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CEll bIOlOGy

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Go to vwr.com for the latest news, special offers and

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EN-072011-VTDSVZ


Issue 27

Autumn 2011

the market source for life science

I Page 10 Real-time PCR analysis of surfactin gene expression I Page 25 Quantitative Western

blotting utilising new horizontal gel system I Page 30 Batch and fed batch cultivation of

different mammalian cell lines


the market source for life science

editorial

Hope you had a great summer and enjoy being back at work

with this autumn edition of the BioMarke Magazine.

New layout, new partner and lots of applications to help you in

your life science work.

Joining us: Polyplus Transfection, a biotechnology company that

develops innovative solutions for in vitro and in vivo delivery of

nucleic acids in reasearch, bioproduction and therapeutics.

Our long standing members have plenty to contribute too with

innovations in some interesting areas such as stem cell therapy

with integrated systems for the expansion of Mesenchymal

stem cells from BD or transfection techniques with jetPRIME ,

a powerful and versatile transfection reagent developed by

Polyplus to deliver DNA and siRNA into adherent cells ...

Also included with this issue is the VWRbioMarke Shop, a

tabloid filled with special offers on key products – often linked

to the magazine articles. This year everyone is feeling the pinch

of rising prices and spending restrictions so make sure that you

have a look through this flyer to help you get the most of your

budget!

Very best regards

The VWRBioMarke team

CONTENTS

genomics

NEW BTX Hybrimune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

The qScript microRNA Quantification System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 - 6

Techne ® thermal cycler performance testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

White PCR disposables for quantitative real-time PCR from BRAND . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 - 9

Real-time PCR analysis of surfactin gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 - 11

DNA/RNA extraction from formalin fixed, paraffin embedded tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 - 13

EvaGreen ® dye - the next generation of DNA binding dye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 - 15

New AgilePulse in vivo system DNA vaccine development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Versatile thermal cycler product range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

DNA and siRNA transfection with jetPRIME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 - 19

PROTEOMICS

Determining the most effective Dialysis MWCO for protein purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 - 21

Pall Life Sciences - centrifugal devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 - 23

OligoClear - removing oligonucleotide contaminations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Quantitative Western blotting with Amersham ECL Gel system & Amersham ECL Prime . . . . . . 25 - 27

Clean, simple and rapid purification of antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 - 29

CELL biology

Batch and fed batch cultivation in the BIOSTAT ® Aplus bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 - 32

Drug screening using Corning ® Osteo Assay Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 - 35

Thermo Scientific Nunclon Vita surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 - 37

ChillProtec ® : New protective medium for cold storage of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 - 40

Building a reliable foundation for stem cell research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 - 43

Petakas´ internal environment reflects the O 2

concentration of tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 - 47

Editor

VWR International Europe bvba

Researchpark Haasrode 2020

Geldenaaksebaan 464

3001 Leuven

Belgium

Copywriting

VWR International Europe bvba

Layout and typesetting

Marketing Services VWR

Printing

Stork, Bruchsal, Germany

No part of this publication may be reproduced

or copied without prior permission by writing of

VWR International Europe.

Run

80 120 copies

Publication date: September 2011

Due to the high sales volume of promoted articles some

items may be temporarily out of stock - VWR Terms and

Conditions of Sale apply.

2 I VWR International I VWRbioMarke Issue 27 I September 2011


genomics

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

NEW BTX Hybrimune System

Large volume hybridoma production

Human antibodies

for Iimunotherapy

development generated

via a human B cell

hybridoma technology.

Human specific monoclonal antibodies, mAbs,

have great potential for safe and targeted

therapy for many diseases. Ideally, therapeutic

Abs are produced in human B cells for optimal

human effector functions and with limited

immunogenicity. This can be done with

hybridomas, generated by fusing antigenspecific

B cells with an immortal B cell line

(myeloma). This work describes hybridoma

production for the generation of fully human

therapeutic mAbs, using electrofusion methods

to align and electroporate the cells with high

fusion efficiency and high cell viability.

Results

1. Hybridomas were produced by electrofusion

of ex vivo immunised primary human B cells

with K6H6 B5 cells at a 1:1 lymphocyte:K6H6

B5 ratio. On average 60% of seeded wells

contained viable hybridoma cells, and 1200

clones were screened after each immunisation.

2. ELISA assay demonstrated specificity of

human GM-CSF mAbs generated from the

hybridomas (Figure 1).

3. A cell-based assay demonstrated the biological

activity of human mAbs against GM-CSF. G9

and G10 mAb inhibit growth of human

GM-CSF-dependent TF1 cells (Figure 2).

4. Hybridomas were stable, showing

homogeneous retention of Ig production after

60 generations.

Conclusions

Electrofusion proved to be an efficient

method used to generate hybridoma lines

secreting mAbs with high binding specificity

and biological activity. One mAb with strong

neutralising activity against human

granulocyte–macrophage colony stimulating

factor was identified.

Figure 1. Antigen panel demonstrates

antigen-specific human mAbs from

hybridomas. Three GM-CSF-specific human

mAbs, E5, G7, and E10, reacted with human

GM-CSF and none of the other antigens in the

panel. The controls reacted as expected: Anti-TT

mAb reacted only to TT, 215, a murine mAb

specific to human GM-CSF, reacted to both

hGM-CSFand mGM-CSF, and anti-IL-3 reacted

only to IL-3.

Figure 2. Biological activity of human mAbs

against GM-CSF. A cell-based assay was

carried out to determine the level of GM-CSF

neutralisation (% of inhibition) mediated by G9 and

E10 mAbs. Human GM-CSF-dependent TF1 cells

were incubated in the presence of 0,1 ng ml

GM-CSF. No inhibition of growth was observed

when normal isotype control IgG was included

in the reaction, regardless o the concentration

used. Both E10 and G9 mAbs neutralised GM-CSF

activity.

VWR International I VWRbioMarke Issue 27 I September 2011 I

3


the market source for life science

qScript microRNA Quantification System:

Superior validation of microRNA profiling data

Ralph Hector, Sistemic UK and David Cheo, Quanta Biosciences

Data obtained from high throughput gene expression profiling platforms is often validated using real-time

RT-qPCR. Recently the application of these technologies has been expanded to include microRNAs. We have

compared the performance of the qScript microRNA Quantification System from Quanta Biosciences to

another commercially available RT-qPCR system. The qScript microRNA Quantification System was found to be

more sensitive than the competitor’s system providing a higher degree of confidence in the accurate detection

and quantification of microRNAs.

Introduction

MicroRNAs (miRNAs) are a unique class of short

(~22 nucleotide) non coding RNA molecules

that are conserved in nature and function

primarily to silence gene expression (1). Targets

for miRNAs consist of specific complementary

sequences within the 3’-untranslated regions

of messenger RNA (mRNA) transcripts. When

miRNAs hybridise to their targets they downregulate

gene expression by enhancing mRNA

degradation or inhibition of protein synthesis.

Since each of the more than 1200 human

miRNAs can potentially regulate multiple

mRNAs, as much as 60% of the human

transcriptome may be regulated by miRNAs as

predicted by recent bioinformatic analyses (2) .

