch, 100 ml) • Zeocin (100 μl @ 100 mg/ml)
• Normocin (1 ml @ 50 mg/ml)
HEK-Blue CD40L Kit • HEK-Blue CD40L Cells (5-7 x 10 6 cells) • Normocin (1 ml @ 50 mg/ml)
• Anti-hCD40L-IgA neutralizing antibody (100 μg) • Blasticidin (100 μl @ 10 mg/ml)
• QUANTI-Blue (1 pouch, 100 ml) • Zeocin (100 μl @ 100 mg/ml)
Contents and Storage
116 www.invivogen.com/cytokine-sensor-cells
hkb-stat6-kit
hkb-il6-kit
hkb-tnfil1-kit
hkb-il1b-kit
hkb-il18-kit
hkb-tgfb-kit
hkb-cd40-kit
HEK-Blue Cytokine cells are shipped on dry ice. Thaw immediately or store in liquid nitrogen. Other products are shipped at room temperature and should
be stored at -20°C.

5
ANTIBODIES & VACCINATION
Antibodies
Vaccination
Antibody Generation 118
Antibody Isotype Collections 120
IgA Antibodies 121-124
IgG Antibodies 125
Vaccine Adjuvants 126-129
OVA Antigens 129
DNA Vaccination 130-131

ANTIBODIES & VACCINATION 5
pFUSE-CLIg and pFUSE-CHIg - Antibody Generation
pFUSE-CLIg and pFUSE-CHIg plasmids are designed to change a monoclonal antibody from one isotype to another human or murine IgG
isotype therefore enabling the generation of antibodies with the same antigen affinity but with different effector functions (increased or reduced
ADCC and CDC). Furthermore, they can be used to produce entire IgG antibodies from fragment antigen-binding (Fab) or single-chain variable
fragment (scFv) fragments that are either chimeric, humanized or fully human depending on the nature of the variable region.
➥ Isotype switch to generate IgG antibodies with different effector functions
➥ Generation of entire IgG antibodies, chimeric, humanized or fully human
Background
Immunoglobulin G (IgG) antibodies are large molecules composed of two
heavy chains g and two light chains, either k or l. They can be separated
in two regions: the Fab (fragment-antigen binding) that contains the
variable domain responsible for the antibody specificity, and the Fc
(fragment crystalline) that binds specific proteins to induce immune
responses such as opsonization and cell lysis.
The IgG class is divided in four isotypes: IgG1, IgG2, IgG3 and IgG4 in humans,
and IgG1, IgG2a, IgG2b and IgG3 in mice. They share more than 95%
homology in the amino acid sequences of the Fc regions but show major
differences in the amino acid composition and structure of the hinge region.
The Fc region mediates effector functions, such as antibody-dependent cellular
cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In
ADCC, the Fc region of an antibody binds to Fc receptors (FcgRs) on the
surface of immune effector cells such as natural killers and macrophages,
leading to the phagocytosis or lysis of the targeted cells. In CDC, the
antibodies kill the targeted cells by triggering the complement cascade at the
cell surface. IgG isoforms exert different levels of effector functions increasing
in the order of IgG4

Antibody generation using pFUSE-CHIg & pFUSE-CLIg
1- Obtaining VH and VL sequences
To obtain the cDNA sequence of the VH and VL regions from an antibody
producing hybridoma, total RNA or mRNA is extracted and reverse
transcribed to cDNA. PCR is performed with 5’ degenerate primers to anneal
to the unknown VH and VL regions and the 3’ primers designed to anneal to
the “known“ CH and CL regions. Alternatively 5’ RACE can be used. The
resulting amplicons are sequenced.
Contents and Storage
pFUSE-CLIg and pFUSE-CHIg plasmids are provided as 20 μg of lyophilized
DNA. Product is shipped at room temperature and should be stored
at -20°C. Each plasmid is provided with 4 pouches of E. coli Fast-Media ®
Zeo (2 TB and 2 Agar, see pages 44-45).
2- Cloning into pFUSE-CHIg and pFUSE-CLIg
Once the VH and VL sequence are known, inserts for cloning into the plasmids
can be generated. When generating the insert for VH, a Nhe I site must be
introduced at the 3’ end to maintain the integrity of the constant region.
Similarly, when generating the insert for VL, a Bsi WI (human VL) or Bst API
(mouse VL) site must be introduced at the 3’ end. There is a choice of
restriction sites at the 5’ end.
PRODUCT
pFUSE-CLIg
ISOTYPE QUANTITY
CAT. CODE
(No IL2ss)
CAT. CODE NEW
(With IL2ss)
pFUSE2-CLIg-hk Human kappa 20 μg pfuse2-hclk pfuse2ss-hclk
pFUSE2-CLIg-hl2 Human lambda 2 20 μg pfuse2-hcll2 pfuse2ss-hcll2
pFUSE2-CLIg-mk Mouse kappa 20 μg pfuse2-mclk pfuse2ss-mclk
pFUSE2-CLIg-ml1 NEW Mouse lambda 1 20 μg pfuse2-mcll1 pfuse2ss-mcll1
pFUSE2-CLIg-ml2 NEW Mouse lambda 2 20 μg pfuse2-mcll2 pfuse2ss-mcll2
pFUSE2-CLIg-rk1 NEW Rabbit kappa 1 20 μg pfuse2-rclk1 pfuse2ss-rclk1
pFUSE2-CLIg-rk2
pFUSE-CHIg
NEW Rabbit kappa 2 20 μg pfuse2-rclk2 pfuse2ss-rclk2
pFUSE-CHIg-hG1 Human IgG1 20 μg pfuse-hchg1 pfusess-hchg1
pFUSE-CHIg-hG2 Human IgG2 20 μg pfuse-hchg2 pfusess-hchg2
pFUSE-CHIg-hG3 Human IgG3 20 μg pfuse-hchg3 pfusess-hchg3
pFUSE-CHIg-hG4 Human IgG4 20 μg pfuse-hchg4 pfusess-hchg4
pFUSE-CHIg-mG1 Mouse IgG1 20 μg pfuse-mchg1 pfusess-mchg1
pFUSE-CHIg-mG2a Mouse IgG2a 20 μg pfuse-mchg2a pfusess-mchg2a
pFUSE-CHIg-mG2b Mouse IgG2b 20 μg pfuse-mchg2b pfusess-mchg2b
pFUSE-CHIg-mG3 Mouse IgG3 20 μg pfuse-mchg3 pfusess-mchg3
pFUSE-CHIg-rG NEW Rabbit IgG 20 μg pfuse-rchg pfusess-rchg
www.invivogen.com/antibody-generation 119
ANTIBODIES & VACCINATION 5

ANTIBODIES & VACCINATION 5
Antibody Isotype Collections NEW
Immunoglobulins (Ig) are divided in isotypes: nine in humans (IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, IgE) and eight in mice (IgG1, IgG2a,
IgG2b, IgG3, IgM, IgA, IgD, IgE). Each isotype displays distinct structural and effector properties. These properties are key features in choosing
the backbone for a therapeutic antibody. To help you decide which Ig isotype is the most suitable for your application, InvivoGen provides two
well-known monoclonal antibodies, anti-hCD20 (rituximab) and anti-hTNF-a (adalimumab), available in the most common human and murine
isotypes. These antibody isotype collections will help you study the effector functions between different isotypes or between the same isotype
of two different species.
Description
The anti-hCD20 and anti-hTNF-a isotype collections consist of
monoclonal antibodies comprising the same variable region, that targets
the human CD20 antigen or human TNF-a cytokine respectively, and the
constant region of different isotypes.
Variable Region
• The Anti-hCD20 isotype collection features the variable region of
rituximab. Rituximab is a mouse/human chimeric monoclonal antibody
that targets the CD20 antigen found on the surface of malignant and
normal B lymphocytes. Binding of rituximab to CD20 results in cell
destruction through different mechanisms including direct signaling of
apoptosis, complement activation and cell-mediated cytotoxicity.
Rituximab has been approved by the FDA for the treatment of various
lymphoid malignancies, including B-cell non-Hodgkin's lymphoma and
B-cell chronic lymphocytic leukaemia.
• The Anti-hTNF-a isotype collection features the variable region of
adalimumab. Adalimumab is a fully human monoclonal antibody against
the pro-inflammatory cytokine TNF-a. Adalimumab binds to TNF-a and
blocks its interaction with TNF receptors thereby downregulating the
inflammatory reactions associated with autoimmune diseases.
Adalimumab has been approved by the FDA for the treatment of various
inflammatory diseases, such as rheumatoid arthritis and Crohn’s disease.
Constant Region
The constant region consists of the human or murine kappa light chain
and the heavy chain of different isotypes. Eight human and three murine
isotypes are available:
• human isotypes
- IgG1, IgG2, IgG3, IgG4
- IgM
- IgA1, IgA2
- IgE
• murine isotypes
- IgG1, IgG2a
- IgA
The anti-hCD20 and anti-hTNF-a isotype collections have been
generated by recombinant DNA technology. They have been produced
in CHO cells and purified by different types of affinity chormatography:
protein G for IgG isotypes, protein L for IgE and IgM, and peptide M for
IgA isotypes.
Contents and Storage
Each anti-hCD20 and anti-hTNF-a antibody is provided lyophilized from a 0.2
μm filtered buffered solution with stabilizers. Product should be reconstituted
in sterile water. Lyophilized antibodies are stable greater than six months when
stored at -20ºC. Reconstituted antibodies are stable 1 month when stored at
4ºC and 6 months when aliquoted and stored at -20ºC.
Name Types Description
IgG 4
IgM 1
IgA 2
IgE 1
120 www.invivogen.com/antibody-isotypes
Major Ig in serum, placental transfer
CDC (hIgG3>hIgG1>hIgG2>hIgG4; mIgG2a>mIgG1)
ADCC (hIgG1≥hIgG3>hIgG2≥IgG4; mIgG2a>mIgG1)
Third most common serum Ig, first Ig to be made
Good CDC, some ADCC
Major class in secretions, second most common serum Ig
monomer in serum, dimer in secretions
No CDC, some ADCC
Least common serum Ig, involved in allergic reaction
Strong binding to Fc receptors on basophils, no CDC
PRODUCT ISOTYPE QTY CAT. CODE
Anti-hCD20 isotype collection
Anti-hCD20-hIgG1 Human IgG1 100 μg hcd20-mab1
Anti-hCD20-hIgG2 Human IgG2 100 μg hcd20-mab2
Anti-hCD20-hIgG3 Human IgG3 100 μg hcd20-mab3
Anti-hCD20-hIgG4 Human IgG4 100 μg hcd20-mab4
Anti-hCD20-hIgM Human IgM 100 μg hcd20-mab5
Anti-hCD20-hIgA1 Human IgA1 100 μg hcd20-mab6
Anti-hCD20-hIgA2 Human IgA2 100 μg hcd20-mab7
Anti-hCD20-hIgE Human IgE 100 μg hcd20-mab8
Anti-hCD20-mIgG1 Mouse IgG1 100 μg hcd20-mab9
Anti-hCD20-mIgG2a Mouse IgG2a 100 μg hcd20-mab10
Anti-hCD20-mIgA Mouse IgA 100 μg hcd20-mab11
Anti-hTNF-a isotype collection
Anti-hTNF-a-hIgG1 Human IgG1 100 μg htnfa-mab1
Anti-hTNF-a-hIgG2 Human IgG2 100 μg htnfa-mab2
Anti-hTNF-a-hIgG3 Human IgG3 100 μg htnfa-mab3
Anti-hTNF-a-hIgG4 Human IgG4 100 μg htnfa-mab4
Anti-hTNF-a-hIgM Human IgM 100 μg htnfa-mab5
Anti-hTNF-a-hIgA1 Human IgA1 100 μg htnfa-mab6
Anti-hTNF-a-hIgA2 Human IgA2 100 μg htnfa-mab7
Anti-hTNF-a-hIgE Human IgE 100 μg htnfa-mab8
Anti-hTNF-a-mIgG1 Mouse IgG1 100 μg htnfa-mab9
Anti-hTNF-a-mIgG2a Mouse IgG2a 100 μg htnfa-mab10
Anti-hTNF-a-mIgA Mouse IgA 100 μg htnfa-mab11

Immunoglobulin A
The mucosal surfaces represent the largest area of exposure of the body
to external pathogens. Immunoglobulin A (IgA), in its secretory form, is the
main effector of the mucosal immune system and provides an important
first line of defense against most pathogens that invade the body at a
mucosal surface 1 . Secretory IgA (SIgA) represents the most abundant
immunoglobulin of body secretions such as saliva, tears, colostrum and
gastrointestinal secretions. The molecular stability and effector immune
functions make SIgA particularly well suited to provide mucosal protection
against pathogens.
Light chain
Heavy chain
J chain
Secretory
component
SIgA is produced by plasma cells predominantly as polymeric IgA (pIgA)
consisting of two or more monomers linked by the J (joining) chain. pIgA
is actively transported by the epithelial polymeric Ig receptor (pIgR) and
released into mucosal secretions with a bound secretory component (the
extracellular domain of the pIgR) that protects the molecule from
proteolytic enzymes. IgA mediates a variety of protective functions 2, 3 .
Luminal SIgA is believed to interfere with pathogen adherence to mucosal
epithelial cells, a process called immune exclusion. In addition, IgA appears
to have two other defense functions: intracellular neutralization, and virus
excretion. In response to continuous stimulation by microbes, the IgA
repertoire is spontaneously diversified by somatic hypermutation (SHM).
IgA that has undergone defective SHM can neither regulate intestinal
microbial homeostasis nor effectively protect the host from pathogens
emphasizing the importance of IgA in maintaining intestinal homeostasis
and efficient mucosal defense 4 . IgA is also found as a monomer in the serum
where it may function as a second line of defence by eliminating pathogens
that have breached the mucosal surface. Serum IgA interacts with an Fc
receptor called FcaR1 triggering antibody-dependent-cell-mediated
cytotoxicity (ADCC).
Due to their specific effector functions, IgA present an interesting therapeutic
potential for mucosal protection against virus and bacteria. Indeed,
monoclonal IgA antibodies have been shown to be efficient in protecting
against infection by various bacteria 5 and viruses, including HIV-1 6-7 .
Despite this great potential and in contrast to monoclonal antibodies
(MAbs) of the IgG isotype, their development as research tools or human
therapeutics has been scarce. This is mostly due to the difficulties
encountered in producing and purifying biologically active IgA. IgA MAbs
can hardly be obtained through the classical hybridoma technique that
involves the fusion between murine splenocytes and myeloma cells 8 . Studies
of IgA would be much facilitated by the availability of a simple method to
isolate and detect IgA.
www.invivogen.com/iga
As a specialist of the Toll-like receptors (TLRs) and innate immunity,
InvivoGen believes that IgA is a new hot topic in this field and therefore is
initiating a vast IgA program. In this regard, we are using two innovative
methods to generate IgA MAbs. The first relies on the use of a transgenic
mouse, named Ca, obtained through insertion of the human a1 gene in
place of the switch sequence Sμ 8 , that allows the isolation of primarily
chimeric IgA MAbs via the classical hybridoma technique. The second
combines hybridoma and recombinant DNA technologies and involves an
IgG-IgA class-switch. In both methods, mice are DNA immunized with a
plasmid expressing the antigen, and IgA- or IgG-producing hybridomas are
screened using a neutralizing assay based on engineered cell lines
(HEK-Blue Cells). IgA antibodies are purified by Protein L affinity
chromatography. Protein L is a bacterial protein that binds antibodies
through k light chain interactions. These techniques have been utilized to
generate a first series of IgA MAbs that target the extra-cellular TLRs and
key cytokines of the innate immune system. These IgA MAbs display potent
neutralizing activities and can be used for flow cytometry, and thus
represent useful research tools. Many more IgA MAbs are in the pipeline,
some with potential therapeutic applications.
1. Cerutti A. et al., 2010. Immunoglobulin Responses at the Mucosal Interfaces. Annu Rev
Immunol. [Epub ahead of print]. 2. Woof JM. & Kerr MA., 2007. The function of
immunoglobulin A in immunity. J Pathol. 208(2):270-82. Review. 3. Fagarasan et al., 2010.
Adaptive immune regulation in the gut: T cell-dependent and T cell-independent IgA
synthesis. Annu. Rev. Immnuol. 28: 740-273. 4. Wei M. et al., 2011. Mice carrying a knockin
mutation of Aicda resulting in a defect in somatic hypermutation have impaired gut
homeostasis and compromised mucosal defense. Nat Immunol. [Epub ahead of print]. 5.
Tokuhara D. et al, 2010. Secretory IgA-mediated protection against V. cholerae and heatlabile
enterotoxin-producing enterotoxigenic Escherichia coli by rice-based vaccine. PNAS
107(19):8794-9. 6. Planque S. et al., 2010. Neutralization of genetically diverse HIV-1 strains
by IgA antibodies to the gp120-CD4-binding site from long-term survivors of HIV infection.
AIDS 24(6): 875-84. 7. Mantis NJ. et al., 2007. Inhibition of HIV-1 Infectivity and Epithelial
Cell Transfer by Human Monoclonal IgG and IgA Antibodies Carrying the b12 V Region. J.
Immunol. 179: 3144 - 3152. 8. Cogne M. et al., 2007. Non-Human Transgenic Mammal for
the Constant Region of the Class a Human Immunoglobulin Heavy Chain and Applications
Thereof. US2007248601.
IgA Product Line
IgA Antibodies
• Anti-Cytokine IgAs
• Anti-TLR IgAs
• Control IgA
IgA Purification
• Peptide M / Agarose
• SSL7 / Agarose
• Jacalin / Agarose
• Protein L / Agarose
IgA Detection & Quantification
• Kappa Light Chain
• IgA Heavy Chain
• J Chain Antiserum
Page
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ANTIBODIES & VACCINATION 5

ANTIBODIES & VACCINATION 5
IgA Antibody Purification
Protein G and Protein A are currently used to purify IgG antibodies, but these supports are not appropriate for IgA antibody purification. For
fast and efficient purification of IgA antibodies from biological samples, InvivoGen provides now four different methods, Peptide M, SSL7, Jacalin,
and Protein L. The correct choice of purification method depends upon the IgA subclass of the antibody, the species in which it was raised and
the intended use of the antibodies. Antibodies from tissue culture supernatant, serum or ascites can be purified using one or more of the
following antibody binding proteins offered by InvivoGen.
Peptide M / Agarose - IgA1 and IgA2 purification
Peptide M is a 50 aa synthetic peptide derived from a streptococcal M protein containing an additional C-terminal cysteine residue. Peptide M binds
monomeric and dimeric human IgA of both subclasses (IgA1 and IgA2) with high specificity and affinity. It also binds bovine IgA but not murine IgA. Peptide
M binding occurs at a site in IgA-Fc conserved in human IgA1 and IgA2 and bovine IgA but not in mouse IgA. Peptide M can be used for single-step affinity
purification of IgA and for specific detection of antigen-bound IgA 1 .
Binding capacity: 6 mg human IgA per ml of gel
SSL7 / Agarose - IgA1 and IgA2 purification
SSL7 (Staphylococcus aureus superantigen-like protein 7; formally known as SET1), is a staphylococcus toxin isolated from Staphylococcus aureus. SSL7 binds
with a high affinity to the monomeric form of human IgA1 and IgA2. SSL7 has no affinity for human IgG, therefore, it can be used to purify human IgA from
human sera, milk and other biological samples 2,3 . SSL7 also binds the secretory form of IgA found in milk from humans, cows, and sheep. SSL7 will bind
bovine IgA antibodies present in milk, but not bovine IgA present in serum. SSL7 does not bind mouse, rabbit, sheep or goat IgA present in serum 2 .
Binding capacity: 1 mg human IgA per ml of gel
Jacalin / Agarose - IgA1 purification
Jacalin is an a D-galactose binding lectin extracted from jack-fruit seeds (Artocarpus integrifolia). Jacalin immobilized on supports such as agarose is useful for the
purification of human serum or secretory IgA1 4,5 . IgA can be separated from human IgG and IgM in human serum or colostrums using Immobilized Jacalin. This
support is also useful for removing contaminating IgA from IgG samples. Additionally, Jacalin binds IgD 4 . Jacalin can also be used to separate IgA1 subclass from IgA2 5 .
Binding capacity: 1-3 mg human IgA per ml of gel
Protein L / Agarose - k light chain specific
Protein L is an immunoglobulin-binding protein expressed by the anaerobic bacterial species Peptostreptococcus magnus 6 . Protein L binds specifically to the variable
domain of Ig k light chain, as a consequence Protein L has the capacity to purifiy k light containing IgA antibodies 7 . It binds strongly to human k light chain subclasses
I, III and IV, and also to most k light chains of other species such as rat and mouse. As it recognizes k light chains, protein L can bind to all classes of Ig, in contrast to
Protein A and Protein G which interact with the Fc region and bind exclusively to IgG heavy chains. Protein L does not bind bovine immunoglobulins which are
present in the fetal bovine serum (FBS) and thus provides a convenient way to purify k light chain-containing monoclonal antibodies from culture supernatant.
Binding capacity >5 mg of human IgA/IgG per ml of gel
PRODUCT
Human k
light chain
Human l
light chain
Human
IgA1
Human
IgA2
Human
IgG
Human
IgM
Human
IgE
Human
IgD
Mouse
IgA
Rat IgA Bovine
IgA
Peptide M - - ++++ ++++ - - n/a n/a - n/a +
SSL7 - - ++++ ++++ - - n/a - - ++++
Jacalin - - ++++ -/+ - n/a n/a +++ - - -
Protein L ++++ - ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ -
1. Sandin C. et al., 2002. Isolation and detection of human IgA using a streptococcal IgAbinding
peptide. J Immunol. 169(3):1357-64. 2. Langley et al., 2005. The staphylococcal
superantigen-like protein 7 binds IgA and complement C5 and inhibits IgA-Fc alpha RI
binding and serum killing of bacteria. J Immunol 174 :2926-2933. 3. Ramsland PA. et al.,
2007. Structural basis for evasion of IgA immunity by Staphylococcus aureus revealed in
the complex of SSL7 with Fc of human IgA1. PNAS 104:15051-15056. 4. Aucouturier P.
et al., 1998. Jacalin, the human IgA1 and IgD precipitating lectin, also binds IgA2 of both
allotypes. J Immnuol Methods 113:185-191. 5. Gregory RL. et al., 1987. Separation of
human IgA1 and IgA2 using jacalin-agarose chromatography. J Immunol Meth 99:101-106.
6. Björck L., 1988. Protein L. A novel bacterial cell wall protein with affinity for Ig L chains.
J Immunol. 1988 Feb 15;140(4): 1194-7. 7. Nilson BH. et al., 1993. Purification of antibodies
using protein L-binding framework structures in the light chain variable domain. J Immunol
Methods. 164(1):33-40.
- (serum)
+ (milk)
PRODUCT QUANTITY CAT. CODE
Peptide M / Agarose
SSL7 / Agarose
Jacalin / Agarose
Protein L / Agarose
122 www.invivogen.com/iga-purification
2 ml
5 ml
2 ml
10 ml
2 ml
5 ml
2 ml
10 ml
gel-pdm-2
gel-pdm-5
gel-ssl-2
gel-ssl-10
gel-jac-2
gel-jac-5
gel-protl-2
gel-protl-10

IgA Detection and Quantification
InvivoGen provides all the reagents necessary to detect and quantify the light and heavy chains of IgA antibodies by sandwich ELISA: capture
antibodies, revelation antibodies and IgA standards. InvivoGen offers also a polyclonal rabbit antiserum to human J chain for the detection of
dimeric IgA.
IgA k Light Chain for Sandwich ELISA
Goat F(ab’)2 anti-human kappa - Capture antibody
Goat F(ab’)2 anti-human kappa was generated from antibodies isolated
from antisera of goats hyperimmunized with human myeloma proteins
containing k light chains. Antibodies were purified by affinity
chromatography and digested by pepsin to remove the Fc portion, to
avoid non-specific binding through Fc receptors.
Goat F(ab’)2 anti-human kappa allows the capture of k light chaincontaining
human IgA antibodies.
Goat anti-human kappa - HRP - Revelation antibody
Goat anti-human kappa-HRP is conjugated with horseradish peroxidase
to allow the detection of human IgA kappa light chain by ELISA.
Human IgA kappa - IgA light chain standard
Human IgA kappa is a human IgAk isotype control purified from human
myeloma serum.
IgA Heavy Chain for Sandwich ELISA
Goat F(ab’)2 anti-human IgA - Capture antibody
Goat F(ab’)2 anti-human IgA was generated from antibodies isolated from
antisera of goats hyperimmunized with human IgA paraproteins. Antibodies
were purified by affinity chromatography and digested by pepsin to remove
the Fc portion, to avoid non-specific binding through Fc receptors.
Goat F(ab’)2 anti-human IgA allows the capture of the heavy chain of
human IgA antibodies.
Goat anti-human IgA - HRP - Revelation antibody
Goat anti-human IgA-HRP is conjugated with horseradish peroxidase to
allow the detection of human IgA heavy chain by ELISA.
Human IgA from colostrum - IgA standard
Human IgA is isolated from pooled normal human colostrum by
fractionation and ion-exchange chromatography.
J Chain Antiserum
J chain antiserum was prepared by injection of purified human J chain
in rabbits. Human J chain is distinct from all other chain components
of polymeric IgA. J chain antiserum can be used for the detection of
dimeric IgA by Western blotting (working dilution 1:1,000).
Contents and Storage
J chain antiserum is provided as 100 μg lyophilized antiserum. J chain
antiserum is sterile and azide-free. Product is shipped at room
temperature and should be stored at -20°C or 4°C once resuspended.
Contents and Storage
Goat F(ab’)2 anti-human kappa is provided as 0.5 mg of purified
immunoglobulin in 1.0 ml of 100 mM borate buffered saline, pH 8.0.
Goat anti-human kappa-HRP is supplied as 1.0 ml of stock solution in
50% glycerol/50% PBS, pH 7.4.
Human IgA kappa is provided filtered sterile at a concentration of 0.5
mg/ml in 1.0 ml of 100 mM borate buffered saline, pH 8.0.
Products are shipped at room temperature and should be stored at 4°C.
PRODUCT QTY CAT. CODE
Goat F(ab’)2 anti-human kappa 0.5 mg fab-igak
Goat anti-human kappa - HRP 1 ml hrp-igak
Human IgA kappa 0.5 mg ctrl-igak
Contents and Storage
Goat F(ab’)2 anti-human IgA is provided as 0.5 mg of purified
immunoglobulin in 1.0 ml of 100 mM borate buffered saline, pH 8.0.
Goat anti-human IgA-HRP is supplied as 1.0 ml of stock solution in 50%
glycerol/50% PBS, pH 7.4.
Human IgA is supplied as an essentially salt-free, lyophilized powder.
Products are shipped at room temperature and should be stored at 4°C.
PRODUCT QTY CAT. CODE
Goat F(ab’)2 anti-human IgA 0.5 mg fab-iga
Goat anti-human IgA - HRP 1 ml hrp-iga
Human IgA 0.5 mg ctrl-iga
PRODUCT QTY CAT. CODE
J Chain Antiserum 100 μg pab-jc
www.invivogen.com/iga-detection 123
ANTIBODIES & VACCINATION 5

ANTIBODIES & VACCINATION 5
124
IgA Antibodies
InvivoGen provides human IgA antibodies that have been generated by recombinant DNA technology. These IgA antibodies are chimeric monoclonal
antibodies composed of the constant domains of the human IgA2 molecule and variable regions of different origins. They have been selected for
their ability to efficiently neutralize the biological activity of selected cytokines and Toll-Like Receptors. The neutralizing activity of these IgA
antibodies was determined using InvivoGen’s HEK-Blue reporter cells. Some of these IgA antibodies can also be used for flow cytometry.
ANTIBODY REACTIVITY APPLICATIONS QUANTITY CATALOG CODE
Anti-Cytokine IgAs
Anti-hCD40L-hIgA2 Human CD40L Neutralization 100 μg maba-hcd40l
Anti-hIFNa-hIgA2 Human IFN-a Neutralization, FC 100 μg maba-hifna
Anti-hIFNg-hIgA2 Human IFN-g Neutralization 100 μg maba-hifng
Anti-hIL-1b-hIgA2 Human IL-1b Neutralization 100 μg maba-hil1b
Anti-hIL-4-hIgA2 Human IL-4 Neutralization 100 μg maba-hil4
Anti-hIL-6-hIgA2 Human IL-6 Neutralization, FC 100 μg maba-hil6
Anti-hIL-10-hIgA2 Human IL-10 Neutralization 100 μg maba-hil10
Anti-hIL-13-hIgA2 Human IL-13 Neutralization, FC 100 μg maba-hil13
Anti-hIL-18-hIgA2 Human IL-18 Neutralization 100 μg maba-hil18
Anti-hTGFb-hIgA2 Human TGF-b Neutralization 100 μg maba-htgfb
Anti-hTNFa-hIgA2 Human TNF-a Neutralization, FC 100 μg htnfa-mab7
Anti-TLR IgAs
Anti-hCD14-hIgA2 Human CD14 Neutralization of TLR2 and TLR4 , FC 100 μg maba-hcd14
Anti-hTLR2-hIgA2 Human TLR2 Neutralization, FC 100 μg maba2-htlr2
Anti-hTLR3-hIgA2 Human TLR3 FC 100 μg maba-htlr3
Anti-hTLR4-hIgA2 Human TLR4 Neutralization, FC 100 μg maba-htlr4
Anti-hTLR5-hIgA2 Human TLR5 Neutralization, FC 100 μg maba2-htlr5
Control IgA
IgA2 Isotype Control Human IgA2 Control 100 μg maba2-ctrl
Contents and Storage
Each IgA antibody is provided lyophilized from a 0.2 μm filtered solution in PBS. Product should be reconstituted in sterile water. Lyophilized antibodies are
stable at least six months when stored at -20ºC. Reconstituted IgAs are stable 1 month when stored at 4ºC and 6 months when aliquoted and stored at -
20ºC.
Anti-Human IgA Secondary Antibodies
InvivoGen provides F(ab’)2 fragment secondary antibodies, to avoid
non-specific binding through Fc receptors, that react with human IgA.
These goat antibodies are conjugated with fluorescein (FITC) or biotin
for immunodetection or cell sorting applications.
Secondary anti-human IgA antibodies are supplied in 1 ml PBS/NaN3.
Store at 4ºC.
www.invivogen.com/iga-antibody
PRODUCT QTY CAT. CODE
Goat F(ab’)2 Anti-Human IgA - Biotin 500 μg chiga-biot
Goat F(ab’)2 Anti-Human IgA - FITC 500 μg chiga-fitc
Goat F(ab’)2 IgG Isotype Control - FITC 100 tests cgig-fitc

