scaricalo in formato PDF - labogen srl

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 .