Acting in concert with complex regulatory

networks, miRNAs participate in the control

of basic cellular processes such as growth,

differentiation and development. Mutation and

dysregulation of miRNAs have been implicated

in the pathogenesis of many human diseases

making miRNA important targets for disease

diagnostics and therapeutics.

High throughput expression profiling of

miRNAs is commonly performed using

microarray and next generation sequencing

platforms. Typically, profiling data is

subsequently validated using real-time RTqPCR.

There are many commercially available

kits designed to quantify the expression level

of miRNAs. Results from different miRNA

quantification systems can vary greatly

depending on multiple factors including the

Figure 1. Comparison of the qScript microRNA Quantification System from Quanta Biosciences (blue lines) to a

competitors system (red lines).

A: Amplification data from hsa-miR-125b representing a log-fold dilution series of cDNA. The qScript microRNA

Quantification System detected hsa-miR-125b with more than ten fold higher sensitivity than the competitors system at each

input amount of cDNA.

B: Amplification data from hsa-miR-1247 representing qPCR amplification of plus-PAP (solid lines) and no-PAP (broken lines)

cDNA synthesis reactions. The qScript microRNA Quantification System detected hsa-miR-1247 with 64 fold (5 Cts) higher

sensitivity and more than 128 fold (7 Cts) difference from the no-PAP control compared to a 2 Ct difference in the competitors

system.

Insets: Dissociation (melt curve) analysis of SYBR Green RT-qPCR products shows specific amplification of a specific product

from the qScript microRNA Quantification System as noted by the predominance of a single peak at the expected Tm.

The melt curve from Lower cDNA yield from the competitor’s system indicated lower yield and the presence of non specific

amplification product.

4 I VWR International I VWRbioMarke Issue 27 I September 2011


genomics

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

amount of RNA available and the abundance

of miRNAs in each sample. When RNA

samples are limiting or miRNAs are present

in low abundance, accurate detection and

measurement of miRNA expression levels

can be challenging. We have compared

the performance of the qScript microRNA

Quantification System from Quanta Biosciences

to a competitor’s miRNA assay system. The

qScript microRNA Quantification System

was determined to be more sensitive in the

detection and quantification of miRNAs and

provided a superior validation of miRNA

expression profiling data.

Materials and methods

Total RNA isolation and QC

Cells were cultured in 6 well plates in

supplemented medium until 80% confluent.

Total RNA was isolated from each sample

using the miRCURY Cell & Plant RNA isolation

kit (Exiqon, Vedbaek, Denmark) following the

manufacturer’s instructions. Total RNA was

quantified using an ND-1000 (NanoDrop,

Wilmington, USA) and RNA integrity was

checked using an Agilent 2100 Bioanalyzer

(Agilent, Santa Clara, USA), following the

manufacturer’s instructions. All total RNA

samples had an RNA integrity number of ≥8,9.

Microarray

Total RNA from each sample (100 ng)

was labelled using the miRNA Complete

Labeling and Hyb Kit (Agilent), following the

manufacturer’s instructions, and hybridised

to Human miRNA Microarrays V3 (Agilent).

Arrays were washed and scanned using the

Agilent Microarray Scanner, following the

manufacturer’s instructions. Feature Extraction

(Agilent) was used to QC the arrays and retrieve

miRNA probe fluorescence data from the

scanned image. Feature Extraction confirmed

that each microarray was of high QC standards.

Real-time RT-qPCR

Specific miRNAs were analysed using two

miRNA assay systems and the results of relative

quantification on the MX3005P Real-Time

PCR System (Stratagene, Santa Clara, USA)

were compared. For the qScript microRNA

Quantification System (Quanta Biosciences,

Gaithersburg, USA), total RNA from each

Figure 2. Two independent sample sets were used to compare RT-qPCR data to microarray data. In the first set

(left panel) hsa-miR-15a was measured in RNA samples from three liver-derived cell lines (A, B and C). In the second set (right

panel) hsa-let-7a was measured in RNA samples from three adipose-derived stem cells (D, E and F). qPCR data (green and red

bars) was plotted against microarray data (black bars) for each specific. For hsa-miR-15, samples B and C were expressed at a

significantly higher level (p


the market source for life science

sample (100 ng) was converted to cDNA using

the qScript microRNA cDNA Synthesis Kit

and qPCR performed using PerfeCTa ® SYBR ®

Green SuperMix, following the manufacturer’s

instructions. For the competitor’s system,

total RNA from each sample (100 ng) was

converted to cDNA using the miRNA-specific

cDNA Synthesis Kit and qPCR performed

using SYBR ® Green reagents following the

manufacturer’s instructions. For each assay

qPCR was performed using triplicate reactions

and appropriate controls which included a notemplate

control, a minus-poly(A) polymerase

(minus-PAP) control and a standard curve to

measure reaction efficiency. In addition, a

dissociation curve was established for each

assay to determine the degree of specificity of

the amplification reaction.

Data analysis

Following Feature Extraction, microarray data

was consolidated using JExpress (Molmine,

Bergen, Norway) and normalised using a two

step approach: normalisation of each array to

the 75th percentile then normalisation across

biological replicates using a locally weighted

linear regression algorithm. qPCR data was

analysed using the MxPro software (Stratagene),

based on comparative quantification and

baseline-corrected, reference dye-normalised

fluorescence (dRn). Dissociation curves

were analysed using the Stratagene MxPro

programme.

.

Results

Microarray data was used to select miRNAs

of high abundance (hsa-miR-125b) or low

abundance (hsa-miR-1247) for use in validation

experiments using RT-qPCR. The results shown

in Figure 1a demonstrate that although

hsa-miR-125b was readily detected by both

systems, the qScript microRNA Quantification

System was more than 16 times more sensitive

(Ct value of


genomics

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

The heat is on!

Seal your PCR plates effectively

Instruments and reagents for PCR are made

to exacting standards to ensure the optimum

performance of DNA amplification, both in

terms of yield and specificity.

Heated lids allow the effective performance of

PCR without old style oil overlays.

Carefully formulated PCR mixes enable

convenient and reproducible set-up of multiple

assays with finely controlled pH values and

precise salt concentrations.

However, all this careful preparation can be

made ineffective if the PCR reactions evaporate

to an appreciable extent during the heating and

cooling cycles of the process by reducing their

volumes.

Salt, primer and template concentrations

will alter. Carefully calculated and controlled

temperature settings are no longer optimal for

the reaction mix in its changed conditions. To

prevent excessive evaporation of samples from

microtitre plates during PCR, Techne ® test and

recommend heat seal films for the effective

control of sample evaporation.

Techne ® thermal cycler

performance testing

During development, all Techne ® PCR

instruments have been tested to ensure the

effective operation of their heated lids.

The increasing use of microtitre plates for

high throughput PCR has led to the need for

effective sealing, especially to prevent edge

wells from evaporation. Techne ® recommend

heat seal films (plastic or aluminium) for the best

blend of convenience and reliability.

Figure 1. Foil seal prevents evaporation.

Testing of a TC5000 style block/lid assembly

with heat sealed 96 well microtitre plates shows

the level of evaporation control (see Table 1).