IgG Antibodies
InvivoGen offers a selection of monoclonal and polyclonal IgG antibodies. Most of them target pattern recognition receptors. These antibodies
have been generated by immunization of mice or rats with DNA or recombinant proteins or peptides. These antibodies can be used for different
applications. They have been tested in our laboratories for neutralization and/or flow cytometry, and for some of them for Western blot.
MAb-TLR antibodies are also available conjugated with FITC.
ANTIBODY DESCRIPTION REACTIVITY APPLICATIONS* QUANTITY CATALOG CODE
Anti-Dectin-1 IgG
MAb-mDectin-1 Mouse IgG2b (clone 2A11) Mouse Dectin-1 Neutralization, FC 100 μg mab-mdect
Anti-Flagellin IgG
Anti-Flagellin FliC Mouse IgG1 S. typhimurium flagellin WB 100 μg mabg-flic
Anti-HA Tag IgG
Anti-HA Tag Mouse IgG1 Influenza HA epitope WB, IP 250 μl ab-hatag
Anti-TLR IgGs
Anti-hTLR1-IgG Mouse IgG1 (H2G2) Human TLR1 Neutralization 100 μg mabg-htlr1
MAb-hTLR1 Mouse IgG1 (GD2.F4) Human TLR1 FC 100 μg mab-htlr1
MAb-hTLR1-FITC Mouse IgG1 (GD2.F4), FITC Human TLR1 FC 100 μg mab-htlr1f
PAb hTLR1 Polyclonal rat IgG Human TLR1 Neutralization 200 μg pab-hstlr1
Anti-mTLR2-IgG Mouse IgG2a (C9A12) Mouse TLR2 Neutralization 100 μg mabg-mtlr2
MAb-hTLR2 Mouse IgG2a (TL2.1) Human TLR2 FC, IHC, WB 100 μg mab-htlr2
MAb-hTLR2-FITC Mouse IgG2a (TL2.1), FITC Human TLR2 FC, IHC 100 μg mab-htlr2f
PAb hTLR2 Polyclonal rat IgG Human TLR2 Neutralization 200 μg pab-hstlr2
MAb-mTLR2 Mouse IgG1 (T2.5) Human/mouse TLR2 Neutralization , FC, IHC 100 μg mab-mtlr2
MAb-mTLR2-FITC Mouse IgG1 (T2.5), FITC Human/mouse TLR2 FC 100 μg mab-mtlr2f
MAb-hTLR3 Mouse IgG1 (TLR3.7) Human TLR3 FC, WB 100 μg mab-htlr3
MAb-hTLR3-FITC Mouse IgG1 (TLR3.7), FITC Human TLR3 FC 100 μg mab-htlr3f
MAb-hTLR4 Mouse IgG2a (HTA125) Human/monkey TLR4 FC, IHC 100 μg mab-htlr4
MAb-hTLR4-FITC Mouse IgG2a (HTA125), FITC Human/monkey TLR4 FC 100 μg mab-htlr4f
PAb hTLR4 Polyclonal rat IgG Human TLR4 Neutralization 200 μg pab-hstlr4
MAb-mTLR4/MD2 Rat IgG2a (MTS510) Mouse TLR4/MD2 FC, IHC 100 μg mab-mtlr4md2
MAb-mTLR4/MD2-FITC Rat IgG2a (MTS510), FITC Mouse TLR4/MD2 FC 100 μg mab-mtlr4md2f
PAb hTLR5 Polyclonal rat IgG Human TLR5 Neutralization 200 μg pab-hstlr5
Anti-mTLR5-IgG Rat IgG2a (Q23D11) Mouse TLR5 Neutralization 100 μg mabg-mtlr5
Anti-hTLR6-IgG Mouse IgG1 (C5C8) Human TLR6 Neutralization 100 μg mabg-htlr6
PAb hTLR6 Polyclonal rat IgG Human TLR6 Neutralization 200 μg pab-hstlr6
MAb-mTLR9 Mouse IgG2a (5G5) Human/mouse TLR9 FC, IHC, WB 100 μg mab-mtlr9
MAb-mTLR9-FITC Mouse IgG2a (5G5), FITC Human/mouse TLR9 FC 100 μg mab-mtlr9f
* FC, flow cytometry; IHC, immunohistochemistry; IP, immunoprecipitation; WB, Western blot
Contents and Storage
All IgG antibodies are provided lyophilized. They are shipped at room temperature. Store at -20°C.
www.invivogen.com/igg-antibody
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ANTIBODIES & VACCINATION 5

ANTIBODIES & VACCINATION 5
Vaccine Adjuvants
Adjuvants are essential for enhancing and directing the adaptative immune
response to vaccine antigens. This response is mediated by two main types
of lymphocytes, B and T cells. Upon activation by cytokines, B cells
differentiate into memory B cells (long-lived antigen-specific B cells) or
plasma cells (effector B cells that secrete large quantities of antibodies).
Most antigens activate B cells using activated T helper (Th) cells, primarily
Th1 and Th2 cells. Th1 cells secrete IFN-g, which activates macrophages
and induces the production of opsonizing antibodies by B cells. The Th1
response leads mainly to a cell-mediated immunity (cellular response),
which protects against intracellular pathogens (invasive bacteria, protozoa
and viruses). The Th1 response activates cytotoxic T lymphocytes (CTL), a
sub-group of T cells, which induce death of cells infected with viruses and
other intracellular pathogens. Natural killer (NK) cells are also activated by
the Th1 response, these cells play a major role in the induction of apoptosis
in tumors and cells infected by viruses. Th2 cells secrete cytokines, including
IL-4, which induces B cells to make neutralizing antibodies. Th2 cells
generally induce a humoral (antibody) response critical in the defense
against extracellular pathogens (helminthes, extracellular microbes and
toxins). The magnitude and type of Th response to a vaccine can be greatly
modulated through the use of adjuvants. For almost 80 years, aluminium
salts (referred to as ‘alum’) have been the only adjuvant in use in human
vaccines. Only in the last two decades, have novel adjuvants (MF59, AS04)
been introduced in the formulation of new licensed vaccines. As our
understanding of the mechanisms of ‘immunogenicity’ and ‘adjuvancy’
increases, new adjuvants and adjuvant formulations are being developed.
Mechanisms of adjuvants
Adjuvants may exert their effects through different mechanisms. Some
adjuvants, such as alum and emulsions (e.g. MF59), function as delivery
systems by generating depots that trap antigens at the injection site,
providing slow release in order to continue the stimulation of the immune
system. These adjuvants enhance the antigen persistence at the injection
site and increase recruitment and activation of antigen presenting cells
(APCs). Particulate adjuvants (e.g. alum) have the capability to bind antigens
to form multi-molecular aggregates which will encourage uptake by APCs 1 .
Some adjuvants are also capable of directing antigen presentation by the
major histocompatibility complexes (MHC) 1 . Other adjuvants, essentially
ligands for pattern recognition receptors (PRR), act by inducing the innate
immunity, predominantly targeting the APCs and consequently influencing
the adaptative immune response. Members of nearly all of the PRR families
are potential targets for adjuvants. These include Toll-like receptors (TLRs),
NOD-like receptors (NLRs), RIG-I-like receptors (RLRs) and C-type lectin
receptors (CLRs). They signal through pathways that involve distinct adaptor
molecules leading to the activation of different transcription factors. These
transcription factors (NF-kB, IRF3) induce the production of cytokines and
chemokines that play a key role in the priming, expansion and polarization
of the immune responses. Activation of some members of the NLR family,
such as NLRP3 and NLRC4, triggers the formation of a protein complex,
called inflammasome, implicated in the induction of the pro-inflammatory
cytokines IL-1b 2 and IL-18. The NLRP3 and NLRC4 inflammasomes have
been involved in the innate immunity induced by certain adjuvants but their
mechanism of action remains unclear.
Alum & emulsions
Alum is the most commonly used adjuvant in human vaccination. It is found
in numerous vaccines, including diphtheria-tetanus-pertussis, human
papillomavirus and hepatitis vaccines 3 . Alum provokes a strong Th2
response, but is rather ineffective against pathogens that require Th1–cellmediated
immunity. Alum induces the immune response by a depot effect
and activation of APCs. Recently, the NLRP3 inflammasome has been linked
to the immunostimulatory properties of alum 2 although its role in adjuvantinduced
antibody responses remains controversial.
126 www.invivogen.com/vaccine-adjuvants
Emulsions (either oil-in-water or water-in-oil), such as Freund’s Incomplete
Adjuvant (IFA) and MF59, can trigger depot generation and induction of
MHC responses IFA induces a predominantly Th2 biased response with
some Th1 cellular response. MF59 is a potent stimulator of both cellular
(Th1) and humoral (Th2) immune responses 4 . However, the precise mode
of action of emulsion-based adjuvants is still unclear. A complication with
emulsion-based adjuvants is their potential to induce autoimmunity.
PRR Ligands
As classical adjuvants induce strong Th2 response with little or no Th1
response, the current challenge is to develop adjuvants which induce a
strong Th1 bias important for vaccines against hepatitis, flu, malaria, and HIV.
New adjuvants are being developed that are natural ligands or synthetic
agonists for PRRs, either alone or with various formulations. PRR activation
stimulates the production of pro-inflammatory cytokines/chemokines and
type I IFNs that increase the host’s ability to eliminate the pathogen. Thus,
the incorporation of pathogens associated molecular patterns (PAMPs) in
vaccine formulations can improve and accelerate the induction of vaccinespecific
responses. A number of these agonists are now in clinical or late
preclinical stages of development for hepatitis and human papillomavirus
vaccines 5, 6 . When used in combination with alum or classical emulsion
adjuvants, the immune response can be biased towards a Th1 response 7 .
TLR3 and RLR Ligands: Double-stranded RNA (dsRNA), which is
produced during the replication of most viruses, is a potent inducer of
innate immunity. Synthetic analogs of dsRNA, such as poly(I:C), have been
tested as adjuvants. They act through TLR3 and RIG-I/MDA-5, inducing
IL-12 and type I IFNs production, facilitating antigen cross-presentation to
MHC class II molecules, and improving generation of cytotoxic T cells 8 .
TLR4 Ligands: Bacterial lipopolysaccharides (LPS), which are ligands for
TLR4, have long been recognized as potent adjuvants, but their pyrogenic
activity have prevented their clinical use. The development of less toxic
derivatives led to the production of MPLA (monophosphoryl lipid A),
which formulated with alum (AS04) triggers a polarized Th1 response and
is approved for clinical use in Europe 8 .
TLR5 Ligands:The TLR5 ligand, bacterial flagellin, is a potent T-cell antigen
and has potential as a vaccine adjuvant. Unlike other TLR agonists, flagellin
tends to produce mixed Th1 and Th2 responses rather than strongly Th1
responses. Flagellin can be used as an adjuvant mixed with the antigen but
it is more frequently fused to a recombinant vaccine antigen 9, 10 .
TLR7/8 Ligands:The TLR7/8 pathway, specialized in the recognition of single
stranded viral RNA, has demonstrated promising pre-clinical results as a
target for potential vaccine adjuvants. Imidazoquinolines (i.e. imiquimod and
R848) are synthetic componds that activate TLR7/8 in multiple subsets of
dendritic cells leading to the production of IFN-a and IL-12 thus promoting
a Th1 response 5 .
TLR9 Ligands: Oligodeoxynucleotides containing specific CpG motifs (CpG
ODNs) are recognized by TLR9. They enhance antibody production and
strongly polarize Th cell responses to Th1 and away from Th2 responses 11,12 .
NOD2 Ligands: Fragments of bacterial cell walls, such as muramyl
dipeptide (MDP), have long been recognized as adjuvants. More recently,
it was discovered that MDP triggers the activation of NOD2 and the
NLRP3 inflammasome 13 .
Adjuvants may be combined to achieve a stronger effect or a more potent
skewing of immune responses. Currently, much effort is devoted to
combining alum with TLR9 agoinsts 14 . In experimental models,
administration of other combinations such as CpG ODNs with MDP or
MPLA has proven very effective 15 . Adjuvants currently in clinical use
enhance humoral responses but new adjuvants in clinical and preclinical
trials are focused on generating multifaceted immune responses. The PRR
pathways are an attractive source of novel adjuvants for vaccines due to
their ability to induce strong cell-mediated immunity.

Mediators?
1. Leroux-Roels G., 2010. Unmet needs in modern vaccinology adjuvants to imporve the immune response. Vaccine. 28S(3):C25-3. 2. Li H. et al., 2008. Cutting edge:
Inflammasome activation by alum and alum's adjuvant effect are mediated by NLRP3. J Immunol. 181(1):17-21. 3. Marrack P. et al., 2009. Towards an understanding of
the adjuvant action of aluminium. Nat Rev Immunol. 9(4):287-93. 4. Ott G. et al., 1995. MF59. Design and evaluation of a safe and potent adjuvant for human vaccines.
Pharm Biotechnol 6: 277-96. 5. Steinhagen F. et al., 2010. TLR-based immune adjuvants. Vaccine [Epub ahead of print]. 6. Mbow ML. et al., 2010. New adjuvants for
human vaccines. Curr Opin Immunol. 22(3):411-6. 7. Didierlaurent A. ,et al., 2009. AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient
localized innate immune response leading to enhanced adaptive immunity. J Immunol 183(10): 6186-97. 8. Coffman R. et al., 2010. Vaccine adjuvants: Putting innate
immunity to work. Immunity 33(4):492-503. 9. Huleatt J. et al., 2007.Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid
cellular and humoral immunity. Vaccine 25(4): 763-75. 10. Mizel S. & Bates JT., 2010. Flagellin as an adjuvant: Cellular mechanisms and potential. J Immunol. 175(10):5677-
82. 11. Kobayashi H. et al., 1999. Immunostimulatory DNA pre-priming: a novel approach for prolonged Th1-biased immunity. Cell Immunol 198(1): 69-75. 12. Tighe H.
et al., 2000. Conjugation of immunostimulatory DNA to the short ragweed allergen amb a 1 enhances its immunogenicity and reduces its allergenicity. J Allergy Clin
Immunol 106: 124-34. 13. Girardin S. et al., 2003. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 278(11):8869-
72. 14. Siegrist C. et al., 2004. Co-administration of CpG oligonucleotides enhances the late affinity maturation process of human anti-hepatitis B vaccine response.
Vaccine 23(5): 615-22. 15. Kim S. et al., 2000. Effect of immunological adjuvant combinations on the antibody and T-cell response to vaccination with MUC1-KLH and
GD3-KLH conjugates. Vaccine 19: 530-7.
www.invivogen.com/vaccine-adjuvants 127
ANTIBODIES & VACCINATION 5

ANTIBODIES & VACCINATION 5
Vaccine Adjuvants NEW
InvivoGen provides different classes of vaccine adjuvants that are either already approved for use in human vaccination, such as alum, or under
investigation such as the TLR agonists gardiquimod and CpG oligonucleotides. InvivoGen adjuvants are VacciGrade (preclinical grade). They
are prepared under strict aseptic conditions and tested for the presence of endotoxins. They are sterile and their endotoxin level is

Poly(I:C)
Synthetic double-stranded RNA, namely poly(I:C), can activate the immune response through two distinct PRRs 2 . Endosomal poly(I:C) activates TLR3 while
cytosolic poly(I:C) activates RIG-I/MDA-5. Triggering the TLR3 pathway induces IL-12 and type I IFNs production, and improves MHC class II expression and
cross-presentation of antigen 2 . Stimulation of MDA-5 enhances the production of type I IFNs that play a critical role in enhancing T and B cell immunity 2 .
Poly(I:C) promotes Th1 biased immunity through its induction of IL-12 and type I IFNs 2 . Promising results have been obtained using poly(I:C) as an adjuvant
in flu vaccine delivered intranasally 9 .
MPLA
The TLR4 agonist, MPLA (monophosphoryl lipid A) is a derivative of lipid A from Salmonella minnesota R595 lipopolysaccharide (LPS or endotoxin). MPLA
is considerably less toxic than LPS whilst maintaining the immunostimulatory activity 10 . When tested in animals models as a vaccine adjuvant, MPLA induces
a strong Th1 response 11 . The adjuvant, AS04, currently approved for use in vaccines against human papilloma virus and hepatitis B, contains MPL (a modified
MPLA) combined with aluminum salt 12 .
N-glycolyl-MDP
MDP (muramyl dipeptide), is the minimal bioactive peptidoglycan motif common to all bacteria and the essential structure required for adjuvant activity in
vaccines. MDP has been shown to be recognized by NOD2, but not TLR2, nor TLR2/1 or TLR2/6 associations 13 . The cell wall of mycobacteria is known to
be extremely immunogenic. This potent activity is attributed to their MDP which is N-glycolylated in contrast to the MDP of most bacteria which is
N-acetylated. N-glycolyl-MDP has been reported to display a stronger NOD2-mediated activity than N-acetyl-MDP and thus to be a more potent vaccine
adjuvant than N-acetyl-MDP 14 . Furthermore MDP leads to the activation of the NLRP3 inflammasome 15 .
ODN 1826 and ODN 2006
Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs), such as ODN 2006 (also known as ODN 7909), have been extensively
studied as adjuvants. CpG ODNs are recognized by TLR9, which is expressed exclusively on human B cells and plasmacytoid dendritic cells (pDCs), thereby
inducing Th1-dominated immune responses. Pre-clinical studies conducted in rodents and non-human primates and human clinical trials have demonstrated that
CpG ODNs can significantly improve vaccine-specific antibody responses 9 . Among the three classes of CpG ODNs identified (see page 86), type B CpG ODNs
are the most studied.
1. Mbow ML. et al., 2010. New adjuvants for human vaccines. Curr Opin Immunol. 22(3):411-6. 2. Coffman RL. et al., 2010. Vaccine adjuvants: Putting innate immunity to work. Immunity
33(4):492-503. 3. Marrack P. et al., 2009. Towards an understanding of adjuvant action of aluminium. Nat Rev Immunol. 9(4): 287-93. 4. Petrovsky N. & Aguilar JC., 2004. Vaccine adjuvants:
Current state and future trends. Immunol Cell Biol. 82(5): 488-96. 5. Moreira L0. et al., 2008. Modulation of adaptive immunity by different adjuvant-antigen combinations in mice lacking
Nod2. Vaccine 26(46): 5808-13. 6. Huleatt J. et al., 2007. Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity.
Vaccine 25(4): 763-75. 7. Skountzou I. et al., 2010. Salmonella flagellins are potent adjuvants for intranasally administered whole inactivated influenza vaccine. Vaccine 28(4): 4103-12. 8. Miao
EA. & Warren SE., 2010. Innate immune detection of bacterial virulence factors via the NLRC4 inflammasome. J Clin Immunol. 30(4): 502-6. 9. Steinhagen F. et al., 2010. TLR-based immune
adjuvants. Vaccine [Epub ahead of print]. 10. Casella CR. et al., 2008. Putting endotoxin to work for us: monophosphoryl lipid A as a safe and effective vaccine adjuvant. Cell Mol Life Sci. 65(20):3231-
40. 11. Fransen F. et al., 2007. Agonists of Toll-like receptors 3, 4, 7, and 9 are candidates for use as adjuvants in an outer membrane vaccine against Neisseria meningitidis serogroup . Infect Immun.
75(12) :5939-46. 12. Didierlaurent A. et al., 2009. AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced
adaptive immunity. J Immunol 183(10): 6186-97. 13. Girardin S. et al., 2003. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 278(11):8869-
72. 14. Coulombe F. et al., 2009. Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide. J Exp Med. 206(8):1709-16. 15. Martinon F. et al., 2004. Identification of bacterial
muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome. Curr Biol 14 (21): 1929-34.
OVA Antigens NEW
Ovalbumin (OVA) is a key reference protein for immunization and biochemical studies (Western blots, ELISA). Commercially available ovalbumin
is often contaminated with endotoxins altering the results in vivo. InvivoGen provides two grades of ovalbumin and two standard OVA peptides.
• EndoFit Ovalbumin: Endotoxin level < 1 EU/mg, for in vivo use,
minimum 98% protein
• Ovalbumin: for detection use (Western blot, ELISA), minimum 98%
protein
• OVA 257-264: an H-2Kb-restricted OVA class I epitope, for
detection use (ELISPOT) - Sequence : SIINFEKL (MW 963.2)
• OVA 323-339: an H-2b-restricted OVA class II epitope, for detection
use (ELISPOT) - Sequence : ISQAVHAAHAEINEAGR (MW 1773.9)
Contents and Storage
Products are provided lyophilized and shipped at room temperature.
Store at 4°C or -20°C according to the product label.
PRODUCT QTY CAT. CODE
EndoFit Ovalbumin 10 mg vac-efova
Ovalbumin 1 g vac-ova
OVA 257-264 1 mg vac-sin
OVA 323-339 1 mg vac-isq
www.invivogen.com/vaccine-adjuvants 129
ANTIBODIES & VACCINATION 5

ANTIBODIES & VACCINATION 5
pBOOST - DNA Vaccine Adjuvants
pBOOST plasmids were developed as genetic adjuvants for DNA vaccines to potentiate the immune response to a specific antigen. The method
of plasmid DNA vaccine delivery is known to bias the immune response to a specific antigen towards aTh1 or Th2 response. These biases can
be further enhanced by the codelivery of interferon regulatory factors (IRFs) orTANK-binding kinase 1 (TBK1) to increase the efficacy of the
vaccination.
Description
pBOOST2-mIRF - Th1 or Th2 polarization
IRF-1, IRF-3 and IRF-7 have been shown to be able to bias T cells towards
type 1 or type 2 immune responses, leading to the activation of cytotoxic
T cells and/or the production of antibodies.
pBOOST2-mIRF plasmids carry the murine IRF-1, IRF-3 or IRF-7 genes.
These genes are either wild-type or mutant.
• Wild-type IRF-1: IRF-1 primarily increases Th2 antibody responses 1 .
Following intramuscular or gene gun injections of DNA vaccines, IRF-1 can
increase the titers of antibodies up to 10-fold.
• Mutant IRF-3: IRF-3 primarily increases Th1 T-cell responses 1 . A
constitutively active form of IRF-3 was generated by creating a single point
mutation of Ser 396 to Asp. This super-activated IRF-3 presents a >10-fold
enhanced transactivating potential over the wild-type IRF-3 for the IFN-a
and IFN-b promoters 2 .
• Chimeric IRF7/3: IRF-3 and IRF-7 increase both Th1 and Th2 responses
by transactivating different target promoters 1 . To exploit the biological
features of both IRFs, a chimeric form of IRF-7 and IRF-3 was generated
by combining the DNA binding specificity of IRF-7 with the strong
transactivation capacity of super-activated IRF-3. IRF-7/3 chimera provides
>10-fold greater induction of IFN-a and IFN-b promoters than superactivated
IRF-3 alone 3 .
pBOOST2-mIRF plasmids express the transgene under the control of the
strong and ubiquitous EF-1a/HTLV composite promoter and are selectable
in E. coli with Zeocin .
pBOOST3-mTBK1 - Enhancement of DNA vaccine immunogenicity
pBOOST3-mTBK1 plasmid expresses the mouse TBK1 gene. TANKbinding
kinase 1 (TBK1), a non-canonical IkB kinase, was recently shown
to mediate the adjuvant effect of DNA vaccines 4 . Administration of DNA
vaccines induces the production of type I interferons and inflammatory
cytokines in a CpG-independent manner but in a TBK1-dependent manner.
Therefore, co-administration of a TBK1-expressing plasmid is expected to
further boost DNA vaccine-induced immunogenicity.
pBOOST3-mTBK1 contains the EF-1a/HTLV composite promoter
coupled to the SV40 enhancer and is selectable in E. coli with Zeocin .
1. Sasaki S. et al., 2002. Regulation of DNA-raised immune responses by cotransfected
interferon regulatory factors. J Virol.76(13):6652-9. 2. Servant MJ. et al., 2003.
Identification of the minimal phosphoacceptor site required for in vivo activation of
interferon regulatory factor 3 in response to virus and double-stranded RNA. J Biol
Chem. 278(11):9441-7. 3. Bramson JL. et al., 2003. Super-activated interferon-regulatory
factors can enhance plasmid immunization. Vaccine. 21(13-14):1363-70. 4. Ishii KJ. et al.,
2008. TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA
vaccines. Nature 451: 725-729
Contents and Storage
130 www.invivogen.com/dna-vaccination
Each pBOOST plasmid is provided as 20 μg of lyophilized DNA. Product
is shipped at room temperature. Store at -20°C. Plasmids are stable up to
one year when properly stored. pBOOST plasmids come with 4 pouches
of E. coli Fast-Media ® Zeo (2 TB and 2 Agar, see pages 44-45).
PRODUCT QTY CAT. CODE
pBOOST2-wtmIRF1 20 μg pbst2-wtmirf1
pBOOST2-samIRF3 20 μg pbst2-samirf3
pBOOST2-samIRF7/3 20 μg pbst2-samirf73
pBOOST2-mcs (control) 20 μg pbst2-mcs
pBOOST3-mTBK1 20 μg pbst3-mtbk1
pBOOST3-mcs (control) 20 μg pbst3-mcs

pVAC - Induction of Neutralizing Antibodies
pVAC is a DNA vaccine plasmid family specifically designed to stimulate a humoral immune response by intramuscular injection. Antigenic proteins
are targeted and anchored to the cell surface by cloning the gene of interest in frame between the IL2 signal sequence and the C-terminal
transmembrane anchoring domain of human placental alkaline phosphatase. The antigenic peptide produced on the surface of muscle cells is thought
to be taken up by antigen presenting cells (APCs) and processed through the major histocompatibility complex (MHC) class II pathway1, 2, 3 .
Description
Cell Surface Expression of the Antigenic Protein
Two pVAC backbones are available:
• pVAC1-mcs is designed for the cloning of an antigenic gene that is not
naturally secreted. The MCS is flanked by the 5’ IL2 signal sequence and
the 3’ glycosylphosphatidylinositol (GPI) anchoring domain of placental
alkaline phosphatase (PLAP).
• pVAC2-mcs is designed for the cloning of an antigenic gene that already
possesses a signal sequence. The MCS is located upstream of the GPI
anchoring domain of PLAP.
High-level Expression of the Antigenic Gene
pVAC plasmids utilize the promoter region of the rhesus monkey EF1a
gene to achieve high levels of expression in skeletal muscle cells and antigen
presenting cells. Expression levels are further increased by the addition of
the SV40 enhancer that heightens the ability of the plasmid to be
transported into the nucleus, especially in non-dividing cells.
Sustained Expression of the Antigenic Gene
The bacterial region required for replication and selection of pVAC in
E. coli contains a reduced number of immunogenic CpG motifs by featuring
a minimized origin of replication (Ori) and a CpG-free Zeocin -resistance
gene (Sh-DCpG).
Convenient Cloning of the Antigenic Gene
pVAC contains a multiple cloning site (MCS) with several commonly used
restriction sites.
1. Corr M. et al. 1999. In vivo priming by DNA injection occurs predominantly by antigen
transfer. J Immunol. 163(9):4721-7. 2. Forns X. et al. 1999. DNA immunization of mice and
macaques with plasmids encoding hepatitis C virus envelope E2 protein expressed
intracellularly and on the cell surface. Vaccine 17:1992-2002. 3. McCluskie MJ et al. 1999.
Route and method of delivery of DNA vaccine influence immune responses in mice and
non-human primates. Mol Med 5:287-300.
Contents and Storage
Each pVAC plasmid is provided as 20 μg of lyophilized DNA. Product is
shipped at room temperature and should be stored at -20°C. Plasmid is
stable up to one year when properly stored.
Each pVAC is provided with 4 pouches of E. coli Fast-Media ® Zeo (2 TB
and 2 Agar, see pages 44-45).
Ori
Ori
www.invivogen.com/dna-vaccination
EM7 Sh∆CpG
EF1 pAn
EM7 Sh∆CpG
SV40
SV40
IL2 ss
rh EF1α
Intron
5’- Bam HI, Eco RV, Bgl II, Eco RI -3’
rh EF1α
5’- Bam HI, Eco RV, Bgl II, Eco RI -3’
PRODUCT QTY CAT. CODE
pVAC1-mcs 20 μg pvac1
pVAC2-mcs 20 μg pvac2
131
ANTIBODIES & VACCINATION 5

6
CpG-FREE DNA
CpG-Free Plasmids 134-137
CpG-Free Genes 138-139

CpG-Free DNA
CpGs and the Immune Response
Bacterial DNA is rich in unmethylated 2’-deoxyribo (cytidine-phosphateguanosine)
(CpG) dinucleotides, in contrast to mammalian DNA which
contains a low frequency of CpG dinucleotides that are mostly
methylated. Unmethylated CpGs in specific sequence contexts activate
the vertebrate immune system via Toll-Like Receptor (TLR) 9. TLR9
recognizes CpG DNA and initiates a signaling cascade leading to the
production of proinflammatory cytokines such as IL-6 and IL-12 1 .
Plasmids used for in vivo experiments are produced in E. coli and therefore
their CpGs are unmethylated and induce immune responses through this
host defense mechanism, which represents a limitation for the clinical
development of DNA vaccines and gene therapy vectors.
CpGs and Gene Silencing
A major limitation of gene delivery vectors for gene therapy applications is
the rapid decline of transgene expression in vivo. Methylation of CpG
dinucleotides within the promoter is a major factor limiting long-lasting gene
expression. Indeed, the transcriptional activity of the widely used and CpGrich
cytomegalovirus immediate early gene promoter (CMV) is highly robust
but prone to inactivation within a few weeks 2 . Replacement of the CMV
promoter with a cellular promoter combined with the use of a CpG-reduced
backbone was shown by our team (figures 1 & 2) and other labs to increase
the duration of expression and lower the inflammatory response 3, 4 . Recently,
CpGs in plasmid DNA (pDNA) were found to enhance the clearance of
PEG-coated pDNA-lipoplexes, a phenomenon that could be reduced by the
use of CpG-free pDNA allowing repeated dosing 5 .
Construction of CpG-free Plasmids
InvivoGen has developed a new family of plasmids that are completely
devoid of CpG dinucleotides, named pCpGfree. pCpGfree plasmids
contain elements that either naturally lack CpG dinucleotides, were
modified to remove all CpGs, or entirely synthesized such as genes
encoding selectable markers or reporters. These plasmids yield high levels
of transgene expression both in vitro and in vivo, and in contrast to CMVbased
plasmids allow sustained expression in vivo (figure 1) 6 .
Applications of CpG-free Plasmids
A major application of CpG-free plasmids is the treatment of inherited
diseases cause by a single gene defect, such as cystic fibrosis and hemophilia,
through gene therapy. Hyde et al. have reported promising results for the
treatment of cystic fibrosis using a pCpGfree-derived plasmid 4 . They show
that a CpG-free pDNA can achieve sustained lung transgene expression in
contrast to a pDNA containing even a single CpG. These results have led
to a clinical trial that has started in February 2009.
pCpGfree plamids represent valuable tools to study the effects of CpGs on
gene expression using cell lines expressing TLR9 7 , as well as their effects on
the innate and acquired immune systems. Furthermore, pCpGfree plasmids
are also useful when analyzing promoter methylation 8, 9 .
1. Bauer S. et al., 2001. Human TLR9 confers responsiveness to bacterial DNA via speciesspecific
CpG motif recognition. PNAS USA. 98(16):9237-42. 2. Scharfmann R. et al., 1991.
Long-term in vivo expression of retrovirus-mediated gene transfer in mouse fibroblast implants.
PNAS U S A. 88(11):4626-30. 3. Yew NS. et al., 2001. High and sustained transgene expression
in vivo from plasmid vectors containing a hybrid ubiquitin promoter. Mol Ther. 4(1):75-82.
4. Hyde SC. et al., 2008. CpG-free plasmids confer reduced inflammation and sustained
pulmonary gene expression. Nat Biotechnol. 26(5):549-51. 5. Tagami T. et al., 2010. CpG motifs
in pDNA-sequences increase anti-PEG IgM production induced by PEG-coated pDNAlipoplexes.
J Control Release. 142(2):160-6 6. Hattori K. et al., 2010. Sustained Exogenous
Expression of Therapeutic Levels of IFN-{gamma} Ameliorates Atopic Dermatitis in NC/Nga
Mice via Th1 Polarization. J Immunol. 184(5):2729-35. 7.Yasuda K. et al., 2009. Requirement for
DNA CpG content in TLR9-dependent dendritic cell activation induced by DNA-containing
immune complexes. J Immunol. 183(5):3109-17. 8. Klug M & Rehli M., 2006. Functional analysis
of promoter CpG methylation using a CpG-free luciferase reporter vector. Epigenetics. 1(3):127-
30. 9. van Straten EM. et al., 2009. The Liver X-Receptor (LXR) gene promoter is
hypermethylated in a mouse model of prenatal protein restriction. Am J Physiol Regul Integr
Comp Physiol. 282(2):R275-82.
Luciferase (μg/ml)
Figure 1. Time course of luciferase expression. Plasmids (30 μg) expressing a CpGfree
luciferase (ssLuc) gene and containing different amounts of CpGs in their
backbone were hydrodynamically injected in Swiss mice. Luciferase expression was
evaluated at different time points. Expression of ssLuc from pcDNA3-CMV (CpGrich
plasmid with CMV promoter) peaked at day 1 after injection but had drastically
fallen at day 4 to reach background levels by day 8. In contrast, ssLuc expression
from pCpGfree-SLS (CpG-free plasmid with a CpG-free engineered albumin
promoter) was high and persisted to day 47. Rapid reduction of luciferase
expression was also observed with pCpGfree-CMV (CpG-free plasmid with CMV
promoter) and pcDNA3-SLS (CpG-rich plasmid with CpG-free promoter).
Figure 2. DNA-induced immune response. Mice were injected i.p. with 500 μg
CpG-rich or CpG-free DNA and the levels of serum TNF-a assessed 4h postinjection.
As expected CpG-free DNA (pCpGfree-mcs and genomic calf DNA)
induced negligeable amounts of TNF-a whereas CpG-rich DNA (pDNA-CMV, CpG
ODN1826 and E. coli DNA) induced significant amounts of TNF-a. All DNAs
injected were endotoxin-free.
A B
Figure 3. LacZ expression using a pCpGfree plasmid. C57Bl6 mice were injected
with 30 μg pCpGfree-mcs (A) or pCpGfree-LacZ (B) plasmids using hydrodynamic
delivery. The livers were harvested 4 days post-inoculation and stained to determine
the expression of the CpG-free LacZ gene.
www.invivogen.com/cpgfree-dna 133
CpG-FREE DNA 6

CpG-FREE DNA 6
pCpGfree - CpG-free Plasmids
Description
CpG-free Plasmid Backbone
pCpGfree is a family of plasmids completely devoid of CpG dinucleotides.
Typically, the elements required for replication and selection of the plasmid
in E. coli and gene expression in mammalian cells are rich in CpG. In the
pCpGfree plasmids these elements are either naturally CpG-free, were
modified to remove all CpGs, or entirely synthesized.
- Origin of replication: The E. coli R6K gamma ori has been modified
to remove all CpGs. This origin is activated by the R6K specific initiator
protein π, encoded by the pir gene 1 .
- Bacterial promoter: EM2K is a CpG-free version of the bacterial EM7
promoter.
- Mammalian promoter: The CpG-free promoter combines the mouse
CMV enhancer, the human elongation factor 1 alpha core promoter and
5’UTR containing a synthetic intron.
- Polyadenylation signal: The polyadenylation signal is a CpG-free form
of the late SV40 polyadenylation signal.
- MAR: Matrix attached regions (MARs) are sequences typically AT-rich
that are able to form barriers between independently regulated domains 2 .
pCpG plasmids contain two MARs, from the 5’ region of the human IFNb
gene or b-globin gene that were chosen because they are naturally CpGfree.
The MARs are placed between the bacterial and mammalian
transcription units.
- Selectable marker: The Zeocin resistance gene is a small gene (

pCpGfree-basic & pCpGfree-promoter NEW
Promoter CpG Methylation Study
Methylation of CpG dinucleotides within the promoter region of genes is often associated with transcriptional
silencing. This epigenetic event plays an important role in the regulation of gene activity in normal and cancer
cells. InvivoGen provides pCpGfree-basic and pCpGfree-promoter, two SEAP reporter plasmids completely devoid
of CpG dinucleotides, that allow to study the effect of promoter CpG methylation in transfection assays. The lack
of CpGs within the plasmid backbone limits in vitro CpG methylation to the CpG dinucleotides present in the
inserted promoter fragment. Thus, the pCpGfree-basic and pCpGfree-promoter plasmids represent useful tools
to analyze the effect of DNA methylation on CpG-containing promoters.
Description
The pCpGfree-basic and pCpGfree-promoter plasmids derive from the
pCpGfree-mSEAP plasmid by deletion of the enhancer/promoter region
or the enhancer sequence alone, respectively.
pCpGfree-basic
The pCpGfree-basic plasmid lacks the entire promoter region (i. e. murine
CMV enhancer and human EF-1a promoter). It contains a multiple cloning
site upstream of the murine secreted embryonic alkaline phosphatase
(mSEAP) reporter gene. Expression of mSEAP in cells transfected with
this plasmid depends on the insertion of a functional promoter or
enhancer/promoter cassette upstream from the mSEAP gene. Thus,
pCpGfree-basic allows to study the effect of CpG methylation on a
promoter, alone or combined with enhancer elements.
pCpGfree-promoter
The pCpGfree-promoter plasmid contains the human EF-1a promoter
and a multiple cloning site in place of the murine CMV enhancer. It is
specifically designed to analyze the effect of methylation on CpG residues
present in enhancer elements.
Procedure
1- Insert promoter and/or enhancer fragment into
pCpGfree-basic or pCpGfree-promoter.
2- Treat recombinant plasmid with CpG methylase (Sss I) or
site specific-methylase (Hha I, Hpa II).
3- Transiently transfect cells with methylase-treated or
untreated plasmid.
4- Analyze promoter CpG methylation by determining
mSEAP expression using QUANTI-Blue , a SEAP detection
medium.
Contents and Storage
pCpGfree-basic and pCpGfree-promoter plasmids are provided as 20 μg
of lyophilized DNA with a paper disk of lyophilized E. coli GT115 strain.
Products are shipped at room temperature and should be stored at -20°C.
Zeo R MAR EF1 prom
Ori EM2K MCS SI126
pCpGfree-promoter
MAR pAn mSEAP
PRODUCT QUANTITY CAT. CODE
pCpGfree-basic NEW 20 μg pcpgf-bas
pCpGfree-promoter NEW 20 μg pcpgf-prom
SdaI
Bsp120I
AvrII
NsiI
Ppu10I
ScaI
BamHI
SpeI
HindIII
BsrGI
AflIII
BbsI
SdaI
Bsp120I
AvrII
NsiI
Ppu10I
ScaI
BamHI
SpeI
HindIII
BsrGI
AflIII
BbsI
Important Licensing Information: These products are covered by a Limited Use License (See Terms and Conditions on page 164). By use of these products you
accept the terms and conditions of all applicable Limited Use Label Licenses.
www.invivogen.com/cpgfree-plasmid 135
CpG-FREE DNA 6

CpG-FREE DNA 6
pCpGfree-vitro - Selectable CpG-free Plasmids
Description
CpG-free Plasmid Backbone
pCpGfree-vitro is a family of expression vectors completely devoid of
CpG dinucleotides that are selectable in mammalian cells. Similarly to
the other pCpGfree plasmids, all the elements required for replication
and selection of the plasmids in bacteria, and gene expression in
mammalian cells have been modified to remove all CpG dinucleotides.
A Choice of Different Backbones
To better suit your needs, the pCpGfree-vitro plasmid comes with a
choice of selection: blasticidin, hygromycin or kanamycin/G418.
Each pCpGfree-vitro is available with a CpG-free allele of the LacZ gene
or a multiple cloning site (MCS). This MCS contains several commonly
used restriction sites, for convenient cloning of a CpG-free gene, such as
the genes provided in the pSELECT plasmid (see page 138) or any open
reading frame or cDNA.
Applications
Study the immunostimulatory effect of CpG motifs
pCpGfree-vitro plasmids represent innovative tools to study the effects
of CpG dinucleotides in a number of applications. DNA vaccination
exploits the immunostimulatory character of certain CpG motifs to prime
and boost the immune response. However, these immunostimulatory
CpG motifs are antagonized by CpG dinucleotides in certain distinct base
contexts, termed neutralizing CpG motifs. Both types of CpG motifs are
usually present in plasmidic DNA, and therefore may lead to an
unfavorable immune response. pCpGfree-vitro is the ideal tool to
overcome this problem, and may be used to study the effects of these
two types of CpG motifs by adding them in different configurations to
the pCpGfree-vitro backbone.
Study CpG methylation
CpG dinucleotides are key elements in a number of cellular functions
associated with chromatin. Several large multisubunit complexes,
consisting of methyl-CpG binding (MBD) proteins and histone
deacetylases, have been implicated in the regulation of chromatin
dynamics. These complexes are recruited to methylated CpG
dinucleotides by DNA methyl transferases (DNMTs) and induce
chromatin remodeling. However the specific roles of these complexes
are still to be explored. Due to the absence of CpG dinucleotides within
its backbone, pCpGfree-vitro is not the target of DNMTs and thus MBD
proteins. Therefore, it provides a useful model to study the other proteins
involved in these complexes, in particular the histone deacetylases. It can
also be used to analyze the effects of CpG methylation on the regulation
and duration of gene expression.
Contents and Storage
Each pCpGfree-vitro plasmid is provided as 20 μg of lyophilized DNA
with a paper disk of lyophilized E. coli GT115 strain. Products are shipped
at room temperature and should be stored at -20°C.
Selections Available in pCpGfree-vitro Plasmids
- Blasticidin
- Hygromycin
- Neomycin (G418)
PRODUCT QUANTITY CAT. CODE
pCpGfree-vitroBmcs 20 μg pcpgvtb-mcsg2
pCpGfree-vitroBLacZ 20 μg pcpgvtb-lz
pCpGfree-vitroHmcs 20 μg pcpgvth-mcsg2
pCpGfree-vitroHLacZ 20 μg pcpgvth-lz
pCpGfree-vitroNmcs 20 μg pcpgvtn-mcsg2
pCpGfree-vitroNLacZ 20 μg pcpgvtn-lz
Related Products
Important Licensing Information: These products are covered by a Limited Use License (See Terms and Conditions on page 164). By use of these products you
accept the terms and conditions of all applicable Limited Use Label Licenses.
136 www.invivogen.com/cpgfree-plasmid
Blasticidin, page 17 Hygromycin B, page 18
G418 Sulfate, page 17 LyoComp GT115, page 43
MCS
BsrGI
ScaI
BglII
ApaLI
Bsp120I
NcoI
NheI
MscI

pCpGfree-siRNA & pCpGfree-siRNADUO
Description
pCpGfree-siRNA and pCpGfree-siRNADUO are CpG-free plasmids that
allow the expression of one or two shRNAs, respectively. The absence of
CpG dinucleotides in their backbone makes these plasmids less
immunogenic and insensitive to CpG methylation thus ideal for long
lasting expression of shRNA(s) in vivo .
pCpGfree-siRNA
The pCpGfree-siRNA plasmid combines the backbone of the pCpGfree
plasmid with the shRNA expression cassette of the psiRNA plasmid.
pCpGfree-siRNA features the human 7SK promoter, in which all CpG
dinucleotides have been removed. The activity of this RNA Pol III
promoter is augmented by the addition of the enhancer of the murine
CMV immediate-early promoter, a Pol II enhancer 1 . The enhancer has
been modified to remove all CpGs.
The pCpGfree-siRNA plasmid uses Bbs I, an unusual restriction enzyme
that generates asymmetric cohesive overhangs that are not compatible
with each other to eliminate the risk of self-ligation of the vector.
A second cloning strategy can be chosen by using Acc 65I and Hind III
restriction sites.
The pCpGfree-siRNA plasmid exploits the white/blue selection system
to facilitate the screening of recombinant clones. It features an EM7-LacZ
a-peptide cassette between the cloning sites to allow the easy
discrimination between blue parental and white recombinant clones.
pCpGfree-siRNADUO
The pCpGfree-siRNADUO plasmid contains two shRNA cassette driven
by a CpG-free version of the human 7SK promoter. The murine CMV
enhancer has been added to increase the activity of both 7SK promoters.
The first cassette features Acc65 I / Hind III cloning sites and a LacZ apeptide
bacterial expression cassette for white/blue selection in E. coli.
The second cassette contains Bbs I / Bbs I cloning sites and a GUS
bacterial expression cassette, another system that allows white/blue
selection in E. coli.
pCpGfree-siRNA and pCpGfree-siRNA-DUO are provided with
LyoComp GT115, a lyophilized competent E. coli mutant strain permissive
with the white and blue color selection from the LacZ and GUS systems.
Contents and Storage
The pCpGfree-siRNA and pCpGfree-siRNADUO plasmids are provided
as 50 μg of lyophilized DNA in a kit that includes the following
components:
- 20 μg control plasmid
- 1 vial of LyoComp GT115
- 10 μg of each sequencing primer
- 4 pouches of Fast-Media ® Zeo X-Gal or,
- 2 pouches of Fast-Media ® Zeo X-Gal and 2 pouches of Fast-Media ®
Zeo X-Gluc
Products are shipped at room temperature. Store Fast-Media ® pouches
at room temperature. Store all other components at -20°C.
Acc 65I
Bbs I
Important Licensing Information: These products are covered by a Limited Use License (See Terms and Conditions on page 164). By use of these products you
accept the terms and conditions of all applicable Limited Use Label Licenses.
Bbs I
Hind III
Bbs I
PRODUCT QUANTITY CAT. CODE
pCpGfree-siRNA 1 kit kcpgf-sirna
pCpGfree-siRNADUO 1 kit kcpgf-sirna2
Related Products
Acc 65I
LyoComp GT115, page 43 Fast-Media ® Zeo-X-Gal, page 45
Zeocin , page 18 Fast-Media ® Zeo-X-Gluc, page 45
www.invivogen.com/cpgfree-plasmid 137
Bbs I
Hind III
CpG-FREE DNA 6

CpG-FREE DNA 6
CpG-Free Genes - Synthetic CpG-free Genes
Many non-mammalian genes are widely used as reporter or suicide genes in molecular and cellular studies. However these genes are recognized
as foreign DNA by the vertebrate host leading to a progressive decline of their expression. To circumvent this limitation, InvivoGen has
synthesized new alleles of these genes completely devoid of CpG dinucleotides. These synthetic CpG-free genes display higher activity and
lower immunogenicity than their wild-type counterparts.
Description
The DNA sequence of the CpG-free genes was modified by optimizing
the codon usage, eliminating the CpG nucleotides and avoiding secondary
DNA structures without changing the amino acid sequence of the wild
type proteins.
CpG-free genes were chemically synthesized by assembling large
oligonucleotides. Sequence integrity was verified by double-stranded
sequencing.
CpG-free genes are provided in the pSELECT-zeo plasmid (see page 32).
pSELECT-zeo is a mammalian expression plasmid selectable in E. coli and
mammalian cells with Zeocin . Each CpG-free gene is flanked by a unique
restriction site at the 5’ and 3’ end to facilitate its subcloning into another
vector.
Contents and Storage
Each pSELECT-zeo- plasmid is provided as 20 μg of
lyophilized DNA. Product is shipped at room temperature and should
be stored at -20°C. Plasmid is stable up to one year when properly stored.
Each plasmid is supplied with 4 pouches of E. coli Fast-Media ® Zeo (2 TB
and 2 Agar, see pages 44-45).
GENE DESCRIPTION
Suicide Genes
Fcy::fur* S. cerevisiae cytosine deaminase-uracil phosphoribosyl transferase fusion 33 20 μg psetz-fcyfur
HSV1-tk Herpes simplex virus 1 (HSV1) thymidine kinase 121 20 μg psetz-hsv1tk
HSV1-tk::Sh* HSV1 thymidine kinase-Zeocin resistance fusion 372 20 μg psetz-hsv1tksh
Reporter Genes
GFP::Sh* Green fluorescent protein-Zeocin resistance fusion 110 20 μg psetz-zgfpsh
GFP::Bsr* NEW Green fluorescent protein-Blasticidin resistance fusion 110 20 μg psetz-zgfpbsr
LacZ b-Galactosidase 289 20 μg psetz-lacz
LacZnls b-Galactosidase with SV40 nuclear localization signal 289 20 μg psetz-lacznls
LacZ::Sh* b-Galactosidase-Zeocin resistance fusion 341 20 μg psetz-laczsh
Luc::Sh* Firefly luciferase-Zeocin resistance fusion 151 20 μg psetz-lucsh
SEAP Mouse secreted embryonic alkaline phosphatase 65 20 μg psetz-mseap
* Fusion gene
138 www.invivogen.com/cpgfree-genes
Note: The pSELECT-zeo plasmid backbone contains CpG dinucleotides.
CpGs IN
NATIVE GENE
QUANTITY CAT. CODE

Custom-Made CpG-Free Genes
InvivoGen is an expert in developing CpG-Free genes and now offers a CpG-Free gene custom service. Send us the sequence of your gene
of interest, and our specialists will design, synthesize and clone into an expression vector a CpG-Free allele of this gene. We guarantee that
the sequence of the synthetic gene will 100% match the designed sequence.
➥ Free Sequence Design
➥ Shor t Turnaround Time
➥ Competitive Price
➥ Confidentiality Guaranteed
Description
Sequence Design
Our experts will design the sequence of a CpG-free allele of your gene
of interest using a proprietary software that will include the following:
- elimination of all CpG dinucleotides
- humanization of the sequence
- elimination of secondary structures such as hairpins and long direct
repeated sequences in order to increase the stability and expression of
the mRNA
- elimination/reduction of dam and dcm methylations to diminish E. coli
signature
Cloning in an Expression Vector
The Custom-made CpG-free Gene is cloned into the pMA plasmid. We
can clone it in the vector of your choice for an additional charge.
Sequencing
The Custom-made CpG-free Gene is 100% sequence verified, both strands
are sequenced.
Processing
Send us the nucleic (Genbank accession number) or proteic sequence
of your gene of interest. The synthesis of the CpG-free allele will start
after you confirm its sequence.
The turnaround time is 2-3 weeks for sequences shorter than 1 kb.
We can process sequences up to 5 kb.
Contents and Storage
Custom-made CpG-free genes are provided as 20 μg of lyophilized DNA.
Products are shipped at room temperature. Store at -20°C.
PRODUCT QTY CAT. CODE
Custom-made CpG-free Gene 20 μg p-custom
Contact us for more information.
info@invivogen.com
www.invivogen.com//cpgfree-genes 139
CpG-FREE DNA 6

7
RNA INTERFERENCE
psiRNA System 142-145
psiTEST System 146
Interferon Response 147
Ready-Made psiRNA 148
Custom-Made psiRNA 150

RNA INTERFERENCE 7
psiRNA System
InvivoGen provides a plasmid-based system developed to knockdown efficiently the expression of a wide variety
of mammalian genes. This system represents a simple and affordable method to generate short hairpin RNAs
(shRNAs) by eliminating the need to synthesize RNA oligonucleotides.
The psiRNA System is designed to assist you in all the steps necessary to obtain efficient silencing of a gene of
interest from the selection of an effective siRNA/shRNA to its prevalidation.
• Selection of Candidate siRNA/shRNA and Design of Hairpin Insert:
siRNA Wizard Software - www.sirnawizard.com
The design of siRNAs and short hairpin RNAs (shRNAs) remains an empirical process since the
molecular mechanisms underlying RNAi are not yet sufficiently understood to allow for their
rational design. However, based on the research from various laboratories including our own,
InvivoGen has been able to develop siRNA Wizard, a free software accessible online from our
homepage, that will help you do the following:
➥ Select Candidate siRNA/shRNAs
The siRNA Wizard algorithm allows to select effective and specific siRNAs/shRNAs against your
gene of interest based on thermodynamic and sequence-related criteria. Two search options are
available:
- Standard Search
“Standard Search” uses default criteria described in the “siRNA/shRNA Design Guidelines”
paragraph to select several candidate siRNAs/shRNAs against your gene of interest.
- Advanced Search
“Advanced Search” lets you manually set the selection criteria.
➥ Design Hairpin Insert
Using your selected siRNA/shRNA sequence, this tool will design two complementary
oligonucleotides necessary to create the hairpin insert for psiRNA cloning vectors and let you
choose the sequence of the loop.
➥ Generate siRNA/shRNA Scramble Sequence
This tool will return a scramble sequence with no match with any mRNA of the selected species
database. This scramble sequence that serves as a negative control contains the same nucleotide
composition as the selected siRNA/shRNA sequence.
• Cloning of siRNA/shRNA Insert: psiRNA-h7SK Plasmids
InvivoGen provides psiRNA-h7SK, a family of cloning vectors featuring the human 7SK RNA
polymerase III promoter and a choice of selection markers (see page 144). psiRNA-h7SK plasmids
exploit the white/blue selection system to facilitate the screening of recombinant bacterial clones.
Furthermore, they feature a GFP::Zeo fusion gene that allows to evaluate transfection efficiency
and normalize silencing experiments.
psiRNA-h7SK plasmids are provided in a kit that contains, in addition to the cloning vector, two
control vectors, and a set of tools designed to facilitate the cloning of shRNAs in the plasmid. The
psiRNA-h7SK tools are the following:
- E. coli GT116 & GT115
- Sequencing Primers
- Fast-Media ® X-Gal & X-Gluc
142 www.invivogen.com/psirna-system
“Standard Search” Selection Criteria
Target gene trimming
> > > > > > > > >
Thermodynamic analysis
GC content analysis
Termination signal exclusion
Secondary structure avoidance
Immunostimulatory motif exclusion
BLAST search
3’ UTR/seed region analysis
(optional)
siRNAs/shRNAs failing the selection
are rejected
Effective and specific
siRNAs/shRNAs

E. coli GT116 & GT115
InvivoGen has engineered two specific E. coli strains, named GT116 and GT115,
to facilitate the cloning of shRNAs into psiRNA plasmids. Hairpin structures are
known to be unstable in E. coli due to their elimination by a protein complex
called SbcCD that recognizes and cleaves hairpins. To increase their stability in
E. coli, we developed GT116 and GT115 by deleting the sbcC and sbcD genes.
Both strains are more compatible with hairpin structures than commonly used
E. coli strains increasing the probability of obtaining the correct clone in a
minimum number of steps. GT115 is further mutated in the uidA gene rendering
this strain deficient for b-glucuronidase (see page 42).
Both strains are provided as lyophilized chemically competent cells, named
LyoComp GT116 and LyoComp GT115 respectively, that can also be purchased
separately (see page 43).
GT116 Genotype: F¯ mcrA D(mrr-hsdRMS-mcrBC) Φ80lacZDM15 DlacX74 recA1
rspL (StrA) endA1 ∆dcm ∆sbcC-sbcD
GT115 Genotype: F¯ mcrA D(mrr-hsdRMS-mcrBC) Φ80lacZDM15 DlacX74 recA1
rspL (StrA) endA1 ∆dcm uidA(∆MluI)::pir-116 ∆sbcC-sbcD
Sequencing Primers
Several sequencing primer pairs have been designed, one for each psiRNA
plasmid backbone, that allow full reading of the insert sequence by using
conventional sequencing methods.
- OL559-OL408 pair, for all psiRNA-h7SK plasmids
- OL178-OL408 and OL906-OL176 pairs for the a-peptide cassette and the
GUS cassette of the psiRNA-DUO plasmid, respectively
Fast-Media ® X-Gal & X-Gluc
Fast-Media ® X-Gal and Fast-Media ® X-Gluc are ready-to-use microwaveable
LB-based medium powder for E. coli selection. They already contain the selective
antibiotic and X-Gal (LacZ) or X-Gluc (Gus) at the appropriate co ncentration.
These media allow to prepare high quality white/blue selection medium in less
than 5 minutes.
Fast-Media ® X-Gal is available with four different selections: blasticidin,
hygromycin, kanamycin and Zeocin . Fast-Media ® X-Gluc is available with
Zeocin .
Fast-Media ® can be purchased separately (see pages 44-45).
• Prevalidation of siRNA/shRNA: psiTEST System
The psiTEST System was developed to provide a rapid, simple, and convenient
method to screen for functional siRNA and shRNA sequences (see pages 146-
147). The silencing efficiency of a given siRNA or shRNA is evaluated by
monitoring the reduction of expression of a chimeric gene consisting of a
secreted alkaline phosphatase (SEAP) reporter gene transcriptionally fused to
the target gene. Transfection of effective siRNAs or shRNAs triggers the RNAi
process resulting in the degradation of the chimeric mRNA and therefore the
absence of SEAP activity.
Strategy for psiRNA-mediated RNA
interference
www.invivogen.com/psirna-system 143
RNA INTERFERENCE 7

RNA INTERFERENCE 7
psiRNA-h7SK - 7SK-based expression of shRNAs
Features and Benefits
RNA Polymerase III Based Plasmid
psiRNA-h7SK is a family of expression vectors designed to generate
shRNA from the human 7SK RNA polymerase III (Pol III) promoter.
7SK is an abundant and evolutionarily conserved small nuclear RNA
transcribed by RNA polymerase III 1 . The human 7SK promoter is ideal
for the production of shRNAs as it can generate high amounts of
shRNAs 1, 2 . In a series of experiments aimed to compare the strength of
the human 7SK, H1 and U6 promoters, we found that the best silencing
efficiencies of various target genes was consistently obtained with the 7SK
promoter. psiRNA-h7SK has been successfully used to achieve gene
silencing in vitro and in vivo 3-5 . Furthermore, the 7SK promoter can be used
in combination with other RNA pol III promoters for multiple shRNA
expression strategies 6 .
Cloning Options
psiRNA-h7SK plasmids offer two cloning strategies for the cloning of an
shRNA insert, either Acc 65I / Hind III or Bbs I / BbsI. The two Bbs I sites
are different although recognized by the same restriction enzyme thus
preventing self-ligation of the plasmid.
White and Blue Selection
psiRNA-h7SK plasmids exploit the white/blue selection system to
facilitate the screening of recombinant clones. The cloning sites flank a
bacterial LacZ a-peptide expression cassette allowing the discrimination
between blue parental clones and white recombinant clones. Although
over 90% of the white clones have integrated a fragment, it is necessary
to sequence the insert to verify the integrity of the sequence since only
one base difference can lead to an inactive shRNA.
Variety of Selectable Markers
psiRNA-h7SK plasmids are available with several selectable markers that
function in E. coli and mammalian cells. They are driven by strong
mammalian promoter in tandem with a constitutive bacterial promoter.
Normalize Your Silencing Experiments with GFP::Zeo
psiRNA-h7SKGFPzeo features a GFP::zeo fusion gene that combines the
GFP reporter gene and the Zeocin resistance gene. This fusion gene
allows the evaluation of transfection efficiency by determining the
percentage of transfected cells by assaying the reporter activity. Therefore,
psiRNA-h7SKGFPzeo use allows to normalize your silencing experiments.
Easily Convertible into an Adenovector
The shRNA expression cassette is flanked by Spe I at the 5’ end and
Hind III at the 3’ end. This cassette can be easily excised from any
psiRNA-h7SK plasmid using these two restriction enzymes and
subcloned into one of the shuttle plasmids commercially available
digested with Nhe I (compatible with Spe I) and Hind III.
1. Czauderna F. et al., 2003. Inducible shRNA expression for application in a prostate
cancer mouse model. Nucleic Acids Res. 31(21):e127. 2. Koper-Emde D. et al.,2004. RNA
interference by small hairpin RNAs synthesised under control of the human 7S K RNA
promoter. Biol Chem. 385(9):791-4. 3. Triantafilou K. et al., 2005. Human cardiac
inflammatory responses triggered by Coxsackie B viruses are mainly Toll-like receptor
(TLR) 8-dependent. Cell Microbiol. 7(8):1117-26. 4. Tajeddine N. et al., 2005. Tumorassociated
antigen preferentially expressed antigen of melanoma (PRAME) induces
caspase-independent cell death in vitro and reduces tumorigenicity in vivo. Cancer
Res.65(16):7348-55. 5. Mazzanti CM. et al., 2004. Early genetic mechanisms underlying
the inhibitory effects of endostatin and fumagillin on human endothelial cells. Genome
Res. 14(8):1585-93. 6. ter Brake O. et al., 2008. Lentiviral Vector Design for Multiple
shRNA Expression and Durable HIV-1 Inhibition. Mol Ther. 16(3):557-64.
Bbs I
Acc 65I
Contents and Storage
144 www.invivogen.com/psirna-system
Bbs I
Hind III
Choose your selectable marker
Blasticidin S
Hygromycin B
Kanamycin / G418
Zeocin
GFP::Zeo
psiRNA-h7SK kits contain the following components:
- 20 μg of the psiRNA-h7SK plasmid of your choice (see list)
- 20 μg of the corresponding control plasmid 1 (psiRNA-h7SKgfp for all
selectable markers and psiRNA-h7SKlacZ for GFP::zeo)
- 20 μg of the corresponding control plasmid 2 (psiRNA-h7SKluc for all
selectable markers)
- 1 vial of LyoComp GT116
- 2 x 10 μg of the corresponding sequencing primer pair
- 4 pouches of the appropriate Fast-Media ® X-Gal
Products are shipped at room temperature. Store Fast-Media ® at room
temperature. Store all other components at -20°C.
PRODUCT QTY CAT. CODE
psiRNA-h7SKblasti 1 kit ksirna3-b21
psiRNA-h7SKhygro 1 kit ksirna3-h21
psiRNA-h7SKneo 1 kit ksirna3-n21
psiRNA-h7SKzeo 1 kit ksirna3-z21
psiRNA-h7SKGFPzeo 1 kit ksirna4-gz21

psiRNA-DUO - Expression of Two shRNAs
Description
psiRNA-DUO generates two shRNAs from the same plasmid for the
silencing of either a single target gene (with/without polymorphisms) or
two different target genes. psiRNA-DUO contains special features
designed to make the cloning of the shRNA inserts and selection of
recombinant clones simple and rapid.
The major features of the psiRNA-DUO plasmid are its two RNA Pol III
cassettes and the GFP-Zeo fusion gene.
7SK RNAi Cassettes
The first cassette contains the following elements:
- Human 7SK promoter (see page 144)
- Acc65 I / Hind III cloning sites
- LacZ a-peptide expression cassette for white and blue selection
The second cassette contains the following elements:
- Human 7SK promoter
- Bbs I / Bbs I cloning sites
- GUS expression cassette for white and blue selection
b-Glucuronidase (GUS, uidA) is an E. coli enzyme capable of hydrolyzing
the chromogenic substrate X-Gluc to generate a blue pigment. Therefore,
similarly to LacZ, the expression of GUS in permissive E. coli allows the
discrimination between blue parental clones and white recombinant
clones.
psiRNA-DUO is provided with the E. coli GT115, a lacZ uidA mutant
strain, enabling the white and blue color selection from the LacZ and
GUS systems.
GFP-Zeo Fusion Gene
The GFP-Zeo gene is a transcriptional fusion between the Green
Fluorescent Protein reporter gene and the Zeocin resistance (Zeo R )
gene. The two moieties are separated by a bacterial promoter (EC2K)
located within an intron. In E. coli, Zeo R is expressed from the EC2K
promoter that is spliced out in mammalian cells. A composite CMV
promoter drives the expression of the GFP-Zeo fusion gene, allowing the
monitoring of transfection efficiency and the selection of stable clones.
Contents and Storage
psiRNA-DUO is provided as a kit composed of following:
- 20 μg of psiRNA-DUO-GFPzeo
- 20 μg of psiRNA-LucLac-GFPzeo (control plasmid)
- 1 vial of LyoComp GT115
- 2 sets of sequencing primers (2 x 10 μg each)
- 2 pouches of Fast-Media ® Zeo X-Gal
- 2 pouches of Fast-Media ® Zeo X-Gluc
For more information on LyoComp GT115, sequencing primers and
Fast-Media ® X-Gal and X-Gluc, see page 45.
Products are shipped at room temperature. Store Fast-Media ® at room
temperature. Store all other components at -20°C.
Procedures
Two Cloning Step Procedure
1- Clone first shRNA insert in first 7SK RNAi cassette
opened with Acc65 I and Hind III.
2- Select white recombinant clones on X-Gal plates prepared
with Fast-Media ® Zeo X-Gal.
3- Clone second shRNA insert in second 7SK RNAi cassette
opened with Bbs I.
4- Select white recombinant clones on X-Gluc plates prepared
with Fast-Media ® Zeo X-Gluc.
One Cloning Step Procedure
1- Digest psiRNA-DUO with Acc65 I, Hind III and Bbs I.
2- Ligate both shRNA inserts with the two psiRNA-DUO
fragments (Hind III/Bbs I and Bbs I/Acc65 I).
3- Select sequentially white recombinant clones on X-Gal
plates and X-Gluc plates.
PRODUCT QTY CAT. CODE
psiRNA-DUO 1 kit ksirna4-gz3
www.invivogen.com/psirna-system 145
RNA INTERFERENCE 7