Plates with 15, 20 and 50 µl volumes of water/

glycerol/dye mix (to simulate PCR reaction

mixes) were put through 40 cycles in a two step

PCR programme (95 ºC for 10 seconds and 60

ºC for 20 seconds). This was preceded by a hot

start step, 95 ºC for 2 minutes.

The microtitre plates used were accurately

weighed before and after thermal cycling, to

estimate the average loss of fluid per well.

Also, the plates were visually inspected for fluid

loss post run, and were tested by pipetting

for remaining volume in wells at positions A1,

A7, A12, D9, E4, H1, H7 and H12. To pass the

evaporation test, no well was accepted that did

not have more then 85% of the original volume

left after the cycling programme. Any other well

positions that seemed reduced in volume by

visual inspection were also tested by pipetting

for residual content, to pass the 85% criterion.

Results for this series of tests for multiple 96

well plates on multiple instruments are shown

in Table 1.

Table 1 : Evaporation losses, 96 well plates.

Average loss/well (% of vol)

50µl/well 1.80%

20µl/well 4.43%

15µl/well 5.90%

Heat sealer unit and plate.

The latest Techne ® thermal cycler, the TC-PLUS

model, has also been through similar testing,

and a successful example of evaporation testing

post run is shown in Figure 1, featuring a foil

sealed 384 well plate.

VWR International I VWRbioMarke Issue 27 I September 2011 I

7


the market source for life science

White PCR disposables for quantitative real-time PCR

from BRAND

Within the last 25 years PCR has become routine in most laboratories. Today

PCR techniques are common in most medical, pharmaceutical and biological

investigations.

BRAND develops, manufactures and supplies

a wide range of high-tech disposables for

storage, cell culture applications, immunology

etc. BRAND is well known for its line of high

quality, thin walled PCR products made of

PP. The wide range of BRAND PCR products

has now been available for more than 12

years. The latest developments include a

comprehensive range of white disposable

products for qPCR.

In 1992/1993 the real-time polymerase chain

reaction (or quantitative real-time polymerase

chain reaction) was developed by Higuchi et.

al. 1), 2) . Many different abbreviations are used

for this technique including Q-PCR, qPCR,

qrt-PCR and sometimes RT-PCR which leads to

confusion because this stands commonly for

reverse transcription PCR. The most significant

difference between PCR and qPCR is that the

amplified target DNA is quantified in real-time

simultaneously with the amplification process.

Quantification of amplified product is obtained

using fluorescent probes.

The new line of white PCR products from

BRAND perfectly fits the requirements of

quantitative real time PCR (qPCR).

Features of qPCR products from BRAND

Formats

Single tubes with caps, strip tubes with separate strip caps and different plate formats (24, 48, 96

and 384 well). In the 96 well format, standard and low profile versions (for the Roche ® LightCycler ®

480) are available.

Colour

All white disposables are uniformly coloured with titanium dioxide (TiO 2

). In combination with

smooth inner wall surfaces they show better amplification results due to the higher reflection of the

fluorescence signal and because of the reduced autofluorescence.

Quality

BRAND qPCR products are manufactured under cleanroom conditions according to ISO 14 644-1

class 8.

Following production, products are tested for molecular biological contamination by an

independent, accredited test laboratory. All PCR products from BRAND are certified DNA, DNase

and RNase-free.

Products

8

I VWR International I VWRbioMarke Issue 27 I September 2011


genomics

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

Additional

highlights

• Suitable for use in most

common thermal cyclers

36.0

V1

V1

V3

V4

• Extra thin walls provide for

the optimal thermal transfer

and short cycle times

Cp‘s

32.0

• 96 well low profile and 384

well PCR plates compatible

with Roche ® LightCycler ®

480 (Figure 1).

28.0

Literature

1.) Higuchi, R., Dollinger, G., Walsh,

P. S., and Griffith, R. 1992.

Simultaneous amplification

and detection of specific DNA

sequences.

Biotechnology 10:413–417.

2.) Higuchi, R., Fockler, C., Dollinger,

G., and Watson, R. 1993. Kinetic

PCR: Real time monitoring of

DNA amplification reactions.

Biotechnology 11:1026–1030.

24.0

10pg

DNA

40pg

DNA

100pg

DNA

400pg

DNA

1000pg

DNA

4000pg

DNA

Sample 1 Sample 2 Sample 3

Figure 1: Comparison between original 96 well Roche ® plate and BRAND white low profile plate using Roche ®

LightCycler ® using SYBR ® Green qPCR. Both plates lead to similar cp-values*. V1, V2 original Roche ® plates, V3, V4

BRAND PCR plates.

*Cp-value: PCR cycles that show statistically significant increases in the product from multiple samples that include

an internal standard. The point at which the fluorescence crosses the threshold is called the Cp or Ct.

Description Pk Cat. No.

Strips of 8 PCR tubes, white 125 strips/bag 731-1272

Strips of 8 PCR caps, flat, transparent 125 strips/bag 731-1250

24 well PCR plates, no skirt, white 40 (5 plates/bag) 731-0165

48 well PCR plates, no skirt, white 20 (5 plates/bag) 731-0166

96 well PCR plates, no skirt, white 50 (5 plates/bag) 731-0167

96 well PCR plates, semi-skirted, white 50 (5 plates/bag) 731-0163

96 well PCR plates, semi-skirted, low profile, white,

for Roche ® LightCycler ® 50 (10 plates/bag) 732-1462

96 well PCR plates, semi-skirted, low profile, white,

for Roche ® LightCycler ® , including qPCR sealing film

50 (10 plates/bag) + 50 sealing films 732-1463

384 well PCR Plates, fully-skirted, white,

for Roche ® LightCycler ® 50 (10 plates/bag) 731-0164

Sealing film, self-adhesive, for qPCR 100 sheets 732-1475

All products are also available in a transparent version.

VWR International I VWRbioMarke Issue 27 I September 2011 I

9


the market source for life science

Real-time PCR analysis of surfactin gene expression

Simon Baker, Research Fellow in Biotechnology, Oxford Brookes University.

The Bioprocess Research lab at Oxford Brookes University is primarily concerned

with new high value bioproducts, and better ways of over-producing them.

Introduction

One of the focus points of research in our

laboratory is the biosynthesis of surfactin, a

lipo heptapeptide with interesting surfactant

properties 1 . Although the majority of our work

has been funded by the UK Engineering &

Physical Sciences Research Council to develop

foam fractionation as a physical method for

separating biosurfactant molecules from culture

broths, we have become increasingly interested

in the way in which surfactin is regulated at

the molecular level. Surfactin is produced by

new high value

bioproducts

bacteria in the genus Bacillus, most notably

Bacillus subtilis. Each of the seven amino acids

that make up surfactin are joined together

by large cytoplasmic enzymes, with no DNA

template. This process of non ribosomal

peptide synthesis (NRPS) has been noted in

the biosynthesis of many small peptides in

both bacteria and eukaryotes. The size and

complexity of the enzyme machinery needed

to catalyse the synthesis of surfactin means

that conventional biochemical assays cannot

give us any insight into the physiological state

of the cells we are growing, so instead we are

using RT-qPCR to monitor the transcription

of key surfactin genes. The rapidity and

accuracy of real-time PCR allow us to monitor

growth in bioreactors, especially important as

we are developing a continuous method of

production 2 .