RNA INTERFERENCE 7
psiTEST System - Prevalidation of siRNAs and shRNAs
The psiTEST system enables you to screen for effective siRNAs and shRNAs without using time-consuming and costly reagents that are specific
to your target gene, such as antibodies. The psiTEST system can be used to evaluate RNAi on virtually any gene of any species for which the
sequence is known. The silencing efficiency of a given siRNA or shRNA is evaluated by monitoring the reduction of expression of a chimeric
gene consisting of a secreted alkaline phosphatase (SEAP) reporter gene transcriptionally fused to the target gene. Transfection of effective
siRNAs or shRNAs triggers the RNAi process resulting in the degradation of the chimeric mRNA and therefore the absence of SEAP activity.
Description
The psiTEST system is based on a transcriptional fusion between a
reporter gene and your target gene. The reporter gene is an optimized
alkaline phosphatase gene engineered to be secreted (SEAP). The efficacy
of an siRNA or shRNA to induce RNAi on your target gene is
determined by measuring the expression levels of the SEAP reporter
gene (see figure). Since SEAP is a secreted protein, the expression level
can be evaluated from supernatants therefore allowing to perform kinetics
of the silencing process.
Features and Benefits
The psiTEST system contains two major components, the psiTEST plasmid
and QUANTI-Blue .
psiTEST plasmid
The psiTEST plasmid features the SEAP gene fused at the 3’ end after its
Stop codon to a multiple cloning site (MCS). This MCS contains many
common restriction sites compatible with many other restriction sites.
psiTEST also features a GFP::zeo fusion gene: the zeo moiety allows to
amplify the plasmid in E. coli and select stable mammalian clones while
the GFP moiety enables the monitoring of transfection efficiency.
psiTEST can be used to clone fragments of genes or entire genes, the
optimum size being 1.5 kb, although genes up to 3.5 kb can be targeted.
QUANTI-Blue
QUANTI-Blue is a SEAP detection medium that turns purple/blue in
the presence of phosphatase activity. Functional siRNAs or shRNAs will
induce the degradation of the SEAP mRNA resulting in the reduction or
absence of SEAP activity.
Silencing efficiencies of the siRNAs or shRNAs can be observed visually
and unlike fluorescent reporters can also be easily quantified using a
microplate reader or spectrophotometer.
Contents and Storage
The psiTEST system is provided with several control plasmids: psiTESThp53,
psiRNA-hp53 and psiRNA-Luc. psiTEST-hp53 contains a fusion
between the SEAP and human p53 genes. psiRNA-hp53 produces an
shRNA that functionally silences the human p53 resulting in the absence
of phosphatase activity. psiRNA-Luc expresses an shRNA insert that
targets luciferase GL3 and therefore will not affect the expression of the
phosphatase reporter gene.
The psiTEST system contains the following components:
- 20 μg of psiTEST and 20 μg of psiTEST-hp53 (control plasmid)
- 20 μg of psiRNA-hp53 and 20 μg of psiRNA-Luc
- 2 pouches of QUANTI-Blue (100 ml each)
- 4 pouches of Fast-Media ® Zeo (2 TB, 2 Agar)
Products are shipped at room temperature. Store Fast-Media ® at room
temperature. Store all other components at -20°C.
Procedure
146 www.invivogen.com/psitest-system
1- Clone your target gene (or a fragment) into the psiTEST
plasmid.
2- Cotransfect mammalian cells with recombinant psiTEST
and siRNA or shRNA-producing plasmid, such as psiRNA .
3- Incubate at 37°C with 5% CO2 for 72 hours.
4- Collect supernatant and add to a QUANTI-Blue -
containing well.
5- Incubate at 37°C for 30 min to 3 hours.
6- Determine the functionality of your siRNA or shRNA by
observing the coloration of QUANTI-Blue visually and/or
with a spectrophotometer at 620-655 nm.
PRODUCT QTY CAT. CODE
psiTEST 1 kit ksitest
QUANTI-Blue 5 pouches rep-qb1

IFNr qRT-Primers - Detection of the IFN Response
Double strand RNA (dsRNA) is a molecular pattern associated with viral infection. It is recognized by the innate immune system leading to the
production of type I interferons and the subsequent induction of a variety of antiviral genes. Recent reports indicate that certain siRNAs and
shRNAs can activate the IFN pathway, potentially leading to severe side effects. To determine whether a given siRNA or shRNA is
immunostimulatory, InvivoGen has developed a set of primers that detect the expression of human genes involved in the IFN response by
real-time quantitative PCR using the SYBR ® Green detection.
➥ The IFNr qRT-Primers provide highly specific and sensitive results in real-time quantitative PCR.
➥ Each IFNr qRT-Primer Pair is carefully designed and tested.
➥ The size of the amplified fragments varies from 80 to 280 bp.
➥ The IFNr qRT-Primers allow to perform 100 x 50 μl reactions (in a 96-well plate) or 250 x 20 μl reactions (in a 384-well plate).
Description
The IFNr qRT-Primers are a set of primer pairs designed to measure
the mRNA expression of 5 genes induced by IFNa:
- IFNb
- OAS1
- MX1 (also known as MxA)
- G1P2 (also known as ISG15)
- IFIT1 (also known as CDKL2 or P56),
and the housekeeping gene GAPDH as a control.
The IFNr qRT-Primers allow one-step or two-step qRT-PCR in
combination with SYBR ® Green I dye.
Interferon Response: An Off-Target Effect
The RNAi reagents, siRNA and shRNA, can be potent inducers of
interferons (IFNs) and inflammatory cytokines both in vivo and in vitro 1-3
due to their recognition by cellular receptors of foreign RNA and
DNA. Much of the IFN response is caused by the activation of the
dsRNA-dependent protein kinase R (PKR), that recognizes long
double-stranded (ds)RNA, leading to a global inhibition of protein
synthesis. siRNAs that are shorter than 30 bp can evade PKR
activation, but are recognized by other receptors, such as the Toll-like
receptors (TLRs). TLR3, which ligand is dsRNA, and TLR7 and TLR8,
that sense single-stranded RNA, have been implicated in the
recognition of siRNAs 1-4 . Recognition by TLR7 and TLR8 appears to
sequence-dependent as several immunostimulatory sequences have
been identified. These sequences are characterized by a high content
of guanosine and uridine residues 1, 5 . They have been integrated in
InvivoGen’s siRNA Wizard algorithm and are systematically excluded
to limit the IFN response.
The IFN response induced by shRNAs results mainly from the DNA
vector that contains non-methylated CpG sequences that are
recognized by TLR9. InvivoGen has designed a CpG-free vector,
pCpGfree-siRNA, to overcome this problem (see page 137).
Contents and Storage
The IFNr qRT-Primers contain 10 μM of each primer. The 5’ sense primer
and the 3’ antisense primer are provided in separate vials. Products are
lyophilized and shipped at room temperature.
PRODUCT QTY CAT. CODE
IFNr qRT-Primers 1 kit rts-hifnr
1. Hornung V. et al., 2005. Sequence-specific potent induction of IFN-alpha by short interfering
RNA in plasmacytoid dendritic cells through TLR7. Nat Med. 11(3):263-70. 2. Sioud M.,
2005. Induction of inflammatory cytokines and interferon responses by double-stranded and
single-stranded siRNAs is sequence-dependent and requires endosomal localization. J Mol
Biol. 348(5):1079-90. 3. Agrawal S. & Kandimalla ER. 2004. Antisense and siRNA as agonists
of Toll-like receptors. Nat. Biotechnol. 22: 1533-1537. 4. Kariko K. et al., 2004. Exogenous
siRNA mediates sequence-independent gene suppression by signaling through toll-like
receptor 3. Cells Tissues Organs. 177(3):132-8. 5. Judge AD. et al., 2005. Sequencedependent
stimulation of the mammalian innate immune response by synthetic siRNA. Nat
Biotechnol. 23(4):457-62.
www.invivogen.com/interferon-response 147
RNA INTERFERENCE 7

RNA INTERFERENCE 7
Ready-Made psiRNA - Plasmids Expressing Functional shRNAs
Ready-made psiRNA is a new family of plasmids expressing a growing list of shRNAs which functionality has been described in the literature
or validated in house. Ready-made psiRNA plasmids eliminate the need to design and clone several siRNA sequences before identifying an
effective one. They express shRNAs that silence the expression of a target gene by >70%.
Features and Benefits
Ready-made psiRNAs are psiRNA-h7SKGFPzeo-derived plasmids. They
feature the human 7SK RNA Pol III promoter that generates high amounts
of short hairpin RNAs. They also feature the GFP::Zeo fusion gene which
confers both reporter and antibiotic resistance activities making these
plasmids very useful for the following applications:
• Monitor transfection efficiency
• Standardize gene silencing efficiency
• Select clones that stably express a validated siRNA
The silencing efficiency of each Ready-made psiRNA plasmid has been
tested using the psiTEST system (see page 146). The genes or fragments
of the genes targeted by the Ready-made psiRNA have been fused to
the SEAP reporter gene within the psiTEST plasmid. Silencing efficiencies
have been confirmed by the absence of SEAP activity after cotransfecting
HEK293 cells with each recombinant psiTEST and corresponding Readymade
psiRNA.
Contents and Storage
Ready-made psiRNA plasmids are available alone or in a kit.
Ready-made psiRNA plasmids are provided as 20 μg of lyophilized DNA.
Each Ready-made psiRNA kit contains the following components:
- 20 μg of a Ready-made psiRNA plasmid
- 20 μg of a control psiRNA plasmid (psiRNA-Luc)
- 1 vial of LyoComp GT116
- 4 pouches of Fast-Media ® Zeo
Products are shipped at room temperature. Store at -20°C.
PRODUCT QTY CAT. CODE*
Ready-made psiRNA plasmid 20 μg psirna42-
Ready-made psiRNA kit 1 kit ksirna42-
*See tables
148 www.invivogen.com/readymade-psirna
GENE NAME/ALIASES SPECIES
CAT. CODE
(Plasmid)**
A20 / TNFAIP3 Human, mouse psirna42-(h/m)a20
ASC / CARD5 Human, mouse psirna42-(h/m)asc
AIM2 / IFI210 Human, mouse psirna42-(h/m)aim2
ATF3 Human psirna42-hatf3
ATG5 Human, mouse psirna42-(h/m)atg5
BAF47 / SMARCB1 Human psirna42-hbaf47
BP1 NEW Human psirna42-hbpi
BCL2 Human psirna42-hbcl2
BCL3 Human, mouse psirna42-(h/m)bcl3
BCL10 / CLAP Human, mouse psirna42-(h/m)bcl10
Beclin-1 Human, mouse psirna42-(h/m)beclin
CARD8 / Cardinal Human psirna42-hcard8
CARD9 Human, mouse psirna42-(h/m)card9
Caspase 1 / ICE Human, mouse psirna42-(h/m)casp1
Caspase 8 / FLICE Human, mouse psirna42-(h/m)casp8
CD14 Human, mouse psirna42-(h/m)cd14
CD36 Human, mouse psirna42-(h/m)cd36
CD44 Human, mouse psirna42-(h/m)cd44
CLEC9A Human, mouse psirna42-(h/m)clec9a
CXCL16 NEW Human, mouse psirna42-(h/m)cxcl16
CXCR4 / CD184 Human psirna42-hcxcr4
CYLD Human psirna42-hcyld
DAI / ZBP1 Human psirna42-hdai
DAK Human, mouse psirna42-(h/m)dak
DC-SIGN / CD209 Human, mouse psirna42-(h/m)dcsign
DDX3 / DDX3X Human psirna42-hddx3x
Dectin-1 Human, mouse psirna42-(h/m)dectin1
DNMT1 Human psirna42-hdnmt1
DNMT2 Human psirna42-hdnmt2
DNMT3A Human psirna42-hdnmt3a
DNMT3B Human psirna42-hdnmt3b
DNMT3L Human psirna42-hdnmt3l
DUBA / OTUD5 Mouse psirna42-mduba
FADD / MORT1 Human, mouse psirna42-(h/m)fadd
FLII / Fliih Human psirna42-hflii
GM-CSF Human, mouse psirna42-(h/m)gmcsf
HDAC1 Human psirna42-hhdac1
HDAC2 Human psirna42- hhdac2
HDAC3 Human psirna42-hhdac3
HDAC6 Human psirna42-hhdac6
HDAC8 Human psirna42-hhdac8
HPRT Human psirna42-hhprt
IFI16 / PYHIN2 NEW Human psirna42-hifi16
IFNAR1 Human psirna42-hifnar1
** For the kit catalog code, replace psirna42 by ksirna42

GENE NAME/ALIASES SPECIES
CAT. CODE
(plasmid)**
IFNAR2 Human, mouse psirna42-(h/m)ifnar2
IKKε / IKBKE / IKK-i Human, mouse psirna42-(h/m)ikke
IL1R1 / IL1RA Human psirna42-hil1r1
IPAF / CARD12 Human psirna42-hipaf
IPS-1 / MAVS / VISA Human, mouse psirna42-(h/m)ips1
IRAK-1 Human, mouse psirna42-(h/m)irak1
IRAK-4 Human, mouse psirna42-(h/m)irak4
IRAK-M Human psirna42-hirakm
IRF1 Human, mouse psirna42-(h/m)irf1
IRF3 Human, mouse psirna42-(h/m)irf3
IRF5 Human, mouse psirna42-(h/m)irf5
IRF7 Human, mouse psirna42-(h/m)irf7
IRF9 Human, mouse psirna42-(h/m)irf9
JAK1 / JAK1A Human, mouse psirna42-(h/m)jak1
KAISO / ZBTB33 Human psirna42-hkaiso
KRAS Human, mouse psirna42-(h/m)kras
Lamin Human psirna42-hlamin
LGP2 / DHX58 Human, mouse psirna42-(h/m)lgp2
LRRFIP2 Human psirna42-hlrrfip2
MBL2 Human psirna42-hmbl2
MBD1 / PCM1 Human psirna42-hmbd1
MD2 NEW Human psirna42-hmd2
MDA-5 / IFIH1 Human, mouse psirna42-(h/m)mda5
MeCP2 Human psirna42-hmecp2
MEKK3 / MAP3K Human, mouse psirna42-(h/m)mekk3
Mincle / CLEC4E Human, mouse psirna42-(h/m)mincle
MNDA / PYHIN3 NEW Human psirna42-hmnda
MSK1 NEW Human, mouse psirna42-(h/m)msk1
MULAN / MUL1 Human, mouse psirna42-(h/m)mul1
MyD88 Human, mouse psirna42-(h/m)myd88
Naip5 / BIRC1E Mouse psirna42-mnaip5
NALP1 / CARD7 Human psirna42-hnalp1
NALP2 / PAN1 Human psirna42-hnalp2
NALP3 / NLRP3 Human, mouse psirna42-(h/m)nalp3
NALP11 / NLRP11 NEW Human psirna42-hnalp11
NALP12 / Monarch1 Human psirna42-hnalp12
NAP1 / AZI2 Human, mouse psirna42-(h/m)nap1
NOD1 / CARD4 Human, mouse psirna42-(h/m)nod1
NOD2 / CARD15 Human, mouse psirna42-(h/m)nod2
NOD9 / NLRX1 Human, mouse psirna42-(h/m)nod9
p53 Human, mouse psirna42-(h/m)p53
PACT / PRKRA Human, mouse psirna42-(h/m)pact
Pannexin 1 / PANX1 Human, mouse psirna42-(h/m)panx1
Pellino 1 / PELI1 Human, mouse psirna42-(h/m)pellino1
Pellino 2 / PELI2 Human, mouse psirna42-(h/m)pellino2
Pellino 3 / PELI3 Human psirna42-hpellino3
PIN1 / DOB Human, mouse psirna42-(h/m)pin1
PKD1 / PRKD1 Human psirna42-hpkd1
PKR / EIFAK2 Human, mouse psirna42-(h/m)pkr
RAC1 Human, mouse psirna42-(h/m)rac1
RAGE / AGER Human, mouse psirna42-(h/m)ager
RIG-I / DDX58 Human, mouse psirna42-(h/m)rigi
RIP1 / RIPK1 Human, mouse psirna42-(h/m)rip1
RIP2 / RIPK2 Human, mouse psirna42-(h/m)rip2
GENE NAME/ALIASES SPECIES
CAT. CODE
(plasmid)**
RNF125 / TRAC-1 Human, mouse psirna42-(h/m)rnf125
RP105 / CD180 Human psirna42-hrp105
S100A8 Mouse psirna42-ms100a8
S100A9 Mouse psirna42-ms100a9
SARM1 Human psirna42-hsarm1
SHIP NEW Human psirna42-hship
SIGIRR / TIR8 Mouse psirna42-msigirr
SIGNR1 Mouse psirna42-msignr1
SIGNR2 Mouse psirna42-msignr2
SIGNR3 Mouse psirna42-msignr3
SIKE Human, mouse psirna42-(h/m)sike
Smad3 Human psirna42-hsmad3
Smad4 Human psirna42-hsmad4
SOCS-1 Human, mouse psirna42-(h/m)socs1
SOCS-3 Human psirna42-hsocs3
ST2 / IL1RL1 / T2 Human psirna42-hst2
STAT1 / STAT91 Human, mouse psirna42-(h/m)stat1
STAT2 / STAT113 Human, mouse psirna42-(h/m)stat2
STAT3 / APRF Human psirna42-hstat3
STING NEW Human, mouse psirna42-(h/m)sting
SUGT1 / SGT1 NEW Human psirna42-hsugt1
SUV39H1 / KMTIA Human psirna42-hsuv39h1
TAK1 / MAP3K7 Human, mouse psirna42-(h/m)tak1
TANK Human, mouse psirna42-(h/m)tank
TBK1 Human, mouse psirna42-(h/m)tbk1
TBKBP1 / SINTBAD NEW Human psirna42-htbkbp1
TICAM1 / TRIF Human, mouse psirna42-(h/m)ticam1
TICAM2 / TRAM Human, mouse psirna42-(h/m)ticam2
TIFA Human, mouse psirna42-(h/m)tifa
TIRAP / MAL Human, mouse psirna42-(h/m)tirap
TLR1 / CD281 Human, mouse psirna42-(h/m)tlr1
TLR2 / CD282 Human, mouse psirna42-(h/m)tlr2
TLR3 / CD283 Human, mouse psirna42-(h/m)tlr3
TLR4 / CD284 Human, mouse psirna42-(h/m)tlr4
TLR5 Human, mouse psirna42-(h/m)tlr5
TLR6 / CD286 Human, mouse psirna42-(h/m)tlr6
TLR7 Human, mouse psirna42-(h/m)tlr7
TLR8 / CD288 Human, mouse psirna42-(h/m)tlr8
TLR9 / CD289 Human, mouse psirna42-(h/m)tlr9
TLR10 / CD290 Human psirna42-htlr10
TNFR1 / TNFRSF1A Human psirna42-htnfr1
TNFR2 / TNFRSF1B Human, mouse psirna42-(h/m)tnfr2
Tollip / IL-1RAcP1P Human psirna42-htollip
TRADD Human, mouse psirna42-(h/m)tradd
TRAF1 NEW Human psirna42-htraf1
TRAF3 / CAP-1 Human, mouse psirna42-(h/m)traf3
TRAF4 NEW Human, mouse psirna42-(h/m)traf4
TRAF6 / RNF85 Human, mouse psirna42-(h/m)traf6
TRAFD1 / FLN29 Human psirna42-htrafd1
Triad3A Human psirna42-htriad3a
Tyk2 / JTK1 Human, mouse psirna42-(h/m)tyk2
UBP43 NEW Human, mouse psirna42-(h/m)ubp43
UNC93B1 Human, mouse psirna42-(h/m)unc93
VEGF Human, mouse psirna42-(h/m)vegf
** For the kit catalog code, replace psirna42 by ksirna42
www.invivogen.com/readymade-psirna 149
RNA INTERFERENCE 7

RNA INTERFERENCE 7
Custom-Made psiRNA
InvivoGen provides a complete siRNA service to help you speed-up your gene silencing experiments. Just give us the accession number of
your gene of interest and we will take care of all the steps, from helping you choose the target sequence on your gene to sequencing the
resulting siRNA insert. InvivoGen provides a choice of plasmidic vectors to better suit your needs.
➥ Full Custom Service
➥ Your siRNA vector Ready-to-Use
➥ Cost-effective
➥ Rapid Processing
Features and Benefits
Choose your siRNA sequence
You can either send us your siRNA sequence or, using the siRNA Wizard
software, InvivoGen will select the best candidate siRNA sequence for
your target gene.
Choose your psiRNA Plasmid
- psiRNA-h7SK (see page 144)
- psiRNA-DUO (see page 145)
Choose your selectable marker
psiRNA plasmids are available with various selectable markers that confer
antibiotic resistance in both E. coli and mammalian cells:
- Blasticidin - Zeocin
- Hygromycin - GFP::Zeo
- Kanamycin / G418
Description
Construction of custom-made psiRNA plasmids includes the following:
- Selection of the siRNA sequence for the target gene
- Synthesis of the siRNA insert oligonucleotides
- Cloning of the annealed oligonucleotides in psiRNA
- Sequencing of the siRNA insert
Contents and Storage
Custom-made psiRNA are provided as 20 µg of lyophilized DNA. They
can also be provided as a kit that contains in addition
- 20 µg of psiRNA-EGFP or psiRNA-LucGL3 as a control
- 1 vial of LyoComp GT116
- 4 pouches of the corresponding Fast-Media ®
Products are shipped at room temperature. Store at -20°C.
150 www.invivogen.com/custom-cloning
Although the siRNA Wizard software can facilitate the selection
of functional shRNAs, there is no guarantee that the resulting
shRNA will be effective enough for your experiment. Thus it is
usually recommended to select 3-5 candidate shRNAs.
Furthermore, most journal article reviewers require that gene
silencing results be confirmed with a second effective siRNA to a
target before an article is accepted for publication.
Orders of multiple psiRNA plasmids targeting the same gene will
be eligible for a volume discount.
PRODUCT QTY CAT. CODE
Custom-made psiRNA 20 μg p-custom
Custom-made psiRNA Kit 1 kit k-custom

8
INHIBITORS
Cytokine Receptor Inhibitor 155
DNA Synthesis Inhibitors 159
Epigenetic Inhibitors 157
Heat Shock Protein Inhibitors 158
Ion Channel Inhibitors 155
Nuclear Export Inhibitor 155
MAPK Inhibitors 156
NF-kB Activation Inhibitors 155
Protein Kinase Inhibitors 157
PRR Signaling Inhibitors 154

INHIBITORS 8
Inhibitors
PRODUCT DESCRIPTION
Pattern Recognition Receptor (PRR) Signaling Inhibitors
BX795 TBK1/IKKε and PDK1 inhibitor 10 nM - 1 μM 5 mg tlrl-bx7 p 154
Chloroquine Endosomal acidification inhibitor 10 μM 250 mg tlrl-chq p 154
CLI-095 TLR4 signaling inhibitor 50 nM - 1 μM 1 mg tlrl-cli95 p 154
Cyclosporin A NEW Calcineurin inhibitor 50 nM - 1.5 μM 100 mg tlrl-cyca p 154
Gefitinib (Iressa) NEW RIP2 tyrosine kinase inhibitor 10 μM 10 mg tlrl-gef p 154
OxPAPC TLR2 and TLR4 inhibitor 30 μg/ml 1 mg tlrl-oxp1 p 154
Pepinh-MYD MyD88 inhibitory peptide 5 - 100 μM 2 mg tlrl-pimyd p 154
Pepinh-TRIF TRIF inhibitory peptide 5 - 100 μM 2 mg tlrl-pitrif p 154
Polymyxin B LPS-induced TLR4 activation inhibitor 10 μg/ml 100 mg tlrl-pmb p 154
Rapamycin (Sirolimus) mTOR inhibitor 10-100 nM 5 mg tlrl-rap p 154
Z-VAD-FMK Pan-caspase Inhibitor 10 μg/ml (20 μM) 1 mg tlrl-vad p 154
Cytokine Receptor Inhibitor
SB431542 TGF-b receptor inhibitor 2 μM 5 mg inh-sb43 p 155
Ion Channel Inhibitors
Bafilomycin A1 V-ATPase inhibitor 0.1 - 1 μM 10 μg tlrl-baf p 155
Glybenclamide (glyburide) ATP-dependent K+ channel inhibitor 25 μg/ml 1 g tlrl-gly p 155
Nuclear Export Inhibitor
WORKING
CONCENTRATION
QUANTITY
CATALOG
CODE
Leptomycin B NEW Nuclear export inhibitor 50 - 200 nM 5 μg tlrl-lep p 155
NF-kB Activation Inhibitors
Bay11-7082 IkB-a inhibitor 1 - 10 μM 10 mg tlrl-b82 p 155
Celastrol NF-kB inhibitor 2.5 - 10 μM 1 mg ant-cls p 155
Triptolide NF-kB activation inhibitor 10 - 100 nM 1 mg ant-tpl p 156
Mitogen-Activated Protein (MAP) Kinase Inhibitors
PD98059 MAP kinase kinase inhibitor 50 - 100 μM 10 mg tlrl-pd98 p 156
PD0325901 MEK inhibitor 0.5 μM 2 mg inh-pd32 p 156
SB203580 p38 MAP kinase inhibitor 1 - 20 μM 5 mg tlrl-sb20 p 156
SP600125 JNK inhibitor 10 - 500 μM 10 mg tlrl-sp60 p 156
U0126 MEK1-MEK2 inhibitor 10 - 50 μM 5 mg tlrl-u0126 p 156
Phosphatidylinositol 3-Kinase Inhibitors
3-Methyladenine PI3K inhibitor 5 mM 50 mg tlrl-3ma p 156
LY294002 PI3K inhibitor 50 - 100 μM 5 mg tlrl-ly29 p 156
Wortmannin PI3K inhibitor 0.1 - 2.5 μM 5 mg tlrl-wtm p 156
152 www.invivogen.com/inhibitors
INFO

PRODUCT DESCRIPTION
Protein Kinase and Protein Tyrosine Kinase Inhibitors
WORKING
CONCENTRATION
QUANTITY
CATALOG
CODE
2-Aminopurine PKR inhibitor 1 - 10 mM 250 mg tlrl-apr p 157
AG490 JAK family tyrosine kinase inhibitor 1 -100 μM 10 mg tlrl-ag4 p 157
H-89 PKA inhibitor 5 - 50 μM 5 mg tlrl-h89 p 157
Epigenetic Inhibitors
5-Aza-cytidine DNA methyltransferase inhibitor 2 μM 100 mg inh-aza p 157
5-Aza-2’-deoxycytidine DNA methyltransferase inhibitor 0.1 - 10 μM 10 mg met-adc-1 p 157
Bix-01294 G9a histone methyltransferase inhibitor 1 μM 2 mg inh-bix p 157
Trichostatin A Histone deacetylase inhibitor 150 - 300 nM 1 mg met-tsa-1 p 157
Valproic Acid Histone deacetylase inhibitor 2 mM 5 g inh-vpa p 157
Heat Shock Protein Inhibitors
Geldanamycin (GA) Hsp90 inhibitor 1 nM - 10 μM 5 mg ant-gl-5 p 158
17-AAG GA analogue 1 nM - 10 μM 5 mg ant-agl-5 p 158
17-DMAG GA analogue 1 nM - 10 μM 5 mg ant-dgl-5 p 158
17-AEP-GA GA analogue 1 nM - 10 μM 1 mg ant-egl-1 p 158
17-DMAP-GA GA analogue 1 nM - 10 μM 1 mg ant-mgl-1 p 158
17-GMB-APA-GA GA analogue for conjugation to MAb 1 nM - 10 μM 1 mg gmbapa-ga p 158
17-NHS-ALA-GA GA analogue 1 nM - 10 μM 1 mg ant-nhgl-1 p 158
Biotin-GA Biotin-labeled GA analogue 1 nM - 10 μM 1 mg ant-bgl-1 p 159
17-AAGH2 GA analogue 1 nM - 10 μM 1 mg ant-agh-1 p 159
17-DMAGH2 GA analogue 1 nM - 10 μM 1 mg ant-dgh-1 p 159
DNA Synthesis Inhibitors
5-Fluorocytosine DNA synthesis inhibitor 200 - 500 μg/ml 250 mg sud-5fc p 159
5-Fluorouracil DNA synthesis inhibitor 1 - 300 μg/ml 250 mg sud-5fu p 159
Ganciclovir DNA synthesis inhibitor 1 - 100 μg/ml 250 mg sud-gcv p 159
Contents and Storage
Products are provided as solids and shipped at room temperature. Store at room temperature, 4°C or -20°C according to the product label. Geldanamycin
and analogues, and triptolide should be kept in the dark. Products are stable at least 6 months.
INFO
www.invivogen.com/inhibitors 153
INHIBITORS 8