qPCR methodology

Although our RT-qPCR methodology is fairly

straightforward (two to three genes of interest,

plus three housekeeping genes give relative

expression levels) we are hampered by several

factors. Firstly, most of the research team are

chemical engineers rather than molecular

biologists, so our reagents must be simple to

use and come with clear instructions. Secondly,

we require a great dynamic range, since we

monitor expression of genes such as srfAA

from effectively zero to being one of the most

numerically dominant mRNA molecules in any

sample. Thirdly, we try to collect data every hour

for between 12 and 36 hours. Gene expression

is not the only measurement that is taken, so the

assay must allow quick error checking. Lastly, we

do not have high throughput RT-qPCR facilities:

all the assays have to be set up manually before

analysis on an ABI 7500.

qPCR consumables

Before undertaking our studies we decided

to test a range of qPCR consumables to

optimise all assays. We tried a variety of

different manufacturer’s reagents before

settling on Thermo Scientific Verso QRT-

PCR Master Mixes. The reproducibility and

dynamic range were comparable to or better

than reagents from other manufacturers

(including those that came with the realtime

PCR machine), but what really sets them

apart is the blue coloration. This means that

under time pressure, any operator, even the

most inexperienced, can set up the reactions

in 96 well plates confidently. As the volumes

are small (25 µl) it is also possible to check by

eye whether all the components have been

Figure 1: Molecular structure of the non ribosomally produced lipoheptapeptide “surfactin”

10 I VWR International I VWRbioMarke Issue 27 I September 2011


genomics

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

Figure 2: Pilot demonstration equipment showing

foam fractionation. A culture is grown in the

vessel (A) and produces surfactin. Air from the

sparger (B) blows foam into the J-tube (C). As

the foam travels up the column, medium drains

downwards by gravity, so that the ‘foamate’

collected in the beaker (D) contains 40 – 50x

more surfactant than the culture broth. The

‘foamate’ also contains a small amount of

biomass. Measuring mRNA levels in (A) provides

information on cellular biosynthetic activity, while

measuring 16S rDNA in (D) gives an accurate

bacterial count of the degree of carryover.

added, particularly useful given the numbers

of different primers used in a single plate.

Most of our work has been done using SYBR ®

Green I, but we have obtained comparable

results using corresponding master mixes for

probe detection too. A further improvement

in ease of use was the recent introduction

of the white ABgene SuperPlate . We tried

them to see by how much the white colour

would improve assay sensitivity. We found

that assay sensitivity improved even at very

low template concentrations. Pipetting clear

reagents into white plates is difficult, which

increases the likelihood of introducing errors.

This may cause more handling problems than

can be justified by the increase in sensitivity.

However, the blue coloured Thermo Scientific

Verso QRT-PCR reagents enabled us to spot

our pipetting errors before they impacted

our results. Consequently, we were able to

develop an extremely reliable assay.

References

1. Bonmartin, JM; Laprevote, O; Peypoux, F. (2003). Diversity

among cyclic lipopeptides: iturins and surfactins. Activitystructure

relationships to design new bioactive agents.

2. Combinatorial Chemistry and High Throughput

Screening 6 (6) 541-546. Chen, CY; Baker, SC; Darton,

RC (2006). Continuous production of biosurfactant with

foam fractionation. Journal of Chemical Technology &

Biotechnology 81(12) 1915-1922.

Description Pack Cat. No.

Verso 1-Step QRT-PCR Kit plus ROX vial 400 x 25 µl 733-1114

Verso SYBR Green 1-Step QRT-PCR kit plus ROX vial 400 x 25 µl 733-1129

ABgene Superplate PCR detection plate 25 plates 732-1056

ABgene Superplate PCR detection plate, white 25 plates 731-0305

ABgene Superplate semi-skirted PCR 25 plates 732-1060

ABgene Superplate semi-skirted PCR, white 25 plates 732-1063

ABgene Superplate skirted PCR plate 25 plates 732-1049

ABgene Superplate skirted PCR plate, white 25 plates 732-1052

ABgene Superplate semi-skirted raised deck, barcoded 25 plates 732-1492

ABgene Superplate semi-skirted raised deck, barcoded, white 25 plates 732-1493

ABgene Superplate semi-skirted flat deck, barcoded 25 plates 732-1494

ABgene Superplate semi-skirted flat deck, barcoded, white 25 plates 732-1495

ABgene Superplate skirted low profile, barcoded 25 plates 732-1496

ABgene Superplate skirted low profile, barcoded, white 25 plates 732-1497

VWR International I VWRbioMarke Issue 27 I September 2011 I

11


the market source for life science

DNA/RNA extraction from formalin fixed,

paraffin embedded tissue

Formalin Fixed, Paraffin

Embedded (FFPE)

tissue is one of the

most widely practiced

methods for clinical

sample preservation and

archiving. It is estimated

that, worldwide, over

a billion tissue samples,

most of them FFPE, are

being stored in numerous

hospitals, tissue banks,

and research laboratories.

These archived samples could potentially

provide a wealth of information in retrospective

molecular studies of diseased tissues. While

standard for histopathology and microscopic

investigation (e.g., Hematoxylin and Eosin

[H&E] staining, ImmunoHistoChemistry

[IHC], andTissue MicroArray [TMA]), FFPE

samples pose a major challenge for molecular

pathologists, because nucleic acids are heavily

modified and trapped by extensive proteinnucleic

acid and protein-protein cross-linking.

Historically, FFPE samples were not considered

to be a viable source for molecular analyses.

Recently, however, it has been discovered

that with appropriate protease digestion, it

is possible to release microgram amounts of

DNA and RNA from FFPE samples. The purified

nucleic acids, although highly fragmented,

are suitable for a variety of downstream

genomic and gene expression analyses, such as

polymerase chain reaction (PCR), quantitative

reverse transcription PCR (qRT-PCR), microarray,

array comparative genomic hybridisation (CGH),

microRNA, and methylation profiling.

Omega Bio-tek offers a complete line of

products for your FFPE extraction needs.

The E.Z.N.A ® spin column-based kits are ideal

for low throughput applications, whereas

our Mag-Bind ® family of magnetic beadsbased

kits are designed specifically for high

throughput users with automation capability.

Together, these kits provide clinical researchers

a comprehensive and robust sample preparation

solution for biomarker discovery, validation and

application.

Figure 1. Representative gel analysis of DNA

extracted from FFPE liver tissue using the Mag-

Bind ® FFPE DNA 96 kit (left) and E.Z.N.A. ® FFPE

DNA Isolation kit (right).

Figure 2. Representative Agilent Bioanalyzer trace

of RNA extracted from FFPE liver tissue using the

E.Z.N.A. ® FFPE RNA Isolation kit.

Table 1. Yield and quality of DNA extracted from FFPE liver tissue using the E.Z.N.A. ® FFPE DNA/RNA Isolation kit

(column) and Mag-Bind ® FFPE DNA/RNA 96 kit (magnetic beads)

DNA

RNA

Method

Column

Beads

Conc.

(μg/μL)

Yield (μg) 260/280 260/230

Conc.