INHIBITORS 8
Pattern Recognition Receptor (PRR) Signaling Inhibitors
BX795 - TBK/IKKε Inhibitor
BX795, an aminopyrimidine compound, was developed as an inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1) 1 . It was recently shown to be a
potent inhibitor of the IKK-related kinases, TANK-binding kinase 1 (TBK1) and IKKε, and hence of IRF3 activation and IFN-β production 2 . BX795 inhibits the
catalytic activity of TBK1/IKKε by blocking their phosphorylation.
Chloroquine - Inhibitor of Endosomal Acidification
Chloroquine is a lysosomotropic agent that prevents endosomal acidification 3 . It accumulates inside the acidic parts of the cell, including endosomes and
lysosomes. This accumulation leads to inhibition of lysosomal enzymes that require an acidic pH, and prevents fusion of endosomes and lysosomes. Chloroquine
is commonly used to study the role of endosomal acidification in cellular processes, such as the signaling of intracellular TLRs 4, 5 .
CLI-095 - TLR4 Signaling Inhibitor
CLI095, also known as TAK-242, is a novel cyclohexene derivative that specifically suppresses TLR4 signaling, inhibiting the production of NO and proinflammatory
cytokines 6 . It acts by blocking the signaling mediated by the intracellular domain of TLR4, but not the extracellular domain. It potently suppresses
both ligand-dependent and -independent signaling of TLR4 7 .
Cyclosporin A - Calcineurin inhibitor NEW
Cyclopsporin A is a calcineurin inhibitor that exerts its immunosuppressive effects through the down-regulation of NFAT (nuclear factor of activated T cells)
transcription factor, thus preventing the transcription of T cell effector cytokines 8 . Moreover, NFAT has been implicated in the downstream signaling of
Dectin-1 which is important in the innate immune response against fungal infection 9 . Conversely, it has been demonstrated that inhibition of calcineurin in
macrophages enhances NF-κB activation 10 and can trigger TLR signaling by activating both the MyD88-dependent and the MyD88-independent pathways 11 .
Gefitinib - RIP2 Tyrosine Kinase inhibitor NEW
Gefitinib (also known as Iressa) is a selective inhibitor of epidermal growth factor, a growth factor that plays a pivotal role in the control of cell growth, apoptosis, and
angiogenesis. Recent studies demonstrated that Gefitinib can inhibit NOD2-induced cytokine release and NF-kB activation by inhibiting RIP2 (receptor-interacting
protein 2) tyrosine phosphylation which is critical for activation of NOD2 downsream signaling pathways 12 . Gefitinib is used in the treatment of advanced nonsmall cell
lung cancer 13 .
OxPAPC - TLR2 and TLR4 Inhibitor
OxPAPC (1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine), is an oxidized phospholipid that has been shown to inhibit the signaling induced by
bacterial lipopeptide and lipopolysaccharide (LPS). It acts by competing with CD14, LBP and MD2, the accessory proteins that interact with bacterial lipids,
thus blocking the signaling of TLR2 and TLR4 14, 15 .
Pepinh-MYD - MyD88 inhibitory peptide
Pepinh-MYD is a 26 aa peptide that blocks MyD88 signaling by inhibiting its homodimerization through binding. Pepinh-MYD contains a sequence from the
MyD88 TIR homodimerization domain (RDVLPGT) 16 preceeded by a protein transduction sequence (RQIKIWFQNRMKWKK) derived from antennapedia
which enables the peptide to translocate through the cell membrane 17 . Pepinh-MYD is provided with a control peptide.
Pepinh-TRIF - TRIF inhibitory peptide
Pepinh-TRIF is a 30 aa peptide that blocks TRIF signaling by interfering with TLR-TRIF interaction. Pepinh-TRIF contains the 14 aa that correspond to the
sequence of the BB loop of TRIF (FCEEFQVPGRGELH) 18 linked to the cell-penetrating segment of the antennapedia homoedomain
(RQIKIWFQNRMKWKK) 17 . Pepinh-TRIF is provided with a control peptide.
Polymyxin B - Inhibitor of LPS-induced TLR4 activation
Polymyxin B (PMB) is a cyclic cationic polypeptide antibiotic produced by the soil bacterium Paenibacillus polymixa. PMB blocks the biological effects of Gram
negative LPS (endotoxin) through binding to lipid A, the toxic component of LPS, which is negatively charged 19, 20 . The neutralizing effect of PMB on LPS is
dose-related and specific for LPS 21 . PMB is widely used to eliminate the effects of endotoxin contamination, both in vitro and in vivo.
Rapamycin - mTOR Inhibitor
Rapamycin is an inhibitor of the Ser/Thr protein kinase named "mammalian target of rapamycin" (mTOR) that regulates cell growth and metabolism in
response to environmental cues. mTOR is a downstream target of PI3K, an important actor in TLR signaling 22 . Rapamycin was shown to block TLR2- and
TLR4-mediated expression of TNF-a and IL-6 in neutrophils stimulated with Pam3CSK4 or LPS 23 .
Z-VAD-FMK - Caspase Inhibitor
Z-VAD-FMK is a cell-permeable pan-caspase inhibitor that irreversibly binds to the catalytic site of caspase proteases 24 . The peptide is O-methylated in the
P1 position on aspartic acid, providing enhanced stability and increased cell permeability. Z-VAD-FMK is used in apoptosis studies and also in inflammasome
studies. It is a potent inhibitor of caspase-1 activation in NLRP3-induced cells 25 .
1. Feldman RI. et al., 2005. Novel Small Molecule Inhibitors of 3-Phosphoinositide-dependent Kinase-1. J. Biol. Chem., 280: 19867 - 19874. 2. Clark K. et al., 2009. Use of the Pharmacological
Inhibitor BX795 to Study the Regulation and Physiological Roles of TBK1 and I{kappa}B Kinase {epsilon}: a distinct upstream kinase mediates Ser-172 phosphorylation and activation. J. Biol.
Chem. 284: 14136 - 14146. 3. Steinman RM. et al., 1983. Endocytosis and the recycling of plasma membrane. J. Cell. Biol. 96:1-27. 4. Rutz, M. et al., 2004. Toll-like receptor 9 binds singlestranded
CpG-DNA in a sequence- and pH-dependent manner. Eur. J. Immunol. 34:2541-2550. 5. Hart OM. et al., 2005.TLR7/8-Mediated Activation of Human NK Cells Results in Accessory
Cell-Dependent IFN-{gamma} Production. J. Immunol., 175: 1636 - 1642. 6. Li M. et al., 2006. A Novel Cyclohexene Derivative, Ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-
1-ene-1-carboxylate (TAK-242), Selectively Inhibits Toll-Like Receptor 4-Mediated Cytokine Production through Suppression of Intracellular Signaling. Mol. Pharmacol., 69: 1288 - 1295.
7. Kawamoto T. et al., 2008. TAK-242 selectively suppresses Toll-like receptor 4-signaling mediated by the intracellular domain. Eur J Pharmacol. 584(1):40-8. 8. Chow CW. et al., 1999.
Requirement for transcription factor NFAT in interleukin-2 expression. Mol Cell Biol. 19(3): 2300-07. 9. Goodridge et al. 2007. Dectin-1stimulation by Candida albicans yeast or zymosan
154 www.invivogen.com/inhibitors

triggers NFAT activation in macrophages and dendritic cells. J Immunol. 178( 5): 3107-15. 10. Asai T. et al., 2003. Activation of transcription factors AP-1 and NF-κB in chronic cyclosporine
A nephrotoxicity: role in beneficial effects of magnesium supplementation. Transplantation 75(7):1040–4. 11. Kang Y. et al., 2007. Calcineurin negatively regulates TLR-mediated activation
pathways. J Immunol 179(7):4598–607. 12. Tigno-Aranjuez J. et al., 2010. Inhibition of RIP2’s tyrosine kinase activity limits NOD2-driven cytokine responses. Genes Dev. 24(23):2666-77.
13. Maemondo M et al. 2010. Gefitinib or Chemotherapy for Non–Small-Cell Lung Cancer with Mutated EGFR N Engl J Med. 362(25):2380-8. 14. Erridge C. et al., 2008. Oxidized Phospholipid
Inhibition of Toll-like Receptor (TLR) Signaling Is Restricted to TLR2 and TLR4: roles for CD14, LPS-binding protein, and MD2 as targets for specificity of inhibition. J. Biol. Chem., 283: 24748 -
24759. 15. von Schlieffen E. et al., 2009. Multi-Hit Inhibition of Circulating and Cell-Associated Components of the Toll-Like Receptor 4 Pathway by Oxidized Phospholipids. Arterioscler Thromb
Vasc Biol, 29: 356 - 362. 16. Loiarro M. et al., 2005. Peptide-mediated Interference of TIR Domain Dimerization in MyD88 Inhibits Interleukin-1-dependent Activation of NF-{kappa}B. J. Biol.
Chem., 280: 15809-14. 17. Derossi D. et al., 1994. The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem., 269: 10444-50. 18. Toshchakov
VU. et al., 2005. Differential Involvement of BB Loops of Toll-IL-1 Resistance (TIR) Domain-Containing Adapter Proteins in TLR4- versus TLR2-Mediated Signal Transduction. J. Immunol., 175:
494 - 500. 19. Palmer JD, Rifkind D. 1974. Neutralization of the hemodynamic effects of endotoxin by polymyxin B. Surg Gynecol Obstet. 138(5):755-9. 20. Lindemann.RA 1988. Bacterial
activation of human natural killer cells: role of cell surface lipopolysaccharide. Infect Immun. 56 (5): 1301–1308. 21. Duff GW, Atkins E. 1982. The inhibitory effect of polymyxin B on endotoxininduced
endogenous pyrogen production. J Immunol Methods. 52(3):333-40. 22. Kuo CC. et al., 2006. Class I and III Phosphatidylinositol 3'-Kinase Play Distinct Roles in TLR Signaling Pathway.
J. Immunol., 176: 5943 - 5949. 23 Lorne E. et al., 2009. Participation of Mammalian Target of Rapamycin Complex 1 in Toll-Like Receptor 2– and 4–Induced Neutrophil Activation and Acute
Lung Injury. Am. J. Respir. Cell Mol. Biol., 41: 237 - 245. 24. Slee EA. et al., 1996. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the
processing of CPP32. Biochem J. 315 ( Pt 1):21-4. 25. Dostert C. et al., 2009. Malarial hemozoin is a Nalp3 inflammasome activating danger signal. PLoS One. 4(8):e6510.
Cytokine Receptor Inhibitor
SB431542 - TGF-b Receptor Inhibitor
SB431542 is a potent and selective inhibitor of transforming growth factor-b (TGF-b) superfamily type I activin receptor-like kinase (ALK) receptors, specifically
ALK4, ALK5 and ALK7 1 . Inhibition of TGF-b signaling is known to induce the de-repression of epithelial fate and thus was hypothesized to benefit the
reprogramming process. Indeed, treatment of OSKM-transduced human primary fibroblasts with a combination of SB431542 and PD0325901, a MEK
inhibitor, was found to improve reprogramming efficiency of human cells 2 .
1. IInman GJ. et al., 2002. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4,
ALK5, and ALK7. Mol Pharmacol. 2002 Jul;62(1):65-74. 2. Lin T. et al., 2009. A chemical platform for improved induction of human iPSCs. Nat Methods. 6(11):805-8.
Ion Channel Inhibitors
Bafilomycin A1 - Proton pump inhibitor
Bafilomycin A1 is a specific inhibitor of the lysosomal proton pump, thus it indirectly inhibits lysosomal enzymes which have acidic pH optima. Bafilomycin
treatment has been shown to inhibit fusion of autophagosomes with both endosomes and lysosomes 1, 2 .
Glybenclamide (glyburide) - Potassium channel inhibitor
Glybenclamide, also known as glyburide, blocks the maturation of caspase-1 and pro-IL-1b by inhibiting the K+ efflux 3 . Glybenclamide was shown to potently
block the activation of the NLRP3 inflammasome induced by PAMPs, DAMPs and crystalline substances 4 . Recent data suggest that glybenclamide works
downstream of the P2X 7 receptor but upstream of NLRP3.
1. Yamamoto A. et al., 1998. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells.
Cell Struct Funct. 23(1):33-42. 2. Mousavi SA. et al., 2001. Effects of inhibitors of the vacuolar proton pump on hepatic heterophagy and autophagy. Biochim Biophys Acta. 1510(1-2):243-57
3. Laliberte RE. et al., 1999. ATP treatment of human monocytes promotes caspase-1 maturation and externalization. J Biol Chem. 274(52):36944-51. 4. Lamkanfi M. et al., 2009. Glyburide
inhibits the Cryopyrin/Nalp3 inflammasome. J. Cell Biol., 187: 61 - 70. .
Nuclear Export Inhibitor
Leptomycin B - Nuclear export inhibitor NEW
Leptomycin B, an inhibitor of nuclear export, can be used to study nucleo-cytoplasmic translocation. It has been demonstrated that Leptomycin B can cause the nuclear
accumulation of proteins that shuttle between the cytosol and nucleus such as IRAK-1 and NLRC5 1, 2 . The cellular target of Leptomycin B has been identified as
CRM1 (exportin 1), an evolutionarily conserved receptor for the nuclear export signal of proteins 3 .
1. Liu G. et al., 2008. Interleukin-1 receptor-associated kinase (IRAK)-1-mediated NF-kB activation requires cytosolic and nuclear activity. FASEB J 22(7): 2285-96. 2. Benko S. et al., 2010.
NLRC5 limits the activation of inflammatory pathways. J Immunol. 185(3):1681-91. 3. Kudo N. et al., 1999. Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine
residue in the central conserved region. PNAS. 96(16):9112-7.
NF-kB Activation Inhibitors
Bay 11-7082 - IkB-a Inhibitor
Bay 11-7082 is an irreversible inhibitor of TNF-a-induced IkB-a phosphorylation resulting in the inactivation of NF-kB 1 . TNF-a-dependent effects of NF-kB
are important for TLR expression and cytokine production 2, 3 .
Celastrol - NF-kB Inhibitor
Celastrol is a triterpenoid compound isolated from the medicinal plant Tripterygium wilfordii known for its anti-inflammatory properties. Its mode of action
and spectrum of cellular targets are still poorly understood. Celastrol was recently shown to act as an effective inhibitor of the transcription factor NF-kB
resulting in the attenuation of nitric oxide and proinflammatory cytokine production 4, 5 .
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INHIBITORS 8

INHIBITORS 8
Triptolide - NF-kB Inhibitor
Triptolide, a diterpenoid isolated from the Chinese herb Tripterygium wilfordii hook F, has been used for centuries in traditional Chinese medicine to treat
immune-related disorders. Current knowledge regarding its mechanism of action is still limited. So far, triptolide is known to block the activation of NF-kB 6,
inhibit the heat shock response 7 and induce caspase activation 8 .
1. Pierce JW. et al., 1997. Novel Inhibitors of Cytokine-induced Ikappa Balpha Phosphorylation and Endothelial Cell Adhesion Molecule Expression Show Anti-inflammatory Effects in Vivo. J.
Biol. Chem., 272: 21096. 2. Phulwani NK. et al., 2008. TLR2 Expression in Astrocytes Is Induced by TNF-{alpha}- and NF-{kappa}B-Dependent Pathways. J. Immunol., 181: 3841-3849.
3. Méndez-Samperio P. et al., 2008. Mycobacterium bovis Bacillus Calmette-Guérin Induces CCL5 Secretion via the Toll-Like Receptor 2-NF-{kappa}B and -Jun N-Terminal Kinase Signaling
Pathways. Clin. Vaccine Immunol. 15: 277 - 283. 4 Sethi G. et al., 2007. Celastrol, a novel triterpene, potentiates TNF-induced apoptosis and suppresses invasion of tumor cells by inhibiting
NF-kB–regulated gene products and TAK1-mediated NF-B activation. Blood, 109: 2727 - 2735. 5. Jung HW. et al., 2007. Celastrol inhibits production of nitric oxide and proinflammatory
cytokines through MAPK signal transduction and NF-kappaB in LPS-stimulated BV-2 microglial cells. Exp Mol Med. 39(6):715-21. 6. Lee KY. et al., 1999. PG490 (triptolide) cooperates with
tumor necrosis factor-alpha to induce apoptosis in tumor cells. J Biol Chem. 274: 13451-13455. 7. Westerheide SD. et al., 2006. Triptolide, an Inhibitor of the Human Heat Shock Response
That Enhances Stress-induced Cell Death. J. Biol. Chem., 281: 9616 - 9622. 8. Carter BZ. et al., 2006. Triptolide induces caspase-dependent cell death mediated via the mitochondrial pathway
in leukemic cells. Blood 108: 630 - 7.
Mitogen-Activated Protein (MAP) Kinase Inhibitors
PD98059 - MAP Kinase Kinase Inhibitor
PD98059 is a potent and selective inhibitor of MAP kinase kinase (also known as MAPK/ERK kinase or MEK kinase) 1 . It mediates its inhibitory properties by
binding to the ERK-specific MAP kinase MEK, therefore preventing phosphorylation of ERK1/2 (p44/p42 MAPK) by MEK1/2.
PD0325901 - MEK Inhibitor
PD0325901 is a selective inhibitor of mitogen-activated protein kinase kinase (MEK), with potential antineoplastic activity 2 . MEK is a key component of the
RAS/RAF/MEK/ERK signaling pathway that is frequently activated in human tumors. This pathway plays also an important role in the self-renewing state of
embryonic stem cells. Treatment with PD0325901 has been shown to enhance reprogramming efficiencies of induced pluripotent stem cells 3 .
SB203580 - p38/RK MAP Kinase Inhibitor
SB203580 is a pyridinyl imidazole inhibitor widely used to elucidate the roles of p38 mitogen-activated protein (MAP) kinase 4 . SB203580 inhibits also the
phosphorylation and activation of protein kinase B (PKB, also known as Akt) 5 .
SP600125 - JNK Inhibitor
SP600125 is a potent, cell-permeable, selective and reversible inhibitor of c-Jun N-terminal kinase (JNK) 6 . It inhibits in a dose-dependent manner the phosphorylation
of JNK. JNK is a member of the mitogen-activated protein kinase (MAPK) family and plays an essential role in TLR-mediated inflammatory responses.
U0126 - MEK1 and MEK2 Inhibitor
U0126 is a selective inhibitor of the MAP kinase kinases, MEK1 and MEK2. It acts by inhibiting the kinase activity of MEK1/2 thus preventing the activation of
MAP kinases p42 and p44 which are encoded by the erk2 and erk1 genes respectively 7 .
1. Alessi DR. et al., 1995. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270, 27489-27494. 2. Kohno M. &
Pouyssegur J. 2006. Targeting the ERK signaling pathway in cancer therapy. Ann Med. 38(3):200-11. 3. Lin T. et al., 2009. A chemical platform for improved induction of human iPSCs.
Nat Methods. 6(11):805-8. 4. Cuenda A. et al., 1995. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett.
364:229-233. 5. Lali FV. et al., 2000.The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma
hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J Biol Chem. 275(10):7395-402. 6. Bennett BL. et al., 2001. SP600125, an
anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc. Natl. Acad. Sci. USA. 98:13681-13686. 7. Favata MF. et al., 1998. Identification of a novel inhibitor of mitogen-activated protein
kinase.J. Biol. Chem. 273(29):18623-32.
Phosphatidylinositol 3-Kinase (PI3K) Inhibitors
3-Methyladenine - PI3K inhibitor
3-Methyladenine is commonly used as a specific inhibitor of autophagic sequestration 1 . It blocks autophagy by inhibition of phosphatidylinositol 3-kinase
(PI3K) activity, an enzyme required for autophagy 2 .
LY294002 - PI3K Inhibitor
LY294002 is a potent, cell permeable inhibitor of PI3K that acts on the ATP binding site of the enzyme 3 . LY294002 is often used to study the role of PI3K
in apoptosis 4 and autophagy 2 .
Wortmannin - PI3K Inhibitor
Wortmannin is a cell-permeable, fungal metabolite that acts as a potent, selective and irrevesible inhibitor of PI3K 5 . Wortmannin has been used to determine
the involvement of PI3K in many cellular processes, such as apoptosis 4 , autophagy 2 and TLR signaling 6 .
1. Seglen PO. & Gordon PB., 1982. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. PNAS. 1982 Mar;79(6):1889-92.
2. Blommaart EF. et al., 1997. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur. J. Biochem. 243: 240–246.
3. Vlahos CJ. et al., 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem 69(7):5241-8. 4. Duronio V.,
2008. The life of a cell: apoptosis regulation by the PI3K/PKB pathway. Biochem J. 415(3):333-44. 5. Arcaro A. and Wymann MP., 1993. Wortmannin is a potent phosphatidylinositol 3-kinase
inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem. J. 296:297-301. 6. Fukao T and Koyasu S., 2003. PI3K and negative regulation of TLR signaling.
Trends Immunol 24: 358-363.
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Protein Kinase and Protein Tyrosine Kinase Inhibitors
2-Aminopurine - PKR Inhibitor
2-aminopurine (2-AP) is a potent inhibitor of double-stranded RNA (dsRNA)-activated protein kinase (PKR), a critical mediator of apoptosis. PKR is
phosphorylated and activated by dsRNA and poly(I:C) 1 and contributes to the induction of type I interferons, such as IFN-b, which can further increase its
expression 2 . PKR plays also a role in TLR-induced antiviral activities as an intermediary in TLR3, TLR4 and TLR9 signaling 3 .
AG490 - JAK2 Inhibitor
AG490 is a specific and potent inhibitor of the Janus kinase 2 protein (JAK2) 4 AG490 is used to inhibit phosphorylation of the tyrosine kinase receptor EGFR
and signal transducer and activator of transcription 3 (STAT-3), and subsequently reduce invasion and adhesion potential of malignant cells 5 .
H-89 - PKA Inhibitor
H-89 is a selective, potent cell permeable inhibitor of cAMP-dependent protein kinase (PKA) 6 . It can be used to determine the role of PKA in TLR and other
PRR mediated signaling. PKA has be shown to participate in the TLR-mediated TREM-1 expression on macrophages following LPS stimulation ,7 .
1. Kaufman RJ., 1999. Double-stranded RNA-activated protein kinase mediated virus-induced apoptosis: a new role for an old actor. Proc. Natl. Acad. Sci. USA 96 (21):11693-5. 2. Samuel
CE., 2001. Antiviral actions of interferons. Clin Microbiol Rev. 14(4):778-809. 3. García MA. et al., 2006. Impact of Protein Kinase PKR in Cell Biology: from Antiviral to Antiproliferative
Action,Microbiol. Mol. Biol. Rev., 70: 1032 - 1060. 4. Levitzki A., 1990. Tyrphostins- potential antiprolierative agents and novel molecular tools. Biochem. Pharmacol. 40:913-918. 5. Caceres-
Cortes JR., 2008. A potent anti-carcinoma and anti-acute myeloblastic leukemia agent, AG490. Anticancer Agents Med Chem. 8(7):717-22. 6. Chijiwa, T., et al.,1990. Inhibition of forskolininduced
neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)
ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J. Biol. Chem. 265: 5267-5272. 7. Murakami Y. et al., 2007. Lipopolysaccharide-Induced Up-Regulation of Triggering
Receptor Expressed on Myeloid Cells-1 Expression on Macrophages Is Regulated by Endogenous Prostaglandin E2. J. Immunol. 178: 44. 2
Epigenetic Inhibitors
5-Aza-cytidine - DNA Methyltransferase Inhibitor
5-aza-cytidine (AZA) is a potent inhibitor of DNA methyltransferase 1 (DNMT1), known to induce demethylation and reactivation of silenced genes. In a
recent article, reactivation of hypermethylated pluripotency genes was suggested to be important for complete reprogramming. Consistent with this notion,
AZA was shown to facilitate the transition to full pluripotency of partially reprogrammed cell line and to increase the number of ES cell-like colonies 1 .
Hangfu et al. confirmed that AZA treatment promotes reprogramming efficiency 2 .
5-Aza-2’-deoxycytidine - DNA Methyltransferase Inhibitor
5-Aza-2’-deoxycytidine (5-AzadCyD, 5-Aza-CdR, decitabine) is a specific inhibitor of DNA methylation. It functions in a similar manner to aza-citidine, although
decitabine can only be incorporated into DNA strands while azacitidine can be incorporated into both DNA and RNA chains. 5-AzadCyD is a prodrug that
requires activation via phosphorylation by deoxycytidine kinase. The nucleotide analog is incorporated into DNA, where it produces an irreversible inactivation
of DNA methyltransferase. 5-AzadCyD was demonstrated to be a potent antineoplastic agent against leukemia and tumors in animal models 3, 4 .
Bix-01294 - G9a Histone Methyltransferase Inhibitor
Bix-01294 is an inhibitor of the G9a histone methyltransferase, a key regulator of DNA methylation and transcriptional silencing in pluripotent cells 5 . Bix-
01294 is believed to facilitate the reactivation of pluripotency genes and induce passive demethylation, thus promoting reprogramming. Indeed, Bix-01294
was found to improve reprogramming efficiencies of Oct4-Klf4-(OK)-infected neural progenitor cells by approximately 8 fold 6 .
Trichostatin A - Histone deacetylase Inhibitor
Trichostatin A (TSA) is a potent and specific inhibitor of histone deacetylase (HDAC). TSA suppresses the activity of HDAC leading to an increase in histone
acetylation. TSA has been shown to induce apoptosis in many cancer cells at submicromolar concentrations with very low toxicity toward normal cells 7 .
Furthermore, TSA is used to improve the genomic reprogramming of embryos generated by somatic cell nuclear transfer 8 .
Valproic acid - Histone deacetylase Inhibitor
Valproic acid (VPA) is a histone deacetylase inhibitor with potent antitumor activity 9 . VPA treatment promotes histone acetylation allowing the chromatin to
adopt a relaxed structure facilitating the binding of ectopically expressed transcription factors or downstream secondary factors. VPA was found to significantly
enhance reprogramming efficiencies of OSKM-, OSK- and OS-infected fibroblasts, eliminating the need for the oncogenes c-Myc or Klf4 2, 10 . VPA is believed
to induce global transcriptional changes, in particular upregulating ES cell-specific genes while downregulating MEF-specific genes.
1. Mikkelsen TS. et al., 2008. Dissecting direct reprogramming through integrative genomic analysis. Nature. 454(7200):49-55. 2. Huangfu D. et al., 2008a. Induction of pluripotent
stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol. 26(7):795-7. 3. Christman JK., 2002. 5-Azacytidine and 5-aza-20-deoxycytidine
as inhibitors of DNA methylation: Mechanistic studies and their implications for cancer therapy. Oncogene 21, 5483–5495. 4. Kulis M. & Esteller M., 2010. DNA methylation and
cancer. Adv Genet. 2010;70:27-56. 5. Epsztejn-Litman S. et al., 2008. De novo DNA methylation promoted by G9a prevents reprogramming of embryonically silenced genes.
Nat Struct Mol Biol. 15(11):1176-83. 6. Shi Y et al., 2008. A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell.
2(6):525-8. 7. David M. et al., 2001. Trichostatin A Is a Histone Deacetylase Inhibitor with Potent Antitumor Activity against Breast Cancer in Vivo. Clin. Cancer Res., Apr 2001;
7: 971 - 976. 8. Bui H-T, et al., 2010. Effect of Trichostatin A on Chromatin Remodeling, Histone Modifications, DNA Replication, and Transcriptional Activity in Cloned Mouse
Embryos. Biol Reprod, 83: 454 - 463. 9. Duenas-Gonzalez A. et al., 2008. Valproic acid as epigenetic cancer drug: preclinical, clinical and transcriptional effects on solid tumors.
Cancer Treat Rev. 34(3):206-22. 10 . Huangfu D. et al., 2008b. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat Biotechnol.
26(11):1269-75.
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INHIBITORS 8