(μg/μL)

Yield (µg) 260/280 260/230

0,51 5,1 1,85 1,6 0,32 44,5 2,13 2,16

0,49 4,9 1,84 1,6 0,32 44,6 2,12 2,16

0,51 5,1 1,83 1,5 0,25 34,9 2,12 2,17

0,67 6,7 1,85 1,7 0,35 34,5 2,11 1,82

0,72 7,2 1,82 1,9 0,34 34,3 2,11 1,81

0,79 7,9 1,83 1,7 0,32 32,4 2,10 1.79

12 I VWR International I VWRbioMarke Issue 27 I September 2011


genomics

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

A

B

C

D

Features and

benefits

Superior yield

Streamlined protocol

completed in as little as

40 minutes

Figure 3. Real-time quantitative RT-PCR analysis of RNA extracted from FFPE liver tissue. A 160-bp ß-actin

gene fragment was amplified using a SYBR Green qRT-PCR kit. Linear amplification was maintained over 5 log range

for RNA extracted using the Mag-Bind ® FFPE RNA 96 kit (A, B) and E.Z.N.A. ® FFPE RNA Isolation kit (C, D).

Standard protocol

requires no xylene

extraction

Magnetic beads-based

kits fully compatible

with most automation

platforms: Hamilton,

Biomek, Tecan,

KingFisher

DNA/RNA suitable for

the most demanding

downstream assays:

real-time PCR, gene

expression microarray,

copy number variation,

whole genome SNP

array, methylation

profiling and more

See BioMarkeShop

for special offer!

Description Pk Cat. No.

E.Z.N.A. ® FFPE DNA Isolation kit

50 tests OMEGD3399-01

100 tests OMEGD3399-02

Mag-Bind ® FFPE DNA 96 kit

50 tests OMEGM6958-01

100 tests OMEGM6958-02

Mag-Bind ® FFPE DNA 96 KF kit

50 tests OMEGM6954-01

100 tests OMEGM6954-02

E.Z.N.A. ® FFPE RNA Isolation kit

50 tests OMEGR6954-01

100 tests OMEGR6954-02

Mag-Bind ® FFPE RNA 96 kit

50 tests OMEGM2551-01

100 tests OMEGM2551-02

Mag-Bind ® FFPE RNA 96 KF kit

50 tests OMEGM6953-01

100 tests OMEGM6953-02

VWR International I VWRbioMarke Issue 27 I September 2011 I

13


the market source for life science

EvaGreen ® - the next generation of DNA binding dyes

EvaGreen ® dyes are ideal for use in

quantitative real-time PCR (qPCR)

and many other applications.

Biotium scientists designed the

dye by taking into consideration

several essential dye properties

relevant to PCR, including PCR

inhibition, safety, and stability

and fluorescence spectra of the

dye. The result is a dye superior

to SYBR ® Green I and other

commercial PCR or high resolution

melt curve (HRM) dyes. 1-4

See BioMarkeShop

for special offer!

PCR performance

A PCR dye emits fluorescence by forming

a dye-DNA complex. The interaction with

DNA inevitably interferes with PCR in a

number of ways, including making dsDNA

more difficult to melt, promoting primerdimer

formation and/or mis-priming,

and interfering with chain extension.

Dye inhibition of PCR can be particularly

serious at the early stage of PCR, where

the dye-to-DNA ratio is high. On the other

hand, having a sufficient amount of dye in

the master mix is important for generating

good fluorescence signal. As a result, an

optimal dye concentration must be used in

order to attain reliable PCR performance.

For many PCR dyes, such as SYBR ® Green

I, the optimal dye concentration can be

quite low, which limits PCR signal and

also makes the dyes unsuitable for high

resolution melt curve (HRM) analysis. 4

Furthermore, a master mix with low

SYBR ® Green concentration may fail to

detect multiple amplicons by melt peaks

due to dye migration from small amplicons

to large amplicons, giving the false result

of a single amplicon for a reaction that

may actually contain several products. 5

EvaGreen ® dye is designed using a

novel concept of DNA binding via

“release-on-demand” mechanism

(Figure 1). The dye is constructed of two

monomeric DNA binding dyes linked

by a flexible spacer that assumes two

different conformations in equilibrium.

In the absence of DNA, the dimeric dye

assumes a looped conformation that

is inactive in DNA binding. When DNA

is available, the looped dye shifts to a

conformation that is capable of binding to

DNA to emit fluorescence. The chemical

equilibrium provides a unique mechanism

to continuously supply the active form

of the dye from the “reserve” (i.e., the

dye in looped conformation), as more

DNA is formed during a PCR process.

Consequently, EvaGreen ® master mix

can be formulated with relative high dye

concentration to maximise fluorescence

signal without PCR inhibition, making

the mix suitable for both qPCR and HRM

applications (Figure 2). * Moreover, the

EvaGreen ® dye in the mix is sufficiently

concentrated to serve as a DNA gel stain

such that PCR product can be directly

analysed by gel electrophoresis without

the need for another gel stain. Visit

Biotium’s website for more information

on the optimally formulated Fast-Plus

EvaGreen ® master mix products.

Dye safety

Figure 1. EvaGreen ® dye binds to dsDNA

via a “release-on-demand” mechanism.

Figure 2. Comparison among Fast-Plus EvaGreen ® master mix from Biotium and two

fast SYBR ® Green master mixes from two leading companies (company A and company

Q) under similar condition. The inset is an enlarged view of the area near the baseline

for better viewing the curve patterns of the much weaker signals of the two SYBR ® -

based master mixes. Amplicon: ATPG fragment of human genomic DNA; instrument:

ABI 7900 Fast.

Another major advantage of EvaGreen ®

dye over other PCR and HRM dyes is its

safety. EvaGreen ® dye is the first and

only PCR dye currently designed to be

environmentally safe. Very few PCR dyes

have been thoroughly studied for their

safety despite the increasing use of PCR

in research and diagnostics and the fact

that DNA binding dyes are inherently

dangerous due to their potential to cause

mutation. As a result, handling and

disposal of PCR master mixes can be a

health and environmental issue. Indeed,

SYBR ® Green I is found to be even more

environmentally toxic than ethidium

bromide, a well known mutagen. 6 SYBR ®

14

I VWR International I VWRbioMarke Issue 27 I September 2011


genomics

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

Green I has been suggested to interfere

with DNA repair mechanisms in cells and

as a result it potentiates the genotoxicity

of other mutagens as well as DNA

damage by UV light. Although no safety

data is available on other PCR and HRM

dyes (e.g., SYTO9, LC Green, BRYT Green

and ResoLight), these dyes are known

to be cell membrane permeable, thus

posing potential genotoxicity risk. With

this in mind, Biotium’s scientists designed

EvaGreen ® dye to be cell membrane

impermeable by increasing the molecular

size and charge of the dye (Figure 3).

Because EvaGreen ® dye is denied the

chance to interact with genomic DNA

in living cells, it is much safer than other

qPCR dyes. Independent laboratory

testing has confirmed that EvaGreen ® is

non mutagenic, non cytotoxic and safe to

aquatic life. The dye meets environmental

hazardous waste regulations in the

state of California (CCR title 22) for easy

disposal down the drain. Visit Biotium’s

website to download the full EvaGreen ®

dye safety report.

Dye stability

EvaGreen ® dye is highly stable during

storage and PCR. SYBR ® Green I, on

the other hand, is known to degrade

following multiple freeze-thaw cycles

and under PCR conditions. Moreover,

decomposed SYBR ® Green I is reported to

be even more inhibitory to PCR than the

intact dye.7 As a result, when assessing

the performance of EvaGreen ® master

mix, you can eliminate the stability of the

dye as a variable.

Spectral compatibility

EvaGreen ® dye is spectrally similar to

FAM or SYBR ® Green I. No change in

optical settings is required when using an

EvaGreen-based master mix (Figure 4).

Figure 4. Excitation and emission spectra

of EvaGreen in the presence of dsDNA

Other applications

EvaGreen ® dye has been applied in

numerous other applications, such as

isothermal amplification, capillary gel

electrophoresis, DNA quantitation in

solution and selective staining of dead

cells in cell viability tests.

Biotium offers several EvaGreen ® dyebased

products, including stand-alone

EvaGreen dye and EvaGreen ® dye master

mix products. Biotium’s EvaGreen ® dye

technologies are available for licensing.

References:

1. Khan, et al. Detection of aacA-aphD, qacE 1, marA,

floR, and tetA genes from multidrug-resistant

bacteria: comparative analysis of real-time multiplex

PCR assays using EvaGreen ® and SYBR ® Green I

dyes, Molecular and Cellular Probes (2011), doi:

10.1016/j.mcp.2011.01.004.

2. Cheng, et al. Detection of hemi/homozygotes

through heteroduplex formation in high-resolution

melting analysis, Anal. Biochem. 410, 158 (2011).

3. White, et al. Methylation-sensitive high-resolution

melt-curve analysis of the SNRPN gene as a

diagnostic screen for Prader-Willi and Angelman

Syndromes. Clin. Chem. 53, 1960 (2007).

4. Mao, et al. Characterization of EvaGreen Dye and

the implication of its physicochemical properties for

qPCR applications. BMC Biotechnology 7, 76 (2007).

5. Giglio S, et al. Demonstration of preferential binding

of SYBR Green I to specific DNA fragments in realtime

multiplex PCR. Nucleic Acids Res. 31(22), e136

(2003).

6. Ohta, et al. Ethidium bromide and SYBR Green I

enhances the genotoxicity of UV-irradiation and

chemical mutagens in E. coli, Mut. Res. 492, 91

(2001).

7. Karsai, et al. Evaluation of a homemade SYBR green

I reaction mixture for real-time PCR quantification of

gene expression. BioTechniques 32(4), 790 (2002).

* Practicing HRM may require a license from Idaho

Technologies, Inc.; SYBR, ResoLight, LC Green and

BRYT Green are trademarks of Invitrogen, Roche,

Idaho Technologies and Promega, respectively;

EvaGreen technologies are covered by US patent

numbers 7,601,498, 7,776,567 and other pending

US and international patents.

Figure 3. Comparison of cell membrane

permeability between EvaGreen ® dye and

SYBR ® Green I. HeLa cells were incubated

with SYBR ® Green I (1,2 uM) or EvaGreen ®

dye (1,2 uM) at 37 °C. Photographs were

taken following incubation for 5 and

30 minutes. SYBR ® Green I entered cells

rapidly while EvaGreen ® dye appeared

membrane impermeable as evident from

the absence of cell nuclear staining.

Image taken with long photo exposure

time revealed that EvaGreen ® dye only

associated with cell membranes.

Description Size Cat. No.

EvaGreen ® dye, 20x in water

1 ml trial size BTIU31000-T

5x 1 ml

BTIU31000

100 rxn trial size BTIU31020-T

Fast-Plus EvaGreen ® Master mix (no ROX)

200 rxn BTIU31020

500 rxn BTIU31020-1

5000 rxn BTIU31020-2

100 rxn trial size BTIU31014-T

Fast-Plus EvaGreen ® Master mix with low ROX

200 rxn BTIU31014

500 rxn BTIU31014-1

5000 rxn BTIU31014-2

100 rxn trial size BTIU31015-T

Fast-Plus EvaGreen ® Master mix with high ROX

200 rxn BTIU31015

500 rxn BTIU31015-1

5000 rxn BTIU31015-2

VWR International I VWRbioMarke Issue 27 I September 2011 I

15


the market source for life science

New AgilePulse in vivo system

DNA vaccine development

Skin electroporation: Effects on transgene expression,

DNA persistence and local tissue environment

Intradermal DNA

injection targets the skin

for efficient delivery

in vaccine research.

The skin is an excellent

target for DNA vaccine

delivery since it is

easily accessible and

has abundant antigenpresenting

cells for a

robust immunological

response. This response

is further increased

when injection is

followed by intradermal

electroporation-- a series

of electrical pulses that

are applied through an

array of small electrodes

pressed onto the tissue.

This paper studies the

effect of intradermal

electroporation on the

kinetics of transgene

expression and DNA

persistence and reports

on the response of the

local tissue environment.

Results

1. Intradermal electroporation speeds the onset

of transgene expression to 24 hours for injection alone (Figure 2b-d).

Closer examination shows especially fast

expression for some of the injection sites, at

less than 17 minutes (Figure 2b).

2. Electroporation enhanced DNA delivery shows

no change in plasmid persistence over several

months (Figure 2a).

3. There are no visible differences in the skin that

might suggest inflammation of the electrovaccinated

mice.

4. Gene profiling of the treated vs. untreated

skin shows an up-regulation of genes that are

associated with an immunological response in

the treated skin (Figure 1). Both DNA injection

and electroporation alone showed an upregulation

of these pro-inflammatory genes

but the combination of DNA injection and

electroporation produced a tenfold further

up-regulation.

Conclusions

Intradermal electroporation enhances DNA

vaccine delivery with faster expression and

stronger immunological response. The

persistence of DNA expression is not affected by

electroporation.

Figure 1. Genes up-regulated at the DNA electrovaccination

site. Histogram showing fold increase

in gene expression, compared to non treated

control skin, of the indicated genes after the

specified treatments. Bars represent mean6SD.

The number of independently analysed samples

varied from 4 - 6; and each sample was run in

duplicates or triplicates for each QPCR. The QPCR

analysis was run three times.

Figure 2. Time kinetics of transgene expression in skin after DNA electrovaccination. (a) Time

course of in vivo luciferase expression after intradermal (i.d.) DNA delivery alone (dotted

line) and after i.d. DNA delivery followed by electroporation (filled line). One representative

experiment of two is shown (n = 8). (b – d) Immediate monitoring of gene expression after

DNA electrovaccination. Representative bioluminescent images showing luciferase expression

in skin at different time points after DNA electrovaccination. N denotes the negative control

(non injected). The scale shows intensity of luminescence (photons/sec/cm2). The experiment

was repeated three times.

16 I VWR International I VWRbioMarke Issue 27 I September 2011


genomics

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

Versatile thermal cycler product range

Thermo Scientific PCR

instrument range

includes two different

thermal cyclers,

Arktik and Piko, both

providing excellent and

reliable performance

covering a broad range

of applications from

individual PCRs through

to high throughput

projects.