INHIBITORS 8
Heat Shock Protein Inhibitors
Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone critical for the folding, assembly and activity of multiple mutated and overexpressed
signaling proteins that promote the growth and/or survival of tumor cells. Hsp90 client proteins include mutated p53, Raf-1, Akt, ErbB2 and hypoxiainducible
factor 1a (HIF-1a). Geldanamycin (GA), a benzoquinone ansamycin antibiotic, selectively inhibits Hsp90 leading to the degradation of its client
proteins. GA inhibits the proliferation of cancer cells and shows anti-cancer activity in experimental animals. However due to poor aqueous solubility and
liver toxicity, GA has not moved forward in clinical trials. To overcome these undesirable properties, numerous GA analogues have been synthesized which
differ only in their 17-substituent.
Geldanamycin - HSP90 Inhibitor
Geldanamycin (GA) is a natural product produced by Streptomyces hygroscopicus.
InvivoGen produces GA from a mutant strain of S. hygroscopicus, inactivated for the
synthesis of nigericin, a common contaminant of GA. GA binds with high affinity into the
ATP binding pocket of Hsp90. Binding of GA to Hsp90 causes the destabilization and
degradation of its client proteins 1 , thereby inhibiting the oncogenic activity of these
proteins 2 .
17-AAG - Less Toxic and More Stable GA Analogue
17-Allylamino-17-demethoxygeldanamycin (17-AAG) is an analogue chemically derived
from GA. 17-AAG is a less toxic and more stable analogue of geldanamycin (GA) 3 . Even
though 17-AAG binding to Hsp90 is weaker than GA, 17-AAG displays similar antitumor
effects as GA and a better toxicity profile. 17-AAG is currently in phase I clinical trial in
several centers worldwide. Preliminary data obtained from these trials demonstrate that
antitumor activity is achieved at concentrations below the maximum tolerated dose 4 .
17-DMAG - Water-soluble GA Analogue
17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG, NSC 707545)
is the first water-soluble analogue of 17-AAG. This Hsp90 inhibitor shows promise in
preclinical models 5 . 17-DMAG has excellent bioavailability, is widely distributed to tissues,
and is quantitatively metabolized much less than is 17-AAG 6 .
The use of 17-DMAG is covered under US Patent 6,890,917 owned and licensed by the NIH to
InvivoGen.
17-AEP-GA - Water-soluble GA Analogue
17-[2-(Pyrrolidin-1-yl)ethyl]amino-17-demethoxygeldanamycin (17-AEP-GA) is a new
geldanamycin (GA) analogue with an alkylamino group in place of the methoxy moiety at
C17. 17-AEP-GA is less cytotoxic than GA and remains biologically active 7 . 17-AEP-GA
was shown to induce similar tumor cell growth inhibition than 17-AAG and, unlike 17-
AAG which is soluble in DMSO, to be water soluble.
17-DMAP-GA - Water-soluble GA Analogue
17-(Dimethylaminopropylamino)-17-demethoxygeldanamycin (17-DMAP-GA) belongs to
a new set of geldanamycin analogues that have been synthesized based on binding affinity
to Hsp90 and water solubility. 17-DMAP-GA was shown to greatly inhibit the growth of
cancer cells (IC50 below 100 nM) 7 . Its binding affinity to Hsp90 was not significantly affected
while its water solubility was highly improved compared to 17-AAG.
17-GMB-APA-GA - GA Analogue for Conjugation to a Monoclonal Antibody
17-GMB-APA-GA is a maleimido derivative of geldanamycin that enables the conjugation
of GA to a monoclonal antibody (mAb) such as Herceptin, the first mAb approved for
therapy of solid tumors. This geldanamycin immunoconjugate induces less systemic toxicity
than GA by being selectively delivered into malignant cells, a property conferr ed by the
mAb that acts as the targeting vehicle. NCI has reported that Herceptin-GA conjugates
deliver a more potent selective cytotoxic impact to Her2-overexpressing tumors than
Herceptin alone 8 . To prepare such conjugates, GA is modified to introduce a latent primary
amine 9 . After deprotection, this primary amine provides a site for introduction of a
maleimide group that enables linkage to proteins.
17-NHS-ALA-GA - GA Analogue for Coupling of NH2-Containing Molecules
17-NHS-ALA-GA is an NHS (N-hydroxysuccinimide) activated geldanamycin analogue
designed for easy coupling of NH2-containing molecules. NHS coupling forms a chemically
stable amide bond with ligands containing primary amino groups such as small proteins
and peptides. Thus, 17-NHS-ALA-GA can be used for conjugation of GA to a monoclonal
antibody, similarly to 17-GMB-APA-GA. It can also be used to perform affinity
chromatography to purify GA binding proteins such as Hsp90.
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Geldanamycin
C29H40N2O9
MW: 560
Purity: 99%
17-AAG
C31H43N3O8
MW 586
Purity: 99%
17-DMAG
C32H48N4O8, HCl
MW: 617
Purity: 99%
17-AEP-GA
C34H50N4O8
MW: 643
Purity: 99%
17-DMAP-GA
C33H50N4O8
MW: 631
Purity: 99%
17-GMB-APA-GA
C39H53N5O11
MW: 768
Purity: 99%
17-NHS-ALA-GA
C44H84N4O12
MW: 841
Purity: 99%
17

Biotin-GA - Labeled GA Analogue
Biotin-GA was generated by biotinylation of geldanamycin at the C17 position. This labeled
GA can be used to identify novel Hsp90 inhibitors through time-resolved fluorescence
resonance energy transfer-based HTS that measures the binding of biotin-GA to the Nterminal
ATP-binding domain of Hsp90 10 .
17-AAGH2 & 17-DMAGH2 - Hydroquinone GA Analogues
The selective toxicity of benzoquinone ansamycins in tumor cells can be explained by
their reduction to hydroquinones. Indeed, many human cancer cells express at high
levels NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoenzyme that catalyzes the
direct reduction of quinones to hydroquinones. NQO1 has been shown to reduce
17-AAG and to increase the sensitivity to 17-AAG of cancer cell lines 11 . Recent studies
have demonstrated that the reduction product of 17-AAG, 17AAGH2, is a more
potent inhibitor of Hsp90 than the 17-AAG itself 12 , as well as 17-DMAGH2 compared
to 17-DMAG 13 .
InvivoGen provides 17-AAGH2 and 17-DMAGH2 produced by reduction of 17-AAG
and 17-DMAG respectively using a chemical reducing agent. Their purity is 99%.
www.invivogen.com/inhibitors
Biotin-GA
C56H88N8O12S
MW: 1097
Purity: 99%
1. Whitesell L. et al., 1994. Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic
transformation. PNAS 91(18):8324-8. 2. Neckers L., 2002. Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol Med 8(4 Suppl):S55-61. 3. Schulte TW. & Neckers LM., 1998.
The benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin binds to HSP90 and shares important biologic activities with geldanamycin. Cancer Chemother Pharmacol 42(4):273-9.
4. Agnew EB. et al. 2001. Measurement of the novel antitumor agent 17-(allylamino)-17-demethoxygeldanamycin in human plasma by high-performance liquid chromatography. J Chromatogr B
Biomed Sci Appl 755:237-43. 5. Workman P., 2003. Overview: translating Hsp90 biology into Hsp90 drugs. Curr Cancer Drug Targets 3(5):297-300. 6. Egorin MJ, et al., 2002. Pharmacokinetics,
tissue distribution, and metabolism of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (NSC 707545) in CD2F1 mice and Fischer 344 rats. Cancer Chemother Pharmacol 49(1):7-
19. 7. Tian ZQ. et al., 2004. Synthesis and biological activities of novel 17-aminogeldanamycin derivatives.Bioorg Med Chem. 12(20):5317-29. 8. Mandler R. et al., 2004. Herceptin-geldanamycin
immunoconjugates: pharmacokinetics, biodistribution, and enhanced antitumor activity. Cancer Res. 64(4):1460-7. 9. Mandler R. et al., 2002. Modifications in synthesis strategy improve the
yield and efficacy of geldanamycin-herceptin immunoconjugates. Bioconjug Chem. 13(4):786-91. 10. Zhou V. et al., 2004. A time-resolved fluorescence resonance energy transfer-based HTS
assay and a surface plasmon resonance-based binding assay for heat shock protein 90 inhibitors. Anal Biochem. 331(2):349-57. 11. Kelland LR et al., 1999. DT-Diaphorase expression and
tumor cell sensitivity to17-allylamino, 17-demethoxygeldanamycin, an inhibitor of heat shock protein 90. J Natl Cancer Inst. 91(22):1940-9. 12. Guo W et al., 2005. Formation of 17-allylaminodemethoxygeldanamycin
(17-AAG) hydroquinone by NAD(P)H:quinone oxidoreductase 1: role of 17-AAG hydroquinone in heat shock protein 90 inhibition. Cancer Res. 65(21):10006-15. 13.
Guo W et al., 2006. The bioreduction of a series of benzoquinone ansamycins by NAD(P)H:quinone oxidoreductase 1 to more potent heat shock protein 90 inhibitors, the hydroquinone
ansamycins. Mol Pharmacol. 70(4):1194-203.
DNA Synthesis Inhibitors
5-Fluorocytosine - DNA Synthesis Inhibitor
5-Fluorocytosine (5-FC) is a fluorinated cytosine analogue, clinically approved as an antifungal agent. 5-FC is nontoxic to mammalian cells due to their lack
of the enzyme cytosine deaminase (CD). CD converts 5-FC into 5-fluorouracil (5-FU), a highly cytotoxic compound routinely used in cancer chemotherapy.
5-FC is used in combination with the E. coli CD gene (codA) or S. cerevisiae CD gene (fcy) in suicide gene therapy protocols 1 .
5-Fluorouracil - Thymidylate Synthase Inhibitor
5-Fluorouracil (5-FU), a fluorinated analogue of uracil, is approved for cancer chemotherapy as an antineoplastic, antimetabolic agent. The cytotoxic effects
of 5-FU occur mainly following its conversion to 5-fluoro-deoxyuridine monophosphate (5-FdUMP), an irreversible inhibitor of thymidylate synthase. This
leads to cell death by DNA synthesis inhibition through deoxythymidine triphosphate deprivation.
Ganciclovir - DNA Synthesis Inhibitor
Ganciclovir (GCV) is a synthetic analogue of 2'-deoxy-guanosine, clinically approved for the treatment of cytomegalovirus (CMV) infections. GCV is used as
a prodrug to obtain a suicide effect in cells transfected with the herpes virus thymidine kinase gene (HSV-tk) 1 . HSV-TK phosphorylates GCV to GCVmonophosphate
which is further converted to GCV-diphosphate and GCV-triphosphate by host kinases. GCV-triphosphate causes premature DNA chain
termination and apoptosis. The HSV-tk gene and ganciclovir are used in molecular biology for negative selection 2 .
1. Aghi M. et al., 2000. Prodrug activation enzymes in cancer gene therapy. J Gene Med. 2(3):148-64. 2. Karreman C., 1998. New positive/negative selectable markers for mammalian cells on
the basis of Blasticidin deaminase-thymidine kinase fusions. Nucleic Acids Res. 26(10):2508-10.
159
INHIBITORS 8

9
CUSTOM SERVICES
TLR/NOD Ligand Screening 161
Custom-Made CpG-free Genes 161
Custom Cloning 162
Custom-Made psiRNA 162
Custom-Made pDRIVE 162
Large Scale Plasmid DNA Production 163

TLR/NOD Ligand Screening
InvivoGen has developed a high-throughput method to determine whether a compound is recognized by Toll-Like Receptors (TLRs) or
NOD1/NOD2 and acts either as an agonist or antagonist. This method is based on TLR- or NOD-induced activation of various transcription
factors including NF-kB and AP1 in HEK293 clones.
Description
TLR Ligand Screening
Two choices of services are offered, level 1 and level 2, that can be
performed sequentially or individually.
Level 1: Single dose testing on a panel of human TLRs
Testing is carried out on seven TLRs: TLR2, TLR3, TLR4, TLR5, TLR7, TLR8
and TLR9.
Screening is performed at a single concentration, typically a 1/10 dilution
of the original compound solution provided, or customer specified.
Duplicate tests (plus controls, see list).
Level 2: Dose response on one or two TLRs
Three concentrations of the compound(s), typically 1/10, 1/100 and
1/1000 dilutions of the original compound solution, are tested on the
TLR(s) recognizing the compound(s) as determined in level 1 or specified
by the customer. Triplicate tests (plus controls).
NOD Ligand Screening
Two cytoplasmic proteins involved in pathogen recognition, NOD1 and
NOD2, can be added to this screening service. Their activation by a given
compound is detected using the same method as the one described above.
A detailed report will be prepared and provided to the customer
electronically and in hard copy. All procedures are performed accordingly
to strict guidelines. Confidentiality is guaranteed.
Custom-Made CpG-Free Genes
www.invivogen.com/custom-ser vices
PRODUCT CAT. CODE
TLR / NOD Ligand Screening tlrl-test
For more information, see page 91
InvivoGen is an expert in developing CpG-Free genes and now offers a CpG-Free gene custom service. Send us the sequence of your gene
of interest, and our specialists will design, synthesize and clone into an expression vector a CpG-Free allele of this gene. We guarantee that
the sequence of the synthetic gene will 100% match the designed sequence.
Description
Our experts will design the sequence of a CpG-free allele of your gene
of interest using a proprietary software that allows the elimination of all
CpG dinucleotides, the humanization of the sequence, the elimination of
secondary structures, and the elimination/reduction of dam and dcm
methylations to diminish E. coli signature
The Custom-made CpG-free Gene is 100% sequence verified and
provided into the pMA plasmid (see map page 139).
Contents and Storage
Custom-made CpG-free genes are provided as 5 μg of lyophilized DNA.
Products are shipped at room temperature. Store at -20°C.
PRODUCT QTY CAT. CODE
Custom-made CpG-free Gene 20 μg p-custom
For more information, see page 139
161
CUSTOM SERVICES 9

CUSTOM SERVICES 9
Custom Cloning : Many Combinations Possible According to Your Aims
At the leading edge of research and development in vector construction, gene delivery, and gene expression systems, our specialists are pleased
to provide an extensive range of customized products. With more than 20 years of experience in cloning, we offer custom cloning services at
very competitive rates and short turn-around time.
➥ Wide Exper tise and Consultation Available
➥ Customized Scientific Suppor t
➥ Rapid Processing
➥ Your Product Ready-to-Use
➥ Cost-Effective
Services
Custom-Made pSELECT
pORF and pMOD plasmids are not selectable in mammalian cells. If you
are planning on stably expressing an open reading frame provided in a
pORF or pMOD, our experts can subclone this open reading frame into
a pSELECT plasmid carrying the selectable marker of your choice:
- pSELECT-blasti - pSELECT-puro
- pSELECT-hygro - pSELECT-zeo
- pSELECT-neo - pSELECT-gfpzeo
Custom-Made pDRIVE
You can create your own composite promoter by combining the enhancer,
core promoter and 5’UTR of your choice. We will assemble this composite
promoter in a pDRIVE plasmid harboring a reporter gene from the list
below:
- pDRIVE-LacZ
- pDRIVE5-GFP
- pDRIVE5-SEAP
Custom-made pDRIVE carry the Zeocin resistance gene for selection
and amplification in E. coli. We can replace the bacterial selection cassette
with a mammalian selection cassette of your choice derived from a
pSELECT plasmid.
Custom-Made psiRNA (see page 150)
You can either send us your siRNA/shRNA sequence or we can select the
best candidate siRNA sequence for your target gene using the siRNA
Wizard software. The insert encoding the expected shRNA sequence will
be cloned in the psiRNA-h7SK plasmid which is available with different
selectable markers:
- psiRNA-h7SKblasti - psiRNA-h7SKzeo
- psiRNA-h7SKhygro - psiRNA-h7SKGFPzeo
- psiRNA-h7SKneo
Contents and Storage
Custom-made plasmids (pSELECT, pDRIVE or psiRNA) are provided as
20 μg of lyophilized DNA.
Custom-made psiRNA plasmids are also available as a kit that contains
20 μg of custom-made psiRNA, 20 µg of a control psiRNA, 1 vial of
LyoComp GT116 (E. coli competent cells, page 43), and 4 pouches of
the corresponding Fast-Media ® (E. coli selection medium, pages 44-45).
Products are shipped at room temperature. Store at -20°C.
162
www.invivogen.com/custom-cloning
Save time and speed up your research by using our
expertise and knowledge. Do not hesitate to inquire
about our custom service department.
PRODUCT QTY CAT. CODE
Custom-made pSELECT 20 μg p-custom
Custom-made pDRIVE 20 μg p-custom
Custom-made psiRNA 20 μg p-custom
Custom-made psiRNA Kit 1 kit k-custom
Contact us for more information
info@invivogen.com

Large Scale Plasmid DNA Production
If you are conducting gene therapy research, DNA vaccination research, transfection or pre-clinical trials, we can provide you with large quantities
of plasmid DNA. We offer a comprehensive custom service. In order to supply the most suitable and ready-to-use product, we manufacture
plasmid DNA at standard research and pre-clinical grades.
➥ Technological Exper tise
➥ Personalized Technical Suppor t
➥ High Quality Plasmid DNA
➥ Cost-Effective
➥ Shor t Turn-Around Time
Two purification grades of plasmid DNA
If you are conducting gene therapy research, DNA vaccination research,
transfection or pre-clinical trials, we can provide you with large quantities
of plasmid DNA. We offer a comprehensive custom-made service. In
order to supply the most suitable and ready-to-use product, we
manufacture plasmid DNA at standard research and pre-clinical grades.
- Standard Research Grade
The level of endotoxin is reduced to less than 0.1 EU/μg plasmid DNA
(FDA specifications).
- Pre-Clinical Grade
The purification is guaranteed with a protein level less than 1% and with
an absence of microorganisms (verified by Bioburden Assay).
High Standard Quality Control
Our processing and our rigorous quality procedures allow us to provide
certified plasmid DNA. Each batch of plasmid DNA is delivered with a
certificate of analysis and meets the characteristics listed below.
CRITERIA TESTED VALUE ASSAY METHOD
Appearance in water solution Clear, colorless Visual inspection
Plasmid identity Restriction pattern Agarose gel electrophoresis
DNA homogeneity >95% supercoiled form Agarose gel electrophoresis
Purity 1.7-2.0 A260/A280 Optical density measurements
Presence of E. coli DNA

CUSTOM SERVICES 9
Prices
164
TERMS AND CONDITIONS
Written price quotes are firm for purchase orders received within 30
days. Prices are subject to change without notice.
Payment Terms
Payment terms are net 30 days from the invoice date. Pre-payments may
be required for initial orders with completion of credit application.
InvivoGen does not require a minimum order quantity.
Shipping
Product is shipped F.O.B. from InvivoGen, San Diego, CA. Domestic
orders are shipped 2-3 day express by our designated carrier. Orders can
be expedited to overnight service for an additional fee. European orders
are shipped from our affiliate in France, Cayla. Please include Value Added
Tax (VAT) registration number when placing the order. For non-U.S.
orders, other charges such as import duties and value added taxes may
apply. Shipping days are Monday through Friday.
Warranty
InvivoGen warrants that the products sold will meet our specifications at
the time of delivery. InvivoGen’s sole liability shall be limited to, at our
option, replacement of material(s) that does not meet our specification
or refund of the purchase price. By acceptance of the product, Buyer
indemnifies and holds InvivoGen harmless against, and assumes all liability
for any direct, incidental, special or consequential loss, damage or expense
directly or indirectly arising from the use of the product, even if InvivoGen
knew of the possibility of such loss, damage or expense.
Purchaser Notification / Patents
All InvivoGen products are intended for research purpose only and not
intended for use in humans. They include technologies for which patents
have been issued to us or other companies, or are pending. Not all
components may be available for commercial license. It is incumbent upon
the interested party to contact the appropriate patent assignees for
specific information regarding license issues. Purchase of InvivoGen
products does not grant rights to reproduce, repackage or modify the
products or any derivative thereof to third parties.
Limited Use License
pCpGfree plasmids & HEK-Blue Products: The purchase of these
product conveys to the buyer the non-transferable right to use the
purchased amount of the product and all replicates and derivatives for
research purposes conducted by the buyer in his laboratory only
(whether the buyer is an academic or for-profit entity). The buyer cannot
sell or otherwise transfer (a) this product (b) its components or (c)
materials made using this product or its components to a third party or
otherwise use this product or its components or materials made using
this product or its components for Commercial Purposes.
The buyer agrees that any activity undertaken with the product and
replicates or derivatives will be conducted in compliance with all
applicable guidelines, laws and regulations.
www.invivogen.com
Commercial Purposes means any activity by a party for consideration
and may include, but is not limited to: (1) use of the product or its
components in manufacturing; (2) use of the product or its components
to provide a service, information, or data; (3) use of the product or its
components for therapeutic, diagnostic or prophylactic purposes; or (4)
resale of the product or its components, whether or not such product
or its components are resold for use in research. "Replicate" means any
biological or chemical material that represents a substantially unmodified
copy of the Material such as, but not limited to, material produced by
growth of cells. "Derivative" means material created from the Material
that is substantially modified to have new properties such as, but not
limited to, recombinant DNA modified clones.
If the purchaser is not willing to accept the limitations of this limited use
statement, InvivoGen is willing to accept return of the product with a full
refund. For information on purchasing a license to this product for purposes
other than research, contact InvivoGen, 3950 Sorrento Valley Blvd. Suite A,
San Diego California 92121. Tel:858-457-5873. Fax: 858-457-5843.
Returns
All product returns must have prior authorization and approval. Contact
our customer service or technical service department for a return
authorization number. Return authorization numbers are valid for 30 days
from issuance. Items that are authorized for return must arrive at
InvivoGen Corporation in resalable condition to be eligible for a product
credit. A restocking charge of 20% will be charged on returns that are
through no error or fault of InvivoGen Corporation and shipping charges
will not be credited. Products may not be returned for credit after 20
days of receipt of material.
Trademarks of InvivoGen
EndoFit - Fast-Media ® - Fungin - Gene A-List - HEK-Blue -
HygroGold - InvivoGen ® - LENTI-Smart - LipoGen - LyoVec -
Normocin - Plasmocin - Plasmocure - PlasmoTest - Primocin -
Prom A-List - PromTest - psiRNA - QUANTI-Blue - VacciGrade
- Zeocin

PRODUCT INFORMATION

166
PRODUCT (QUANTITY) CAT. CODE PAGE
17-AAG (5 mg) ant-agl-5 158
17-AAG (25 mg) ant-agl-25 158
17-AAGH2 (1 mg) ant-agh-1 159
17-AEP-GA (1 mg) ant-egl-1 158
17-AEP-GA (5 mg) ant-egl-5 158
17-DMAG (5 mg) ant-dgl-5 158
17-DMAG (25 mg) ant-dgl-25 158
17-DMAGH2 (1 mg) ant-dgh-1 159
17-DMAP-GA (1 mg) ant-mgl-1 158
17-DMAP-GA (5 mg) ant-mgl-5 158
17-GMB-APA-GA (1 mg) gmbapa-ga 158
17-NHS-ALA-GA (1 mg) ant-nhgl-1 158
293/hMD2-CD14 (5-7 x 10 6 cells) 293-hmd2cd14 65
293/hNOD1 (5-7 x 10 6 cells) 293-hnod1 65
293/hNOD2 (5-7 x 10 6 cells) 293-hnod2 65
293/hTLR1-HA (5-7 x 10 6 cells) 293-htlr1ha 65
293/hTLR2 (5-7 x 10 6 cells) 293-htlr2 65
293/hTLR2-HA (5-7 x 10 6 cells) 293-htlr2ha 65
293/hTLR2/6 (5-7 x 10 6 cells) 293-htlr2/6 65
293/hTLR2-CD14 (5-7 x 10 6 cells) 293-htlr2cd14 65
293/hTLR3 (5-7 x 10 6 cells) 293-htlr3 65
293/hTLR3-HA (5-7 x 10 6 cells) 293-htlr3ha 65
293/hTLR4A (5-7 x 10 6 cells) 293-htlr4a 65
293/hTLR4-HA (5-7 x 10 6 cells) 293-htlr4ha 65
293/hTLR4A-MD2-CD14 (5-7 x 10 6 cells) 293-htlr4md2cd14 65
293/hTLR5 (5-7 x 10 6 cells) 293-htlr5 65
293/hTLR5-HA (5-7 x 10 6 cells) 293-htlr5ha 65
293/hTLR5-CD14 (5-7 x 10 6 cells) 293-htlr5cd14 65
293/hTLR6-HA (5-7 x 10 6 cells) 293-htlr6ha 65
293/hTLR10-HA (5-7 x 10 6 cells) 293-htlr10ha 65
293/LacZ (5-7 x 10 6 cells) 293-lacz 65
293/mNOD1 (5-7 x 10 6 cells) 293-mnod1 65
293/mNOD2 (5-7 x 10 6 cells) 293-mnod2 65
293/mTLR1 (5-7 x 10 6 cells) 293-mtlr1 65
293/mTLR1/2 (5-7 x 10 6 cells) 293-mtlr1/2 65
293/mTLR2 (5-7 x 10 6 cells) 293-mtlr2 65
293/mTLR2/6 (5-7 x 10 6 cells) 293-mtlr2/6 65
293/mTLR3 (5-7 x 10 6 cells) 293-mtlr3 65
293/mTLR4 (5-7 x 10 6 cells) 293-mtlr4 65
293/mTLR4-MD2-CD14 (5-7 x 10 6 cells) 293-mtlr4md2cd14 65
293/mTLR5 (5-7 x 10 6 cells) 293-mtlr5 65
293/mTLR6 (5-7 x 10 6 cells) 293-mtlr6 65
293/mTLR9 (5-7 x 10 6 cells) 293-mtlr9 65
293/null (5-7 x 10 6 cells) 293-null 65
293XL/hTLR7 (5-7 x 10 6 cells) 293xl-htlr7 65
293XL/hTLR7-HA (5-7 x 10 6 cells) 293xl-htlr7ha 65
293XL/hTLR8 (5-7 x 10 6 cells) 293xl-htlr8 65
293XL/hTLR8-HA (5-7 x 10 6 cells) 293xl-htlr8ha 65
293XL/hTLR9 (5-7 x 10 6 cells) 293xl-htlr9 65
293XL/hTLR9-HA (5-7 x 10 6 cells) 293xl-htlr9ha 65
293XL/mTLR7 (5-7 x 10 6 cells) 293xl-mtlr7 65
293XL/null (5-7 x 10 6 cells) 293xl-null 65
2-Aminopurine (250 mg) tlrl-apr 93
3-Methyladenine (50 mg) tlrl-3ma 102
5’ppp-dsRNA (25 μg) tlrl-3prna 80
5’ppp-dsRNA (100 μg) tlrl-3prna-100 80
5’ppp-dsRNA Control (25 μg) tlrl-3prnac 80
5’ppp-dsRNA Control (100 μg) tlrl-3prnac-100 80
PRODUCT INFORMATION
PRODUCT (QUANTITY) CAT. CODE PAGE
5-Aza-2’-deoxycytidine (10 mg) met-adc-1 157
5-Aza-2’-deoxycytidine (50 mg) met-adc-5 157
5-Aza-cytidine (100 mg) inh-aza 21
5-Fluorocytosine (250 mg) sud-5fc 159
5-Fluorouracil (250 mg) sud-5fu 159
Adju59 (2 ml) vac-ad59-2 128
Adju59 (5 x 2 ml) vac-ad59-10 128
AG490 (10 mg) tlrl-ag4 93
Alhydrogel 2% (50 ml) vac-alu-50 128
Alhydrogel 2% (250 ml) vac-alu-250 128
Alum Crystals (1 g) tlrl-alk 98
Anti-Flagellin FliC (100 μg) mabg-flic 73
Anti-HA Tag (250 μl) ab-hatag 73
Anti-hCD14-IgA (100 μg) maba-hcd14 72
Anti-hCD20-hIgA1 (100 μg) hcd20-mab6 120
Anti-hCD20-hIgA2 (100 μg) hcd20-mab7 120
Anti-hCD20-hIgE (100 μg) hcd20-mab8 120
Anti-hCD20-hIgG1 (100 μg) hcd20-mab1 120
Anti-hCD20-hIgG2 (100 μg) hcd20-mab2 120
Anti-hCD20-hIgG3 (100 μg) hcd20-mab3 120
Anti-hCD20-hIgG4 (100 μg) hcd20-mab4 120
Anti-hCD20-hIgM (100 μg) hcd20-mab5 120
Anti-hCD20-mIgG1 (100 μg) hcd20-mab9 120
Anti-hCD20-mIgG2a (100 μg) hcd20-mab10 120
Anti-hCD20-mIgA (100 μg) hcd20-mab11 120
Anti-hCD40L-IgA2 (100 μg) maba-h40l 104
Anti-hIFN-a-IgA2 (100 μg) maba-hifna 104
Anti-hIFN-g-IgA2 (100 μg) maba-hifng 104
Anti-hIL-1b-IgA2 (100 μg) maba-hil1b 104
Anti-hIL-4-IgA2 (100 μg) maba-hil4 104
Anti-hIL-6-IgA2 (100 μg) maba-hil6 104
Anti-hIL-13-IgA2 (100 μg) maba-hil13 104
Anti-hIL-18-IgA2 (100 μg) maba-hil18 104
Anti-hTGFb-IgA2 (100 μg) maba-htgfb 104
Anti-hTLR1-IgG (100 μg) mabg-htlr1 72
Anti-hTLR2-IgA (100 μg) maba2-htlr2 72
Anti-hTLR3-IgA (100 μg) maba-htlr3 73
Anti-hTLR4-IgA (100 μg) maba2-htlr4 72
Anti-hTLR5-IgA (100 μg) maba2-htlr5 72
Anti-hTLR6-IgG (100 μg) mabg-htlr6 72
Anti-hTNF-a-hIgA1 (100 μg) htnfa-mab6 120
Anti-hTNF-a-hIgA2 (100 μg) htnfa-mab7 120
Anti-hTNF-a-hIgE (100 μg) htnfa-mab8 120
Anti-hTNF-a-hIgG1 (100 μg) htnfa-mab1 120
Anti-hTNF-a-hIgG2 (100 μg) htnfa-mab2 120
Anti-hTNF-a-hIgG3 (100 μg) htnfa-mab3 120
Anti-hTNF-a-hIgG4 (100 μg) htnfa-mab4 120
Anti-hTNF-a-hIgM (100 μg) htnfa-mab5 120
Anti-hTNF-a-mIgG1 (100 μg) htnfa-mab9 120
Anti-hTNF-a-mIgG2 (100 μg) htnfa-mab10 120
Anti-hTNF-a-mIgA (100 μg) htnfa-mab11 120
Anti-mTLR2-IgG (100 μg) mabg-mtlr2 72
Anti-mTLR5-IgG (100 μg) mabg-mtlr5 72
ATP (1 g) tlrl-atp 98
B16-Blue IFNa/b Cells (5-7 x 10 6 cells) bb-ifnab 106
Bafilomycin A1 (10 μg) tlrl-baf 102
Bay11-7082 (10 mg) tlrl-b82 93
Biotin-GA (1 mg) ant-bgl-1 159

PRODUCT (QUANTITY) CAT. CODE PAGE
Biotin-GA (5 mg) ant-bgl-5 159
Bix-01294 (2 mg) inh-bix 21
Blasticidin (100 mg) ant-bl-1 17
Blasticidin (500 mg) ant-bl-5 17
Blasticidin (500 mg, bottle) ant-bl-5b 17
Blasticidin (1 g powder) ant-bl-10p 17
BX795 (5 mg) tlrl-bx7 93
C12-iE-DAP (1 mg) tlrl-c12dap 80
C3H/TLR4mut MEFs (5-7 x 10 6 cells) mef-c3h4m 71
C3H/WT MEFs (5-7 x 10 6 cells) mef-c3hwt 71
C57/WT MEFs (5-7 x 10 6 cells) mef-c57wt 71
Celastrol (1 mg) ant-cls 155
ChemiComp GT115 (5 x 0.1 ml) gt115-11 43
ChemiComp GT115 (5 x 0.2 ml) gt115-21 43
ChemiComp GT116 (5 x 0.1 ml) gt116-11 43
ChemiComp GT116 (5 x 0.2 ml) gt116-21 43
Chloroquine (250 mg) tlrl-chq 93
CL075 (500 μg) tlrl-c75 78
CL075 (5 mg) tlrl-c75-5 78
CL097 (500 μg) tlrl-c97 78
CL097 (5 mg) tlrl-c97-5 78
CL264 (500 μg) tlrl-c264s 78
CL264 (5 mg) tlrl-c264-5 78
CL264 Biotin (100 μg) tlrl-bc264 78
CL264 FITC (100 μg) tlrl-fc264 78
CL264 Rhodamine (100 μg) tlrl-rc264 78
CLI-095 (1 mg) tlrl-cli95 93
CPPD crystals (5 mg) tlrl-cppd 98
Curdlan (1 g) tlrl-curd 81
Custom Cloning (20 μg) p-custom 162
Custom-made CpG-free Gene (20 μg) p-custom 139
Custom-made psiRNA (20 μg) p-custom 150
Custom-made psiRNA kit k-custom 150
Cyclosporin A (100 mg) tlrl-cyca 93
DNA Standard Research Grade p-custom 163
DNA Pre-Clinical Grade p-custom 163
E. coli DNA ef (1 mg) tlrl-ednaef 78
E. coli ssDNA / LyoVec (200 μg) tlrl-ssec 78
EndoFit Ovalbumin (10 mg) vac-efova 129
Fast-Media ® Amp Agar (30 pouches) fas-am-s 45
Fast-Media ® Amp Agar (500 pouches) fas-am-s500 45
Fast-Media ® Amp TB (30 pouches) fas-am-l 45
Fast-Media ® Amp TB (500 pouches) fas-am-l500 45
Fast-Media ® Amp XGal (20 pouches) fas-am-x 45
Fast-Media ® Base Agar (30 pouches) fas-s 45
Fast-Media ® Base Agar (500 pouches) fas-s500 45
Fast-Media ® Base TB (30 pouches) fas-l 45
Fast-Media ® Base TB (500 pouches) fas-l500 45
Fast-Media ® Blas Agar (20 pouches) fas-bl-s 45
Fast-Media ® Blas TB (20 pouches) fas-bl-l 45
Fast-Media ® Blas XGal (20 pouches) fas-bl-x 45
Fast-Media ® Hygro Agar (20 pouches) fas-hg-s 45
Fast-Media ® Hygro TB (20 pouches) fas-hg-l 45
Fast-Media ® Hygro XGal (20 pouches) fas-hg-x 45
Fast-Media ® Kan Agar (30 pouches) fas-kn-s 45
Fast-Media ® Kan Agar (500 pouches) fas-kn-s500 45
Fast-Media ® Kan TB (30 pouches) fas-kn-l 45
Fast-Media ® Kan TB (500 pouches) fas-kn-l500 45
PRODUCT INFORMATION
PRODUCT (QUANTITY) CAT. CODE PAGE
Fast-Media ® Kan XGal (20 pouches) fas-kn-x 45
Fast-Media ® Puro Agar (20 pouches) fas-pr-s 45
Fast-Media ® Puro TB (20 pouches) fas-pr-l 45
Fast-Media ® Zeo Agar (20 pouches) fas-zn-s 45
Fast-Media ® Zeo TB (20 pouches) fas-zn-l 45
Fast-Media ® Zeo XGal (20 pouches) fas-zn-x 45
Fast-Media ® Zeo X-Gluc (10 pouches) fas-zn-g 45
FLA-BS (100 μg) tlrl-bsfla 78
Flagellin FliC VacciGrade (50 μg) vac-fla 128
FLA-ST (100 μg) tlrl-stfla 78
FLA-ST Ultrapure (10 μg) tlrl-pstfla 78
FLA-ST recombinant (1 μg) tlrl-flic 78
FLA-ST recombinant (10 μg) tlrl-flic-10 78
FSL-1 (100 μg) tlrl-fsl 77
Fungin (75 mg) ant-fn-1 11
Fungin (200 mg) ant-fn-2 11
G418 (1 g) ant-gn-1 17
G418 (5 g) ant-gn-5 17
Ganciclovir (250 mg) sud-gcv 159
Gardiquimod (500 μg) tlrl-gdqs 78
Gardiquimod (5 mg) tlrl-gdq-5 78
Gardiquimod VacciGrade (5 mg) vac-gdq 128
Gefitinib (10 mg) tlrl-gef 93
Geldanamycin (5 mg) ant-gl-5 158
Geldanamycin (25 mg) ant-gl-25 158
Glybenclamide (1 g) tlrl-gly 98
Goat anti-human IgA - HRP (1 ml) hrp-iga 123
Goat anti-human kappa - HRP (1 ml) hrp-igak 123
Goat F(ab’)2 anti-human IgA (0.5 mg) fab-iga 123
Goat F(ab’)2 anti-human IgA - Biotin (0.5 mg) chiga-biot 73
Goat F(ab’)2 anti-human IgA - FITC (0.5 mg) chiga-fitc 73
Goat F(ab’)2 anti-human kappa (0.5 mg) fab-igak 123
Goat F(ab’)2 IgG isotype control - FITC (100 tests) cgig-fitc 73
G-ODN (200 μg) tlrl-godn 80
H-89 (5 mg) tlrl-h89 93
HEK-Blue CD40L Cells (5-7 x 10 6 cells) hkb-cd40 115
HEK-Blue CD40L Kit hkb-cd40-kit 115
HEK-Blue Detection (2 pouches) hb-det1 15
HEK-Blue Detection (5 pouches) hb-det2 15
HEK-Blue hMD2-CD14 Cells (5-7 x 10 6 cells) hkb-hmdcd 66
HEK-Blue hNOD1 Cells (5-7 x 10 6 cells) hkb-hnod1 66
HEK-Blue hNOD2 Cells (5-7 x 10 6 cells) hkb-hnod2 66
HEK-Blue hTLR2 Cells (5-7 x 10 6 cells) hkb-htlr2 66
HEK-Blue hTLR3 Cells (5-7 x 10 6 cells) hkb-htlr3 66
HEK-Blue hTLR4 Cells (5-7 x 10 6 cells) hkb-htlr4 66
HEK-Blue hTLR5 Cells (5-7 x 10 6 cells) hkb-htlr5 66
HEK-Blue hTLR7 Cells (5-7 x 10 6 cells) hkb-htlr7 66
HEK-Blue hTLR8 Cells (5-7 x 10 6 cells) hkb-htlr8 66
HEK-Blue hTLR9 Cells (5-7 x 10 6 cells) hkb-htlr9 66
HEK-Blue IFN-a/b Cells (5-7 x 10 6 cells) hkb-ifnab 107
HEK-Blue IL-1b Cells (5-7 x 10 6 cells) hkb-il1b 100
HEK-Blue IL-1b Kit hkb-il1b-kit 111
HEK-Blue IL-4/IL-13 Cells (5-7 x 10 6 cells) hkb-stat6 108
HEK-Blue IL-4/IL-13 Kit hkb-stat6-kit 108
HEK-Blue IL-6 Cells (5-7 x 10 6 cells) hkb-il6 109
HEK-Blue IL-6 Kit hkb-il6-kit 109
HEK-Blue IL-18/IL-1b Cells (5-7 x 10 6 cells) hkb-il18 112
HEK-Blue IL-18/IL-1b Kit hkb-il18-kit 112
167

168
PRODUCT (QUANTITY) CAT. CODE PAGE
HEK-Blue IL-33/IL-1b Cells (5-7 x 10 6 cells) hkb-il33 113
HEK-Blue mNOD1 Cells (5-7 x 10 6 cells) hkb-mnod1 66
HEK-Blue mNOD2 Cells (5-7 x 10 6 cells) hkb-mnod2 66
HEK-Blue mTLR2 Cells (5-7 x 10 6 cells) hkb-mtlr2 66
HEK-Blue mTLR3 Cells (5-7 x 10 6 cells) hkb-mtlr3 66
HEK-Blue mTLR4 Cells (5-7 x 10 6 cells) hkb-mtlr4 66
HEK-Blue mTLR5 Cells (5-7 x 10 6 cells) hkb-mtlr5 66
HEK-Blue mTLR7 Cells (5-7 x 10 6 cells) hkb-mtlr7 66
HEK-Blue mTLR8 Cells (5-7 x 10 6 cells) hkb-mtlr8 66
HEK-Blue mTLR9 Cells (5-7 x 10 6 cells) hkb-mtlr9 66
HEK-Blue Null1 Cells (5-7 x 10 6 cells) hkb-null1 66
HEK-Blue Null1-k Cells (5-7 x 10 6 cells) hkb-null1k 66
HEK-Blue Null1-v Cells (5-7 x 10 6 cells) hkb-null1v 66
HEK-Blue Null2 Cells (5-7 x 10 6 cells) hkb-null2 66
HEK-Blue Null2-k Cells (5-7 x 10 6 cells) hkb-null2k 66
HEK-Blue TNF-a/IL-1b Cells (5-7 x 10 6 cells) hkb-tnfil1 110
HEK-Blue TNF-a/IL-1b Kit hkb-tnfil1-kit 110
HEK-Blue TGF-b Cells (5-7 x 10 6 cells) hkb-tgfb 114
HEK-Blue TGF-b Kit hkb-tgfb-kit 114
HEK-Blue LPS Detection Kit rep-lps 90
HEK-Blue Selection (5 x 2 ml) hb-sel 8
Hemozoin (5 mg) tlrl-hz 98
HKAL (10 9 cells) tlrl-hkal 77
HKCA (10 9 cells) tlrl-hkca 81
HKEB (10 10 cells) tlrl-hkeb 77
HKHP (10 9 cells) tlrl-hkhp 77
HKLM (10 10 cells) tlrl-hklm 77
HKLP (10 9 cells) tlrl-hklp 77
HKLR (10 10 cells) tlrl-hklr 77
HKMF (10 9 cells) tlrl-hkmf 77
HKPA (10 10 cells) tlrl-hkpa 77
HKPG (10 10 cells) tlrl-hkpg 77
HKSA (10 10 cells) tlrl-hksa 77
HKSC (10 9 cells) tlrl-hksc 81
HKSP (10 10 cells) tlrl-hksp 77
Human IgA (0.5 mg) ctrl-iga 123
Human IgA kappa (0.5 mg) ctrl-igak 123
HygroGold (1 g) ant-hg-1 18
HygroGold (5 g) ant-hg-5 18
HygroGold (10 g powder) ant-hg-10p 18
Hygromycin B (1 g) ant-hm-1 18
Hygromycin B (5 g) ant-hm-5 18
iE-DAP (5 mg) tlrl-dap 80
iE-Lys (5 mg) tlrl-lys 80
IFA (10 ml) vac-ifa-10 128
IFA (6 x 10 ml) vac-ifa-60 128
IFNr qRT-Primers (kit) rts-hifnr 147
IgA2 Isotype Control (100 μg) maba2-ctrl 124
Imiquimod (500 μg) tlrl-imqs 78
Imiquimod (5 mg) tlrl-imq 78
Imiquimod VacciGrade (5 mg) vac-imq 128
Jacalin / Agarose (2 ml) gel-jac-2 122
Jacalin / Agarose (5 ml) gel-jac-5 122
J Chain Antiserum (100 μg) pab-jc 123
L18-MDP (1 mg) tlrl-lmdp 80
LacZ Cell Staining Kit rep-lz-c 12
LacZ Tissue Staining Kit rep-lz-t 12
LAM-MS (500 μg) tlrl-lams 77
PRODUCT INFORMATION
www.invivogen.com
PRODUCT (QUANTITY) CAT. CODE PAGE
LENTI-Smart (INT) (5 vials) ltsint-5 24
LENTI-Smart (INT) (10 vials) ltsint-10 24
LENTI-Smart (INT) (h/m)OSKM (5 vials) ltsint-oskm-5 25
LENTI-Smart (INT) (h/m)OSKM (10 vials) ltsint-oskm-10 25
LENTI-Smart NIL (5 vials) ltsnil-5 24
LENTI-Smart NIL (10 vials) ltsnil-10 24
LENTI-Smart NIL (h/m)OSKM (5 vials) ltsnil-oskm-5 25
LENTI-Smart NIL (h/m)OSKM (10 vials) ltsnil-oskm-10 25
LENTI-Smart Starter Kit (10 vials) lts-str 24
Leptomycin B (5 μg) tlrl-lep 93
LM-MS (250 μg) tlrl-lmms2 77
Loxoribine (50 mg) tlrl-lox 78
LPS-EB (5 mg) tlrl-eblps 77
LPS-EB Biotin (500 μg) tlrl-bblps 77
LPS-EB Ultrapure (5 mg) tlrl-pelps 77
LPS-EK (5 mg) tlrl-eklps 77
LPS-EK Ultrapure (1 mg) tlrl-peklps 77
LPS-PG Ultrapure (1 mg) tlrl-pglps 77
LPS-RS (5 mg) tlrl-rslps 78
LPS-SM Ultrapure (5 mg) tlrl-smlps 77
LTA-BS (5 mg) tlrl-lta 77
LTA-SA (5 mg) tlrl-slta 77
LTA-SA Purified (5 mg) tlrl-pslta 77
LY294002 (5 mg) tlrl-ly29 93
LyoComp GT115 (5 x 0.1 ml) lyo-115-11 43
LyoComp GT115 (5 x 0.2 ml) lyo-115-21 43
LyoComp GT116 (4 x 0.5 ml) lyo-116-11 43
LyoComp GT116 (4 x 1 ml) lyo-116-21 43
LyoVec (8 ml, 160 reactions) lyec-12 19
LyoVec (18 ml, 360 reactions) lyec-22 19
MAb-hTLR1 (100 μg) mab-htlr1 73
MAb-hTLR1-FITC (100 μg) mab-htlr1f 73
MAb-hTLR2 (100 μg) mab-htlr2 73
MAb-hTLR2-FITC (100 μg) mab-htlr2f 73
MAb-hTLR3 (100 μg) mab-htlr3 73
MAb-hTLR3-FITC (100 μg) mab-htlr3f 73
MAb-hTLR4 (100 μg) mab-htlr4 73
MAb-hTLR4-FITC (100 μg) mab-htlr4f 73
MAb-mDectin-1 (100 μg) mab-mdect 72
MAb-mTLR2 (100 μg) mab-mtlr2 72
MAb-mTLR2-FITC (100 μg) mab-mtlr2f 73
MAb-mTLR4/MD2 (100 μg) mab-mtlr4md2 73
MAb-mTLR4/MD2-FITC (100 μg) mab-mtlr4md2f 73
MAb-mTLR9 (100 μg) mab-mtlr9 73
MAb-mTLR9-FITC (100 μg) mab-mtlr9f 73
MDP (5 mg) tlrl-mdp 80
MDP Biotin (500 μg) tlrl-bmdp 80
MDP control (5 mg) tlrl-mdpc 80
MDP FITC (500 μg) tlrl-fmdp 80
MDP Rhodamine (500 μg) tlrl-rmdp 80
MPLA (1 mg) tlrl-mpl 77
MPLAs (500 μg) tlrl-mpls 77
MPLA VacciGrade (1 mg) vac-mpl 128
MSU Crystals (5 mg) tlrl-msu 98
M-TriDAP (1 mg) tlrl-mtd 80
Murabutide (5 mg) tlrl-mbt 80
Murabutide control (5 mg) tlrl-mbtc 80
N-Glycolyl-MDP (5 mg) tlrl-gmdp 80

PRODUCT (QUANTITY) CAT. CODE PAGE
N-Glycolyl-MDP VacciGrade (5 mg) vac-gmdp 128
Nigericin (10 mg) tlrl-nig 98
Nigericin (50 mg) tlrl-nig-5 98
NOD1/2 Agonist Kit (10 ligands) tlrl-nodkit 81
Normocin (500 mg) ant-nr-1 11
Normocin (1 g) ant-nr-2 11
ODN 1585 (200 μg) tlrl-1585 78
ODN 1585 (1 mg) tlrl-1585-1 78
ODN 1585 (5 mg) tlrl-1585-5 78
ODN 1585 control (200 μg) tlrl-1585c 78
ODN 1585 control (1 mg) tlrl-1585c-1 78
ODN 1585 control (5 mg) tlrl-1585c-5 78
ODN 1585 FITC (50 μg) tlrl-1585f 78
ODN 1668 (200 μg) tlrl-1668 78
ODN 1668 (1 mg) tlrl-1668-1 78
ODN 1668 (5 mg) tlrl-1668-5 78
ODN 1668 control (200 μg) tlrl-1668c 78
ODN 1668 control (1 mg) tlrl-1668c-1 78
ODN 1668 control 5 mg) tlrl-1668c-5 78
ODN 1668 FITC (50 μg) tlrl-1668f 79
ODN 1826 (200 μg) tlrl-1826 79
ODN 1826 (1 mg) tlrl-1826-1 79
ODN 1826 (5 mg) tlrl-1826-5 79
ODN 1826 Biotin (50 μg) tlrl-1826b 79
ODN 1826 control (200 μg) tlrl-1826c 79
ODN 1826 control (1 mg) tlrl-1826c-1 79
ODN 1826 control (5 mg) tlrl-1826c-5 79
ODN 1826 FITC (50 μg) tlrl-1826f 79
ODN 1826 VacciGrade (1 mg) vac-1826 128
ODN 2006 (200 μg) tlrl-2006 79
ODN 2006 (1 mg) tlrl-2006-1 79
ODN 2006 (5 mg) tlrl-2006-5 79
ODN 2006 Biotin (50 μg) tlrl-2006b 79
ODN 2006 control (200 μg) tlrl-2006c 79
ODN 2006 control (1 mg) tlrl-2006c-1 79
ODN 2006 control (5 mg) tlrl-2006c-5 79
ODN 2006 FITC (50 μg) tlrl-2006f 79
ODN 2006-G5 (200 μg) tlrl-2006g5 79
ODN 2006-G5 (1 mg) tlrl-2006g5-1 79
ODN 2006-G5 (5 mg) tlrl-2006g5-5 79
ODN 2006 VacciGrade (1 mg) vac-2006 128
ODN 2007 (200 μg) tlrl-2007 79
ODN 2007 (1 mg) tlrl-2007-1 79
ODN 2007 (5 mg) tlrl-2007-5 79
ODN 2007 Control (200 μg) tlrl-2007c 79
ODN 2007 Control (1 mg) tlrl-2007c-1 79
ODN 2007 Control (5 mg) tlrl-2007c-5 79
ODN 2088 (200 μg) tlrl-2088 80
ODN 2088 (1 mg) tlrl-2088-1 80
ODN 2088 control (200 μg) tlrl-2088c 80
ODN 2088 control (1 mg) tlrl-2088c-1 80
ODN 2216 (200 μg) tlrl-2216 79
ODN 2216 (1 mg) tlrl-2216-1 79
ODN 2216 (5 mg) tlrl-2216-5 79
ODN 2216 Biotin (50 μg) tlrl-2216b 79
ODN 2216 control (200 μg) tlrl-2216c 79
ODN 2216 control (1 mg) tlrl-2216c-1 79
ODN 2216 control (5 mg) tlrl-2216c-5 79
PRODUCT INFORMATION
www.invivogen.com
PRODUCT (QUANTITY) CAT. CODE PAGE
ODN 2216 FITC (50 μg) tlrl-2216f 79
ODN 2336 (200 μg) tlrl-2336 79
ODN 2336 (1 mg) tlrl-2336-1 79
ODN 2336 (5 mg) tlrl-2336-5 79
ODN 2336 control (200 μg) tlrl-2336c 79
ODN 2336 control (1 mg) tlrl-2336c-1 79
ODN 2336 control (5 mg) tlrl-2336c-5 79
ODN 2336 FITC (50 μg) tlrl-2336f 79
ODN 2395 (200 μg) tlrl-2395 79
ODN 2395 (1 mg) tlrl-2395-1 79
ODN 2395 (5 mg) tlrl-2395-5 79
ODN 2395 control (200 μg) tlrl-2395c 79
ODN 2395 control (1 mg) tlrl-2395c-1 79
ODN 2395 control (5 mg) tlrl-2395c-5 79
ODN 2395 FITC (50 μg) tlrl-2395f 79
ODN 4084-F (200 μg) tlrl-4084 80
ODN INH-1 (200 μg) tlrl-inh1 80
ODN INH-47 (200 μg) tlrl-inh47 80
ODN M362 (200 μg) tlrl-m362 79
ODN M362 (1 mg) tlrl-m362-1 79
ODN M362 (5 mg) tlrl-m362-5 79
ODN M362 control (200 μg) tlrl-m362c 79
ODN M362 control (1 mg) tlrl-m362c-1 79
ODN M362 control (5 mg) tlrl-m362c-5 79
ODN M362 FITC (50 μg) tlrl-m362f 79
ODN TTAGGG (200 μg) tlrl-ttag 80
ODN TTAGGG (1 mg) tlrl-ttag-1 80
ODN TTAGGG control (200 μg) tlrl-ttagc 80
ODN TTAGGG control (1 mg) tlrl-ttagc-1 80
ORN02 / LyoVec (4 x 25 μg) tlrl-orn2 78
ORN06 / LyoVec (4 x 25 μg) tlrl-orn6 78
OVA 257-264 (1 mg) vac-sin 129
OVA 323-339 (1 mg) vac-isq 129
Ovalbumin (1 g) vac-ova 129
Ovalbumin EndoFit (10 mg) vac-efova 129
OxPAPC (1 mg) tlrl-oxp1 93
PAb-hTLR1 (200 μg) pab-hstlr1 72
PAb-hTLR2 (200 μg) pab-hstlr2 72
PAb-hTLR4 (200 μg) pab-hstlr4 72
PAb-hTLR5 (200 μg) pab-hstlr5 72
PAb-hTLR6 (200 μg) pab-hstlr6 72
Pam2CSK4 (100 μg) tlrl-pam2 77
Pam2CSK4 (1 mg) tlrl-pam2-1 77
Pam2CSK4 Biotin (50 μg) tlrl-bpam2 77
Pam2CSK4 Rhodamine (50 μg) tlrl-rpam2 77
Pam3CSK4 (1 mg) tlrl-pms 77
Pam3CSK4 Biotin (50 μg) tlrl-bpms 77
Pam3CSK4 Rhodamine (50 μg) tlrl-rpms 77
pBLAST- (20 μg) pblast- 37
pBLAST-mcs (20 μg) pbla-mcs 37
(if purchased with another pBLAST) pbla-mcs 37
pBOOST2-mcs (20 μg) pbst2-mcs 130
pBOOST2-msaIRF3 (20 μg) pbst2-samirf3 130
pBOOST2-msaIRF7/3 (20 μg) pbst2-samirf73 130
pBOOST2-wtmIRF1 (20 μg) pbst2-wtmirf1 130
pBOOST3-mTBK1 (20 μg) pbst3-mtbk1 130
pBOOST3-mcs (20 μg) pbst3-mcs 130
pBROAD2 Kit kbroad2 31
169

170
PRODUCT (QUANTITY) CAT. CODE PAGE
pBROAD2-LacZnls (20 μg) pbroad2-lacz 31
pBROAD2-mcs (20μg) pbroad2 31
pBROAD3 Kit kbroad3 31
pBROAD3-LacZnls (20 μg) pbroad3-lacz 31
pBROAD3-mcs (20μg) pbroad3 31
pCpG-Giant (1 mg) tlrl-cpgg 80
pCpGfree-basic (20 μg) pcpgf-bas 135
pCpGfree-LacZ (20 μg) pcpgf-lacz 134
pCpGfree-mcs (20 μg) pcpgf-mcs 134
pCpGfree-mSEAP (20 μg) pcpgf-mseap 134
pCpGfree-promoter (20 μg) pcpgf-pro 135
pCpGfree-siRNA (kit) kcpgf-sirna 137
pCpGfree-siRNADUO (kit) kcpg-sirna2 137
pCpGfree-vitroBLacZ (20 μg) pcpgvtb-lz 136
pCpGfree-vitroBmcs (20 μg) pcpgvtb-mcsg2 136
pCpGfree-vitroHLacZ (20 μg) pcpgvth-lz 136
pCpGfree-vitroHmcs (20 μg) pcpgvth-mcsg2 136
pCpGfree-vitroNLacZ (20 μg) pcpgvtn-lz 136
pCpGfree-vitro-Nmcs (20 μg) pcpgvtn-mcsg2 136
pCpGrich-mcs (20 μg) pcpgr-mcs 134
PD0325901 (2 mg) inh-pd32 21
PD98059 (10 mg) tlrl-pd98 93
pDeNy- (E.coli disk) pdn- 63
pDRIVE--LacZ (E.coli disk) pdrive- 39
pDRIVE--LacZ (E.coli disk) pdrive- 39
pDRIVE-custom (20 μg) p-custom 162
pDRIVE5-GFP-n (20 μg) pdv5-gfp-n 38
pDRIVE5--SEAP (E.coli disk) pdrive5s- 39
pDRIVE5--SEAP (E.coli disk) pdrive5s- 39
pDUO- (E.coli disk) pduo- 61
pDUO2- (E.coli disk) pduo2- 61
Pepinh-Control (2 mg) tlrl-pictrl 93
Pepinh-MYD (2 mg) tlrl-pimyd 93
Pepinh-TRIF (2 mg) tlrl-pitrif 93
Peptide M / Agarose (2 ml) gel-pdm-2 122
Peptide M / Agarose (5 ml) gel-pdm-5 122
pFUSE-CHIg (20 μg) see page 27 27
pFUSEss-CHIg (20 μg) see page 27 27
pFUSE2-CLIg (20 μg) see page 27 27
pFUSE2ss-CLIg (20 μg) see page 27 27
pFUSE-Fc1 native (20 μg) see page 30 28
pFUSE-Fc2 native (20 μg) see page 30 28
pFUSE-Fc1 engineered (20 μg) see page 30 28
pFUSE-Fc2 engineered (20 μg) see page 30 28
pFUSE-SEAP-Fc (20 μg) see page 30 28
PGN-BS (5 mg) tlrl-pgnbs 77
PGN-EB (1 mg) tlrl-pgnec 77
PGN-ECndi ultrapure, insoluble (5 mg) tlrl-kipgn 80
PGN-ECndss ultrapure, soluble (1 mg) tlrl-ksspgn 80
PGN-EK (1 mg) tlrl-pgnek 77
PGN-SA (5 mg) tlrl-pgnsa 77
PGN-SAndi ultrapure, insoluble (5 mg) tlrl-sipgn 80
Phleomycin (100 mg) ant-ph-1 18
Phleomycin (500 mg) ant-ph-5 18
Phleomycin (250 mg powder) ant-ph-2p 18
Phleomycin (500 mg powder) ant-ph-5p 18
Phleomycin (1 g powder) ant-ph-10p 18
pINFUSE-Fc1 (20 μg) see page 30 28
pINFUSE-Fc2 (20 μg) see page 30 28
PRODUCT INFORMATION
www.invivogen.com
PRODUCT (QUANTITY) CAT. CODE PAGE
Plasmocin prophylactic (25 mg) ant-mpp 10
Plasmocin treatment (50 mg) ant-mpt 10
Plasmocure (100 mg) ant-pc 10
PlasmoTest (kit) rep-pt2 8
PlasmoTest Controls (200 tests) pt-ctr2 9
PlasmoTest Reagent Kit (500 samples) rep-ptrk 9
PMA (5 mg) tlrl-pma 81
pMONO-blasti/GFP (20 μg) pmonob-gfp 32
pMONO-blasti/mcs (20 μg) pmonob-mcs 32
pMONO-hygro/GFP (20 μg) pmonoh-gfp 32
pMONO-hygro/mcs (20 μg) pmonoh-mcs 32
pMONO-neo/GFP (20 μg) pmonon-gfp 32
pMONO-neo/mcs (20 μg) pmonon-mcs 32
pMONO-zeo/GFP (20 μg) pmonoz-gfp 32
pMONO-zeo/mcs (20 μg) pmonoz-mcs 32
pNiFty-Luc (E.coli disk) pnifty-luc 74
pNiFty-SEAP (E.coli disk) pnifty-seap 74
pNiFty2-56K-SEAP (E.coli disk) pnf2-56ksp 74
pNiFty2-Luc (E.coli disk) pnifty2-luc 74
pNiFty2-SEAP (E.coli disk) pnifty2-seap 74
pNiFty3-SEAP (E.coli disk) pnf3-sp1 74
pNiFty3-N-SEAP (E.coli disk) pnf3-sp2 74
pNiFty3-A-SEAP (E.coli disk) pnf3-sp3 74
pNiFty3-I-SEAP (E.coli disk) pnf3-sp4 74
pNiFty3-T-SEAP (E.coli disk) pnf3-sp5 74
pNiFty3-AN-SEAP (E.coli disk) pnf3-sp6 74
pNiFty3-IAN-SEAP (E.coli disk) pnf3-sp7 74
pNiFty3-TAN-SEAP (E.coli disk) pnf3-sp8 74
Poly(A:U) (10 mg) tlrl-pau 77
Poly(dA:dT) / LyoVec (100 μg) tlrl-patc 80
Poly(dA:dT) Naked (200 μg) tlrl-patn 80
Poly(dA:dT) Naked (1 mg) tlrl-patn-1 80
Poly(dG:dC) / LyoVec (100 μg) tlrl-pgcc 80
Poly(dG:dC) Naked (200 μg) tlrl-pgcn 80
Poly(dT) (100 nmol) tlrl-pt17 78
Poly(I:C) (HMW) (10 mg) tlrl-pic 77
Poly(I:C) (HMW) (50 mg) tlrl-pic-5 77
Poly(I:C) (HMW) / LyoVec (100 μg) tlrl-piclv 80
Poly(I:C) (LMW) (25 mg) tlrl-picw 77
Poly(I:C) (LMW) (250 mg) tlrl-picw-250 7
Poly(I:C) (LMW) / LyoVec (100 μg) tlrl-picwlv 80
Poly(I:C) Fluorescein (10 μg) tlrl-picf 77
Poly(I:C) Rhodamine (10 μg) tlrl-picr 77
Poly(I:C) VacciGrade (10 mg) vac-pic 128
Polymyxin B (100 mg) tlrl-pmb 93
pORF- (E.coli disk) porf- 37
pORF-mcs (E.coli disk) porf-mcs 37
(if purchased with another pORF) porf-mcs 73
Primocin (500 mg) ant-pm-1 11
Primocin (1 g) ant-pm-2 11
PromTest (10 x 5 μg) prom-test 38
Protein L / Agarose (2 ml) gel-protl-2 122
Protein L / Agarose (10 ml) gel-protl-10 122
pSELECT-blasti/LacZ (20 μg) psetb-lacz 32
pSELECT-blasti/mcs (20 μg) psetb-mcs 32
pSELECT-CGFP-blasti (20 μg) psetb-cgfp 33
pSELECT-CGFP-zeo (20 μg) psetz-cgfp 33
pSELECT-CHA-blasti (20 μg) psetb-cha 33
pSELECT-CHA-zeo (20 μg) psetz-cha 33