Thermo Scientific Arktik thermal

cyclers

reliability and flexibility for

everyday use in any laboratory

Interchangeable heating blocks

Arktik thermal cyclers offer three different

interchangeable heating blocks: 96 well,

384 well and dual blocks with two 48 well

units. The blocks can be changed in seconds

without any additional tools. Dual 48 well

blocks allow two users to run two independent

protocols at the same time or create a new

protocol while running the other. The blocks

allow use of virtually all standard PCR plastics.

High performance

A broad range, up to 30 ºC, and accurate

temperature gradient* is available for protocol

optimisation and simplifies the user operation.

Excellent thermal precision is a result of the

unique heat sink design, as well as from the

heated lid with over tightening protection

system.

Ease of use and convenience

The Arktik thermal cycler features a highly

intuitive user interface with clear protocol

display for highly efficient programming and

operation. In addition, the instrument has the

benefit of a low noise level.

Thermo Scientific Piko thermal

cyclers

high performance in a compact

package

Superior performance

Although only half the size of other instruments,

the Piko thermal cycler provides superior

thermal performance, fast ramping rates and

quick settling times, offering excellent PCR

efficiency and well-to-well consistency. It

enables the user to complete PCR protocols in

less than 15 minutes. The automatic heated lid

function ensures consistent and tight sealing

from run-to-run. High thermal performance

makes the Piko thermal cycler suitable for

conventional and fast PCR applications.

Two formats – 24 and 96 well blocks

Available in 24 and 96 well formats, the Piko

thermal cycler can use low profile PCR tubes,

8 tube strips as well as specialised 24 and 96

well Piko PCR plates. The 24 and 96 well Ultra-

Thin Walled (UTW) Piko plates allow better

temperature transfer and are only ¼ of the size

of conventional microplates. Despite this they

maintain industry standard well spacing and

are compatible with multichannel pipettes and

liquid handling instruments.

Reduced consumption

The unique design of the heating block and small

plate format allow significant savings both in

energy and plastics consumption so helping to

reduce the environmental impact of your PCR.

* Gradient feature not available in the U.S, Germany and

Japan

Description

Cat. No.

Arktik thermal cycler, base with gradient* 731-0231

Arktik thermal cycler, base without gradient 731-0232

Arktik thermal cycler, 96 well block 731-0233

Arktik thermal cycler, 48 well dual block 2 731-0234

Piko thermal cycler, 24 well system 731-0235

Piko thermal cycler, 96 well system 731-0236

See BioMarkeShop

for special offer!

VWR International I VWRbioMarke Issue 27 I September 2011 I

17


the market source for life science

THE DELIVERY EXPERTS

DNA and siRNA transfection with

jetPRIME

Transfection is a widely

used technique for

which numerous cationic

lipid carriers have been

developed over the last

two decades. However,

while improving

efficiency over calcium

phosphate, cationic

lipids destabilise the

cell membrane making

the process toxic.

Polyplus’ researchers

have developed one

of the first non lipidic

transfection reagents,

polyethylenimine (PEI) 1

which works through

a “proton sponge” cell

entry mechanism. This

has been recognised as

a major breakthrough in

transfection techniques 2 .

Polyplus-transfection

continues product

innovation offering

jetPRIME, a powerful

and versatile transfection

reagent.

jetPRIME is an entirely new reagent developed

by Polyplus-transfection; it combines the best

advantages of lipids, i.e. strong interaction

with the nucleic acid cargo, with a natural

cell entry pathway (endocytosis) followed by

efficient proton sponge-mediated endosome

escape (Figure 1). As a consequence of tight

cargo binding, it can be used equally well for

delivery of large plasmids or small siRNA and

the combination of both. As a consequence of

the cell entry mechanism, jetPRIME is efficient

while remaining gentle to cells.

Figure 1 – Transfection mechanism with

jetPRIME .

Plasmid DNA transfection

A comparison of manufacturer’s transfection

charts (Table 1) shows that jetPRIME

requires only half the amount of plasmid and

reagent used by competitor L2K. Even with

lower amounts of reagent, the percentage of

transfected cells is much higher with jetPRIME

(70 - 90%) in all common cell lines tested

(Figure 2). Moreover, an intact cell membrane

and less transfection reagent in the cells leave

the cells healthy with jetPRIME , as can be

observed under a microscope (Figure 3).

Figure 2. Transfection efficiency assessed by FACS

analysis in various cell lines 24 hours following

transfection in 24 well plates according to the

manufacturer’s recommendation for competitor

L2K and 0,5 µg plasmid, 1 µl reagent per well for

jetPRIME .

Figure 3. Phase contrast microscopy of HeLa cells

24 hours after transfections performed according

to the manufacturer’s recommendations for each

reagent.

Many other cell lines of various origins, as well

as primary cells, are transfected with unusually

high percentages (Table 2, see also first

customers’ results 3, 4, 5 ). Moreover, jetPRIME

is recommended for virus production in classical

media.

siRNA transfection and cotransfection

of DNA and siRNA

Gene silencing using siRNA is somehow

complementary to gene expression using

plasmid DNA, and both approaches are being

used widely to unravel complex biological

mechanisms. This is why having a single multipurpose

transfection reagent on the bench is

convenient. jetPRIME leads to over 80% knock

down of even highly expressed endogenous

genes in a variety of cell lines 6 (Table 3) and

can also be used for plasmid DNA and siRNA

co-transfection experiments, with over 90%

silencing using 10 nM siRNA (data not shown).

18

I VWR International I VWRbioMarke Issue 27 I September 2011


genomics

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

Reagent

Volume of reagent

per well

Amount of DNA

per well

Number of transfections

per 1,5 ml vial

jetPRIME 4 μl 2 µg 375

L2K 10 μl 4 µg 150

Table 1 – Amount of reagent and DNA per well for optimal transfection according to the

manufacturers in a 6 well plate.

Cell type Cell line Description

Transfection

efficiency (%)

B16-F10 Murine melanoma 80 - 90

BNL-Cl2 Murine normal embryonic hepatocyte 60 - 70

CaCO2 Human colon carcinoma epithelial 20 - 30

CHO-K1 Chinese hamster ovary 70 - 80

HCT-116 Human colon carcinoma 70 - 80

HeLa Human cervix epitheloid carcinoma >90

Epithelial

HepG2 Human hepatocarcinoma 50 - 60

Huh-7 Human hepatocarcinoma 50 - 60

MCF-7 Human breast adenocarcinoma 35 - 45

MCF-10A Human breast adenocarcinoma 40 - 50

MDCK Canine kidney epithelial 20 - 30

PC-3 Human prostate carcinoma 70 - 80

Vero African green monkey kidney 50 - 60

COS-7 African green monkey kidney 70 - 80

Fibroblast

HEK-293 Human embryonic kidney fibroblast 80 - 90

MRC-5 Human lung fibroblast 50 - 60

NIH-3T3 Murine embryonic fibroblast 60 - 70

Macrophage Raw 264.7 Murine monocyte/macrophage 50 - 60

Myoblast C2C12 Murine myoblast 60 - 70

Neuronal SH-SY5Y Human neuroblastoma 80 - 90

Primary Hepatocytes Human primary hepatocytes cell 20 - 30

Primary Melanocytes Human primary melanocyte cell 40 - 50

Table 2 –Transfection efficiency of various cell types. The percentage of GFP positive cells was

determined by microcapillary cytometry 24 hours after transfection using jetPRIME (0,5 µg

DNA per well in 24 well plates).