PRODUCT (QUANTITY) CAT. CODE PAGE
pSELECT-CHis-blasti (20 μg) psetb-chis 33
pSELECT-CHis-zeo (20 μg) psetz-chis 33
pSELECT-GFP-LC3 (20 μg) psetz-gfplc3 102
pSELECT-GFPzeo-LacZ (20 μg) psetgz-lacz 32
pSELECT-GFPzeo-mcs (20 μg) psetgz-mcs 32
pSELECT-hygro-LacZ (20 μg) pseth-lacz 32
pSELECT-hygro-mcs (20 μg) pseth-mcs 32
pSELECT-neo-LacZ (20 μg) psetn-lacz 32
pSELECT-neo-mcs (20 μg) psetn-mcs 32
pSELECT-NGFP-blasti (20 μg) psetb-ngfp 33
pSELECT-NGFP-zeo (20 μg) psetz-ngfp 33
pSELECT-NHA-blasti (20 μg) psetb-nha 33
pSELECT-NHA-zeo (20 μg) psetz-nha 33
pSELECT-NHis-blasti (20 μg) psetb-nhis 33
pSELECT-NHis-zeo (20 μg) psetz-nhis 33
pSELECT-puro-LacZ (20 μg) psetp-lacz 32
pSELECT-puro-mcs (20 μg) psetp-mcs 32
pSELECT-zeo- (20 μg) psetz- 138
pSELECT-zeo-LacZ (20 μg) psetz-lacz 12
pSELECT-zeo-mcs (20 μg) psetz-mcs 32
pSELECT-zeo-seap (20 μg) psetz-seap 13
psiRNA-DUO Kit ksirna4-gz3 145
psiRNA-h7SKblasti Kit ksirna3-b21 144
psiRNA-h7SKGFPzeo Kit ksirna4-gz21 144
psiRNA-h7SKhygro Kit ksirna3-h21 144
psiRNA-h7SKneo Kit ksirna3-n21 144
psiRNA-h7SKzeo Kit ksirna3-z21 144
psiTEST Kit ksitest 146
pUNO1- (E.coli disk) puno1- 58
pUNO1--11RHA (E. coli disk) puno1rha- 21
pUNO3- (E.coli disk) puno3- 58
pUNO- (E.coli disk) puno- 58
pUNO--HA (E.coli disk) punoha- 62
pUNO-hTLR1-GFP (E.coli disk) phtlr1-gfp 62
pUNO-hTLR2-GFP (E.coli disk) phtlr2-gfp 62
pUNO-hTLR3-GFP (E.coli disk) phtlr3-gfp 62
pUNO-hTLR4-GFP (E.coli disk) phtlr4-gfp 62
pUNO-hTLR5-GFP (E.coli disk) phtlr5-gfp 62
pUNO-hTLR6-GFP (E.coli disk) phtlr6-gfp 62
pUNO-mcs (E.coli disk) puno-mcs 58
(if purchased with another pUNO) puno-mcs 58
Puromycin (100 mg) ant-pr-1 17
Puromycin (500 mg) ant-pr-5 17
pVAC1-mcs (20 μg) pvac1 131
pVAC2-mcs (20 μg) pvac2 131
pVITRO1-blasti-GFP/LacZ (20 μg) pvitro1-bgfplacz 34
pVITRO1-blasti-mcs (20 μg) pvitro1-bmcs 34
pVITRO1-hygro-GFP/LacZ (20 μg) pvitro1-gfplacz 34
pVITRO1-hygro-mcs (20 μg) pvitro1-mcs 34
pVITRO1-neo-GFP/LacZ (20 μg) pvitro1-ngfplacz 34
pVITRO1-neo-mcs (20 μg) pvitro1-nmcs 34
pVITRO2-blasti-GFP/LacZ (20 μg) pvitro2-bgfplacz 34
pVITRO2-blasti-mcs (20 μg) pvitro2-bmcs 34
pVITRO2-hygro-GFP/LacZ (20 μg) pvitro2-gfplacz 34
pVITRO2-hygro-mcs (20 μg) pvitro2-mcs 34
pVITRO2-neo-GFP/LacZ (20 μg) pvitro2-ngfplacz 34
pVITRO2-neo-mcs (20 μg) pvitro2-nmcs 34
pVIVO1-GFP/LacZ (20 μg) pvivo1-gfplacz 35
pVIVO1-mcs (20 μg) pvivo1-mcs 35
PRODUCT INFORMATION
www.invivogen.com
PRODUCT (QUANTITY) CAT. CODE PAGE
pVIVO2-GFP/LacZ (20 μg) pvivo2-gfplacz 35
pVIVO2-mcs (20 μg) pvivo2-mcs 35
pWHERE (20 μg) pwhere 31
pWHERE Kit kwhere 31
pZERO- (E.coli disk) pzero- 63
pZERO--HA (E.coli disk) pzero--ha 63
QUANTI-Blue (5 pouches) rep-qb1 14
QUANTI-Blue (10 pouches) rep-qb2 14
R848 (500 μg) tlrl-r848 78
R848 (5 mg) tlrl-r848-5 78
R848 VacciGrade (5 mg) vac-r848 128
Ramos-Blue Cells (5-7 x 10 6 cells) rms-sp 69
Ramos-Blue -defMyD Cells (5-7 x 10 6 cells) rms-dmyd 69
Rapamycin (5 mg) tlrl-rap 93
RAW-Blue Cells (5-7 x 10 6 cells) raw-sp 70
Ready-Made psiRNA plasmid (20 μg) psirna42- 148
Ready-Made psiRNA kit ksirna42- 148
RecFLA-ST (1 μg) tlrl-flic 78
RecFLA-ST (10 μg) tlrl-flic-10 78
Recombinant human TNF-a (20 μg) rhtnf-a 81
Salmon sperm DNA (50 mg) tlrl-sdef 80
SB203580 (5 mg) tlrl-sb20 93
SB431542 (5 mg) inh-sb43 21
SEAP Reporter Assay Kit rep-sap 13
SP600125 (10 mg) tlrl-sp60 93
SSL7 / Agarose (2 ml) gel-ssl-2 122
SSL7 / Agarose (10 ml) gel-ssl-10 122
ssPolyU / LyoVec (100 μg) tlrl-lpu 78
ssPolyU Naked (10 mg) tlrl-sspu 78
ssPolyU Naked (10 x 10 mg) tlrl-sspu-100 78
ssRNA40 / LyoVec (100 μg) tlrl-lrna40 78
ssRNA41 / LyoVec (100 μg) tlrl-lrna41 78
ssRNA-DR / LyoVec (100 μg) tlrl-ssdr 78
Tamoxifen (200 mg) tlrl-txf 102
THP1-XBlue Cells (5-7 x 10 6 cells) thpx-sp 68
THP1-XBlue -defMyD Cells (5-7 x 10 6 cells) thpx-dmyd 68
THP1-XBlue -MD2-CD14 Cells (5-7 x 10 6 cells) thpx-mdcdsp 68
TLR / NOD Ligand Screening tlrl-test 91
TLR1-9 Agonist Kit-Human (10 ligands) tlrl-kit1hw 81
TLR1-9 Agonist Kit-Mouse (9 ligands) tlrl-kit1mw 81
TLR2 Agonist Kit (7 ligands) tlrl-kit2 81
TLR3/7/8/9 Agonist Kit (14 ligands) tlrl-kit3hw2 81
Trichostatin A (1 mg) met-tsa-1 153
Trichostatin A (5 mg) met-tsa-5 153
Tri-DAP (1 mg) tlrl-tdap 80
Tri-Lys (1 mg) tlrl-tlys 80
Triptolide (1 mg) ant-tpl 152
U0126 (5 mg) tlrl-u0126 93
Valproic Acid (5 g) inh-vpa 21
Wortmannin (5 mg) tlrl-wtm 93
Zeocin (1 g) ant-zn-1 18
Zeocin (1 g powder) ant-zn-1p 18
Zeocin (5 g) ant-zn-5 18
Zeocin (5 g, bottle) ant-zn-5b 18
Zeocin (5 g powder) ant-zn-5p 18
Z-VAD-FMK (1 mg) tlrl-vad 98
Zymosan (100 mg) tlrl-zyn 77
Zymosan Depleted (10 mg) tlrl-dzn 81
171

A
17-AAG
17-AAGH2
17-AEP-GA
2-Aminopurine
Adju59
Adjuvants
AG490
Alhydrogel
Alkaline Phosphatase Detection
- HEK-Blue Detection
- QUANTI-Blue
- SEAP Reporter Assay Kit
Alum crystals
Angiogenic genes
Angiostatic genes
Antiapoptotic genes
Antibodies
- Anti-CD14 antibody
- Anti-CD20 antibody
- Anti-CD40L antibody
- Anti-Dectin-1 antibody
- Anti-Flagellin FliC antibody
- Anti-HA Tag antibody
- Anti-IFN-a antibody
- Anti-IFN-g antibody
- Anti-IL-1b antibody
- Anti-IL-4 antibody
- Anti-IL-6 antibody
- Anti-IL-13 antibody
- Anti-IL-18 antibody
- Anti-TGF-b antibody
- Anti-TLR1 antibody
- Anti-TLR2 antibody
- Anti-TLR3 antibody
- Anti-TLR4 antibody
- Anti-TLR5 antibody
- Anti-TLR6 antibody
- Anti-TLR9 antibody
- Anti-TNF-a antibody
Antibiotics, see Selective antibiotics
Antimycoplasma agents
Antimycotic agent
Apoptotic genes
ATP
Autophagy
5-aza-2’-deoxycytidine
5-Aza-cytidine
B
B16-Blue IFNa/b Cells
Bafilomycin A1
Bay11-7082
Biotin-GA
Bix-01294
Blasticidin S
BX795
C
C12-iE-DAP
C3H/TLR4 mut MEFs
172
A
B
C
158
159
158
93, 157
128
126
93, 157
128
13
15
14
13
98
36
36
36
72, 120, 124
72
120
104, 124
72
73
73
104
104
104
104
104
104
104
104
72
72
72
72
72
72
72
104, 120
16
10
11
36
98
101
157
21, 157
106
102
93
159
21, 157
17
93, 154
80
71
C3H/WT MEFs
C57/WT MEFs
CD genes
Celastrol
Cells
- Cytokine sensor cells
- Dectin reporter cells
- NOD reporter cells
- RLR reporter cells
- TLR reporter cells
Cellular matrix genes
Cellular promoters
- Native promoters
- Composite promoters
- Custom-made promoters
- PromTest
ChemiComp E. coli
Chemokine genes
Chemokine receptor genes
Chloroquine
Chromatin-remodeling genes
CL075
CL097
CL264, CL264 Biotin/FITC/Rhodamine
CLI-095
Connexin genes
Co-stimulatory genes
CPPD crystals
Curdlan
CpG oligonucleotides (ODNs
CpG-free DNA
CpG-free genes
CpG-free plasmids
- pCpGfree
- pCpGfree-basic
- pCpGfree-promoter
- pCpGfree-siRNA
- pCpGfree-siRNA-DUO
- pCpGfree-vitro
C-type Lectin Receptors (CLRs)
- CLR genes
- CLR shRNAs
Custom services
- Custom cloning
- Custom-made CpG-free genes
- Custom-made psiRNA
- Custom-made pDRIVE
- Large scale plasmid DNA production
- TLR ligand screening
Cyclosporin A
Cytokine signaling
- Anti-cytokine antibodies
- Cytokine and related genes
- Cytokine sensor cells
Cytokine suppressor genes
Cytolytic genes
D
INDEX
DNA production
DNA vaccination
17-DMAG
17-DMAGH2
17-DMAP-GA
Dectin-1
- Anti-Dectin-1 antibody
- Dectin-1-expressing cell line
- Dectin-1 gene
D
www.invivogen.com
71
71
37
93, 155
105
70
65,66
71, 106
65-71
36
39
40-41
40-41
162
38
43
36
36
93, 154
36
78
78
78
93, 154
36
36
98
81
78, 86
133
138
134
135
135
137
137
136
55
57
92
161-163
162
161
162
162
163
161
93
103
104
104
105
36
36
163
130-131
158
159
158
55
72
70
59
- Dectin-1 shRNA
Double stranded B DNA
E
E. coli competent cells
- ChemiComp
- LyoComp
E. coli DNA endotoxin free
E. coli selection media
E. coli ssDNA
EndoFit - endotoxin level
Epigenetic inhibitors
F
5-Fluorocytosine (5-FC)
5-Fluorouracil (5-FU)
Fast-Media ®
Fc-fusions
FITC TLR Agonist
Flagellin
- FLA-BS (B. subtilis)
- FLA-ST (S. typhimurium)
- FLA-ST Ultrapure
- Flagellin FliC VacciGrade
- RecFLA-ST (recombinant)
FSL-1
Fungin
G
E
G
G418
Ganciclovir (GCV)
Gardiquimod
Gardiquimod VacciGrade
Gefitinib
Geldanamycin
Geldanamycin analogues
- 17-AAG
- 17-AAGH2
- 17-DMAG
- 17-DMAGH2
- 17-DMAP-GA
- 17-AEP-GA
- 17-GMB-APA-GA
- 17-NHS-ALA-GA
- Biotin-GA
Gene A-List
Genes (pBLAST)
- Angiogenic/angiostatic genes
- Growth factor genes
- Inhibitors of differentiation
Genes (pORF)
- Apoptotic and antiapoptotic genes
- Autophagy genes
- Cellular matrix genes
- Chemokine and chemokine receptor genes
- Chromatin-remodelling genes
- Connexin genes
- Costimulatory / CD genes
- Cytokine genes
- Cytokine suppressor genes
92
98
42
43
43
78
44-45
78
76
157
159
159
44-45
28
78-80
84
78, 84
78, 84
78, 84
128
78, 84
77
11
17
159
78
128
93,154
158
158-159
158
159
158
159
158
158
158
158
159
36
37
36
36
36
37
36
36
36
36
36
36
36
36
36

- Cytolytic genes
- Hematopoietic genes
- Immune receptor genes
- Interferon genes
- Interferon signaling genes
- Reprogramming factors
- Suicide genes
- Tumor antigen genes
- Tumor suppressor genes
Genes (CpG-free)
- Reporter genes
- Suicide genes
Genes (pUNO)
- Toll-like receptor genes
- NOD-like receptor genes
- RIG-I-like receptor genes
- Other pathogen sensor genes
- Adaptor genes
- Co-receptor genes
- Signaling effectors
- Signaling inhibitors
Glybenclamide / Glyburide
G-ODN
Goat antibodies
GT115 E. coli
GT116 E. coli
H
H-89
HEK-Blue cells
- HEK-Blue CD40L Cells
- HEK-Blue IFN-a/b Cells
- HEK-Blue IL-1b Cells
- HEK-Blue IL-4/IL-13 Cells
- HEK-Blue IL-6 Cells
- HEK-Blue IL-18/IL-1b Cells
- HEK-Blue IL-33/IL-1b Cells
- HEK-Blue MD2-CD14 Cells
- HEK-Blue NOD Cells
- HEK-Blue Null Cells
- HEK-Blue TLR Cells
- HEK-Blue TNF-a/IL-1b Cells
- HEK-Blue TGF-b Cells
HEK-Blue Detection
HEK-Blue LPS Detection Kit
HEK-Blue Selection
Hematopoietic genes
Hemozoin
Heat killed Acholeplasma laidlawii (HKAL)
Heat killed Candida albicans (HKCA)
Heat killed Escherichia coli (HKEB)
Heat killed Helibacter pylori (HKHP)
Heat killed Listeria monocytogenes (HKLM)
Heat killed Legionella pneumophila (HKLP)
Heat killed Lactobacillus rhamnos (HKLR)
Heat killed Mycoplasma fermentans (HKMF)
Heat killed Pseudomonas aeruginosa (HKPA)
Heat killed Porphyromonas gingivalis (HKPG)
Heat killed Staphylococcus aureus (HKSA)
Heat killed Saccharomyces cerevisiae (HKSC)
Heat killed Streptococcus pneumoniae (HKSP)
Human IgA
Human IgA kappa
HygroGold
Hygromycin B
36
36
36
36
36
36
36
36
36
138
138
138
58
58
59
59
59
59
59
59-60
60
98, 155
80
73, 123
42
42
H 93, 157,
66, 107-115
115
107
100, 111
108
109
112
113
66
66
66
66
110
114
15
90
8
36
98
77
81
77
77
77
77
77
77
77
77
77
81
77
123
123
18
18
I
iE-DAP
iE-Lys
Incomplete Freund’s Adjuvant (IFA)
IFNr qRT-Primers
IgA2 isotype control
Imiquimod
Imiquimod VacciGrade
Immune receptor genes
Immunoglobulin A
- IgA antibodies
- IgA purification
- IgA detection & quantification
Inflammasomes
Inhibitors
Interferons (IFNs)
- Anti-IFN antibodies
- IFN genes
- IFN reporter cells
- Inducible promoter
Interferon Response
Interferon Regulatory Factors (IRFs)
Interleukin genes
Internal Ribosome Entry Site (IRES)
iPS (induced pluripotent stem) cells
J
Jacalin / Agarose
J chain antiserum
L
INDEX
I
J
L
L18-MDP
LacZ Cell Staining Kit
LacZ Tissue Staining Kit
LENTI-Smart , lentiviral vectors
- LENTI-Smart (INT)
- LENTI-Smart NIL
- LENTI-Smart OSKM
Leptomycin B
Lipoarabinomannan M. smegmatis (LAM-MS)
Lipomannan M. smegmatis (LM-MS)
Lipopolysaccharide (LPS)
- Biotin-LPS
- LPS EB (E. coli 0111:B4), standard
- LPS EB (E. coli 0111:B4), ultrapure
- LPS EK (E. coli K12), standard
- LPS EK (E. coli K12), ultrapure
- LPS-PG (P. gingivalis), ultrapure
- LPS-RS (R. sphaeroides), ultrapure
- LPS SM (S. minnesota), ultrapure
Lipopolysaccharide (LPS) Detection Kit
Lipoteichoic acid (LTA)
- LTA-BS (B. subtilis)
- LTA-SA (S. aureus)
- LTA-SA purified
Loxoribine
Luciferase CpGfree gene
LY294002
LyoComp E. coli
LyoVec
www.invivogen.com
80
80
128
147
124
78
128
36
121
124
122
123
96
152
104
104
106-107
74
147
130
104
32, 34
20
122
123
80
12
12
23
24
24
25
93
77
77
84
77
77
77
77
77
77
78
77
90
82
77
77
77
78
138
93, 156
43
19
M
MAb-mDectin-1
MAb-TLR
MDP - Muramyldipeptide
- L18-MDP
- N-Glycolyl-MDP
- MDP control
- MDP biotin, MDP FITC, MDP rhodamine
MEFs (murine embryonic fibroblast)
- C3H MEFs
- C57 MEFs
3-Methyladenine
Monoclonal antibodies
MPLA - Monophosphoryl lipid A
MPLAs - Synthetic monophosphoryl lipid A
MPLA VacciGrade
MSU (monosodium urate) crystals
M-TriDAP
Murabutide
Murabutide control
Mycoplasma
- Detection
- Removal agents
N
NALP3/NLRP3 inflammasome inducers
NF-kB
- Activators
- Reporter plasmids
- NF-kB reporter cells
N-Glycolyl-MDP
N-Glycolyl-MDP VacciGrade
Nigericin
NOD-like receptors (NLRs)
- NLR genes
- NOD agonist kit
- NOD reporter cells
- NOD ligands
- NOD ligand screening
Normocin
O
M
N
O
Oligonucleotides (ODNs) - TLR9 ligands
- ODN1585, ODN1585 control
- ODN1585 FITC
- ODN1668, ODN1668 control
- ODN1668 ,FITC
- ODN1826, ODN1826 control
- ODN1826 Biotin/FITC
- ODN2006, ODN2006 control
- ODN2006 Biotin/FITC
- ODN2006-G5
- ODN2007, ODN2007 control,
- ODN2088, ODN2088 control
- ODN2216, ODN2216 control
- ODN2216 Biotin/FITC
- ODN2336, ODN2336 control
- ODN2336 FITC
- ODN2395, ODN2395 control
- ODN2395 FITC
72
73
80
80
80
80
80
71
71
71
102
72, 124
77
77
128
98
80
80
80
7
8
10
98
80
74
108
80
128
98
52
57
81
65, 66
80
91
11
86
78
78
78
79
79
79
79
79
79
79
80
79
79
79
79
79
79
173

- ODN 4084-F
- ODN INH-1
- ODN INH-47
- ODN M362, ODNM362 control
- ODN M362 FITC
- ODN TTAGGG, ODN TTAGGG control
Open Reading Frames
ORN02, ORN06
Ovalbumin
OVA peptides
OxPAPC
P
174
P
Pam2CSK4, Pam2CSK4 Biotin/Rhodamine
Pam3CSK4, Pam3CSK4 Biotin/Rhodamine
Pathogen-associated molecular patterns (PAMPs)
pCpG Giant
PD0325901
PD98059
Pepinh-MYD, Pepinh-TRIF, Pepinh-Control
Peptide M / Agarose
Peptidoglycan (PGN)
- PGN-BS (B. subtilis)
- PGN-EB (E. coli 0111:B4)
- PGN-ECndi (E. coli K12) insoluble, ultrapure
- PGN-ECndss (E. coli K12) soluble, ultrapure
- PGN-EK (E. coli K12)
- PGN SA (S. aureus)
- PGN-SAndi (S. aureus) insoluble, ultrapure
Phleomycin
Plasmids
- pBLAST
- pBOOST2 / pBOOST3
- pBROAD
- pCpGfree
- pCpGfree-basic
- pCpGfree-promoter
- pCpGfree-siRNA
- pCpGfree-siRNADUO
- pCpGfree-vitro
- pCpGrich
- pDeNy
- pDRIVE
- pDRIVE-Custom
- pDUO
- pFUSE-CHIg
- pFUSE2-CLIg
- pFUSE-Fc
- pINFUSE
- pMONO
- pNiFty
- pORF
- pSELECT
- pSELECT-Tag
- psiRNA-Custom
- psiRNA-DUO
- psiRNA-h7SK
- psiTEST
- pUNO / pUNO1
- pUNO1-11RHA
- pUNO-HA
- pUNO-TLR-GFP
- pVAC
- pVITRO
- pVIVO
- pWHERE
- pZERO-TLR
- pZERO-TLR-HA
80
80
80
79
79
80
36
80
129
129
93, 154
77
77
76
80
21, 156
93, 156
93, 154
122
83
77
77
80
80
77
77
80
18
37
130
31
134
135
135
137
137
136
134
63
39
162
61
26
26
27
27
32
74
37
32
33
150
145
144
146
58
21
62
62
131
34
35
31
63
63
- Ready-made psiRNA
Plasmocin
Plasmocure
PlasmoTest
PlasmoTest Reagent Kit
PMA - Phorbol myristate acetate
Poly(A:U)
Poly(dA:dT)
Poly(dG:dC),
Poly(dT)
Poly(I:C) HMW, poly(I:C) LMW
Poly(I:C) Fluorescein/Rhodamine
Poly(I:C)/LyoVec Complexes
Poly(I:C) VacciGrade
Polyclonal antibodies
Polymyxin B
Primers
- IFNr qRT-Primers
- Sequencing primers for psiRNA
Primocin
Prodrugs
Prom A-List
Promoters
- Ubiquitous promoters
- Tissue-specific promoters
- Tumor-specific promoters
PromTest
Protein L / Agarose
psiRNA system
psiTEST system
Puromycin
Q
QUANTI-Blue
R
R848
R848 VacciGrade
Ramos-Blue Cells
Ramos-Blue -defMyD Cells
Rapamycin
RAW-Blue Cells
Ready-made psiRNA
Recombinant flagellin
Recombinant human TNF-a
Reporter genes
RLRs (RIG-I-like receptors)
- Genes
- Ligands
- Reporter cells
- shRNAs
RNA interference
Rodent Transgenesis
S
INDEX
Salmon sperm DNA
SB203850
SB431542
Selective antibiotics
- Blasticidin S
- G418
Q
R
S
www.invivogen.com
148
10
10
8
9
81
77
80
80
78
77
77
80
128
72
93, 154
147
143
11
159
39
40
40
40
41
38
122
142
146
17
14
78
128
69
69
93, 102, 154
70
148
78
81
12-13, 138
54
57
80
71, 106
148
141
31
80
93, 156
21, 155
16
17
17
- Hygromycin B / HygroGold
- Phleomycin
- Puromycin
- Zeocin
Short hairpin RNA (shRNA)
- Custom-made
- Design
- Evaluation
- Plasmids
- Ready-made
- Tools
siRNA Wizard
Small interfering RNA (siRNA)
SP600125
ssDNA (E. coli)
SSL7 / Agarose
ssPolyU
ssRNA40, ssRNA41
ssRNA-DR
Staining kits
Synthetic genes
T
Tamoxifen
Terpenoids
THP1-XBlue Cells
THP1-XBlue -MD2-CD14 Cells
THP1-XBlue -defMyD Cells
Toll-like Receptors (TLRs)
- Agonist kits
- Antibodies
- Genes
- Inhibitors of TLR signaling
- Ligands
- Reporter cell lines
- Reporter plasmids
- shRNAs
TLR-GFP fusions
TLR ligand screening
TLR-related genes
Tumor necrosis factor alpha (TNF-a)
- Anti-TNF-a antibody
- TNF-a-related shRNAs
- TNF-a reporter cells
- Recombinant TNF-a
Transfection reagents
Transgenesis
Trichostatin A
Tri-DAP
Tri-Lys
Triptolide
Tumor antigen genes
Tumor suppressor genes
U
U0126
V
Vaccination
Vaccine adjuvants
Valproic acid
T
18
18
17
18
141
150
142
146
137, 144-145
148
143
142
141
93, 156
78
122
78
78
78
12
138
102
155-156
68
68
68
49
81
72-73
36
93
77-80
65-71
74
148
62
91
59
104
149
110
81
19
31
157
80
80
156
36
36
U 93, 156
V 126, 130
126-129
21, 157

Vectors, Cloning/Expression
W
Wortmannin
Z
Zeocin
Z-VAD-FMK
Zymosan
Zymosan depleted
27-35
W 93, 156
W
18
98, 154
77, 81
81
www.invivogen.com
175

ARGENTINA
Genbiotech S.R.L.
Tel: +54 11.4541.5544
Fax: +54 11.4541.5544
info@genbiotech.com.ar
176
INTERNATIONAL DISTRIBUTORS
AUSTRALIA
Integrated Sciences Pty. Ltd.
Toll-free: 1800.252.204
Tel: +61 2.9417.7866
Fax: +61 2.9417.5066
tech@integratedsci.com.au
www.integratedsci.com.au
AUSTRIA
Eubio
Tel: +43 1.895.0145
Fax: +43 1.895.0145/14
koeck@eubio.at
www.eubio.at
BELGIUM
Cayla SAS
Tel: +33 5.62.71.69.39
Fax: +33 5.62.71.69.30
info@invivogen.fr
BRAZIL
Biotika
Tel: +55 11.3876.1004
Fax: +55 11.3826.6996
commercial@biotika.com.br
www.biotika.com.br
CANADA
Cedarlane Laboratories Limited
Toll-free: 800.268.5058
Tel: (905) 878.8891
Fax: (905) 878.7800
info@cedarlanelabs.com
www.cedarlanelabs.com
CANADA
Medicorp. Inc
Toll free: 877.733.1900
Tel: (514) 733.1900
Fax: (514) 733.1212
mktg@medicorp.com
www.medicorp.com
CHINA
ChinaGen, Inc
Tel: +86 755.2601.4623
Fax: +86 755.2601.4527
chinagen@chinagen.com.cn
Genetimes Technology Inc.
Toll free: 800.820.5565
Tel: +86 21.3367.6611
Fax: +86 21.3367.6155
order@genetimes.com.cn
www.genetimes.com.cn
M&C Gene Technology
Tel: +86 010.8205.7786
Fax: +86 010.8205.9875
order@macgene.com
NeoBioscience Technology
Company
Tel: +86 755.2675.5677
Fax: +86 755.2675.5877
info@neobioscience.com
Ming Rui Biotech
Tel: +86 021.6418.1584
Fax: +86 021.5116.3850
marketing@mrbiotech.com.cn
Tin Hang Tech
Tel: +86 852.28172121
Fax: +86 852.25807763
www.tinhangtech.com
DENMARK
Sigma-Aldrich Denmark
Tel: +45 43.565.900
Fax: +45 43.565.905
DENorder@eurnotes.sial.com
www.invivogen.com
FINLAND
YA-Kemia Oy
Tel: +358 9.350.9250
Fax: +358 9.350.92555
FINorder@europe.sial.com
FRANCE
Cayla SAS
Tel: +33 5.62.71.69.39
Fax: +33 5.62.71.69.30
info@invivogen.fr
www.invivogen.fr
GERMANY
Cayla SAS
Tel: +33 5.62.71.69.39
Fax: +33 5.62.71.69.30
info@invivogen.fr
INDIA
Genetix Biotech Asia Pvt. Ltd.
Tel: +91.11.5142.7031
Fax: +91.11.2541.9631
genetix@giasdl01.vsnl.net.in
ISRAEL
Tamar Laboratories
Supplies Ltd.
Tel: +972 2.533.6070
Fax: +972 2.579.9777
mail@tamar.co.il
www.tamar.co.il
ITALY
Labogen S.r.l.
Tel: +39 02.9390.7515
Fax: +39 02.9390.9417
info@labogen-srl.it
www.labogen-srl.it

JAPAN
Nacalai Tesque, Inc.
Tel: +81 75.211.2703
Fax: +81 75.211.2673
info-tech@nacalai.co.jp
www.nacalai.co.jp
KOREA
Daeil Bio Co., Ltd
Tel: +82 2.577.0123
Fax: +82 2.578.1425
info@daeilbio.co.kr
www.daeilbio.com
MALAYSIA
Ace Technoscience Sdn Bhd
Tel: +60 3.6273.1031
Fax: +60 3.6274.5842
acets@pd.jaring.my
MEXICO
ConsuLAB-BQ SOS
Tel: +52-665-521-2151
Fax: +52-665-521-2151
info@consulab-bqsos.com
INTERNATIONAL DISTRIBUTORS
THE NETHERLANDS
Cayla SAS
Tel: +33 5.62.71.69.39
Fax: +33 5.62.71.69.30
info@invivogen.fr
NORWAY
Sigma-Aldrich Norway AS
Tel: +47 23.17.60.60
Fax: +47 23.17.60.10
nororder@sial.com
SINGAPORE
TWC/BIO Pte Ltd
Tel: +65 873.5997
Fax: +65 873.5996
twcbio@singnet.com.sg
SPAIN
Nucliber
Tel: +34 915.062.940
Fax: +34 915.394.330
info@nucliber.com
www.nucliber.com
IBIAN Technologies
Tel: +34 976 901 645
Fax: 976 141 693
info@ibiantech.com
SWEDEN
Labkemi
Tel: +46 8.742.42.00
Fax: +46 8.742.42.43
sweorder@eurnotes.sial.com
SWITZERLAND
LabForce AG
Tel: +41 61.795.96.20
Fax: +41 61.795.96.21
info@labforce.ch
www.labforce.ch
TAIWAN
Hong Jing Co. Ltd.
Tel: +886 2.3233.8585
Fax: +886 2.3233.8686
hongjing@ms10.hinet.net
Watson Biotechnology co., Ltd
Tel: +886-2-89881951
Fax: +886-2-89881953
tensci.gene@msa.hinet.net
TURKEY
Tokra Medikal
Tel: 0090 312 3956009
Fax: 0090 312 3953961
tokra@tokra.com.tr
UNITED KINGDOM
Autogen Bioclear UK Ltd.
Tel: +44 1249.819008
Freephone: 0.800.6526774
Fax: +44 1249.817266
info@autogenbioclear.com
www.autogenbioclear.com