Conclusion

jetPRIME is a new versatile compound

developed by Polyplus-transfection to deliver

DNA as well as siRNA into adherent cells. It

provides higher transfection efficiency, uses

less reagent, allows for gentle transfection

and is cost effective. Despite its outstanding

properties, jetPRIME ’s protocol remains simple

and robust: the reagent is ready-to-use and no

need to change for a serum or antibiotic-free

culture medium.

The 1,5 ml size provides enough reagent to

perform at least 375 transfections in 6 well

plates.

Trial sizes of jetPRIME are available upon

request.

References

1. Boussif et al.1995. A versatile vector for gene and

oligonucleotide transfer into cells in culture and in vivo:

polyethylenimine. PNAS: 92: 7297-7301.

2. Akinc et al. 2005. Exploring polyethylenimine-mediated

DNA transfection and the proton sponge hypothesis. J

Gene Medicine 7: 657-663.

3. Allaire et al. 2010. The Connecdenn DENN domain: a

GEF for Rab35 mediating cargo-specific exit from early

endosomes. Molecular Cell 37:370-382.

4. Behren et al. 2010. Phenotype-assisted transcriptome

analysis identifies FOXM1 downstream from Ras-

MKK3-p38 to regulate in vitro cellular invasion. Oncogene

29:1519-1530.

5. Huet et al. 2010. Nuclear import and assembly of

influenza A virus RNA polymerase studied in live cells by

fluorescence cross-correlation spectroscopy. J Virology

84:1254-1264.

6. Henson et al. 2009. DNA C-circles are specific and

quantifiable markers of alternative-lengthening-oftelomeres

activity. Nature Biotechnology 27:1181-1185.

Cells Target siRNA concentration % of inhibition

A549-Luc Luciferase 10 nM 85 - 90

HeLa GAPDH 20 nM 80 - 85

3T3 Vimentin 20 nM 85 - 90

SH-SY5Y GAPDH 25 nM 90 - 95

MEF Vimentin 25 nM 80 - 85

Primary Melanocytes GAPDH 25 nM 85 - 90

CaCo2 GAPDH 50 nM 75 - 80

C2C12 Vimentin 50 nM 75 - 80

Table 3 – Endogenous gene silencing efficiency in various cell lines using jetPRIME. Cells

were transfected in 24 well plates using 10 to 50 nM siRNA and 2 µl of jetPRIME. Negative

controls were performed for each siRNA concentration. Silencing activity was measured 48

hours after transfection.

Description Reagent size (ml) Buffer size (ml) Cat. No.

0,1 5 PPLU114-01

jetPRIME

transfection

reagent

0,75 60 PPLU114-07

1,5 2x 60 PPLU114-15

5x 1,5 10x 60 PPLU114-75

5x 1,5 120 (5x conc.) PPLU114-75C

VWR International I VWRbioMarke Issue 27 I September 2011 I

19


the market source for life science

Determining the most effective Dialysis MWCO

for protein purification

Objective

Background

Spectrum Laboratories offer a low proteinbinding

Biotech Cellulose Ester (CE), dialysis

tubing and Float-A-Lyzer ® G2 ready-to-use

dialysis devices in 9 concise Molecular Weight

Cut-Off’s (MWCO) in the range of 100 –

1 000 000 Daltons (D). Since the membrane

MWCO is based on the molecular size

retained by 90%, the challenge in purification

applications is knowing which MWCO is the

most effective for removing unwanted low

Molecular Weight (MW) contaminants.

Since the ionic species in desalting, pH change

and buffer exchange applications are sufficiently

smaller than the membrane pores and at least

1000 times smaller than macromolecules,

most MWCO’s between 3,5 and 100 kD will

achieve the desired separation within 1 - 2

days. The only consideration required is that the

MWCO should be smaller than the MW of the

macromolecular species to be retained.

Dialysis is also a preferred method for the gentle

purification of labile proteins, sensitive enzymes

and delicate complexes in order to maintain

activity, functionality or a stable aqueous

environment. However, since the lower MW

contaminants to be eliminated are often only 10

- 100 times smaller than the target protein, not

all MWCO’s are equally effective in achieving the

desired separation. Consequently, selecting the

optimal membrane MWCO is much more critical

in macromolecular purification applications.

Using Float-A-Lyzer ® G2 dialysis devices

(FAL-G2), helps determine the relative

effectiveness of 20, 50 and 100 kD MWCO’s

to retain antibody (IgG, 166 kD) and eliminate

unwanted low MW species 100 times smaller

(Vitamin B12, 1,35 kD) and 10 times smaller

(Cytochrome C, 12,4 kD) in a 24 hour period.

Secondly, to establish a general rule for

determining the most effective membrane MWCO

for dialysis applications like protein purification.

Method

1) 3 different MW solutions are prepared

representing the target protein

(IgG – 166 kD: 1 mg/ml 0,9% PBS), the 1/10X

MW contaminant (Cytochrome C – 12,4 kD:

0,2 mg/ml 0,9% PBS) and the 1/100X MW

contaminant (Vitamin B12 – 1,35 kD: 0,2 mg/

ml 0,9% PBS).

2) 9 FAL-G2 units (5 ml size) are loaded with 4

ml of solution as follows: IgG in 20 kD, 50

and 100 kD MWCO’s; Cytochrome C in 20,

50 and 100 kD MWCO’s; and Vitamin B12 in

20, 50 and 100 kD MWCO’s.

3) Each of the 9 FAL-G2’s units are dialysed for

24 hours at room temperature in 9 separate

dialysate reservoirs each containing 1 litre of

0,9% PBS buffer; exchanging the buffer after

3 and 8 hours with fresh buffer.

4) At 4 time points (0, 3, 8 and 24 hours), 200

µl test samples are taken from each of the 9

FAL-G2; diluted 1:4 in 0,9% PBS and measured

on a UV/VIS spectrophotometer (IgG at 280

nm, Cytochrome C at 406 nm and Vitamin

B12 at 366 nm) to determine the MW solution

concentration in each diluted test sample.

20 I VWR International I VWRbioMarke Issue 27 I September 2011


Proteomics

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send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

See BioMarkeShop

for special offer

and FREE samples!

5) The undiluted concentrations for all 36 test

samples are calculated and compared to the

original MW solution concentration before

dialysis (time point 0) to determine the

percent retention of each MW solute (IgG,

Cytochrome C and Vitamin B12) by each

FAL-G2 MWCO (20, 50 and 100 kD) at 3 time

points (3, 8 and 24 hours).

Results and discussion

The Float-A-Lyzer ® G2 dialysis device with screw

cap lid makes in-process testing possible so that

real-time retention of IgG, Cytochrome C and

Vitamin B12 are measured at 3, 8 and 24 hours

(Table 1) without disrupting dialysis. The percent

retentions are also compared in Table 1.

A graph for each MWCO (20, 50 and 100 kD) is

plotted to trend and compare the retention of

the target protein, IgG (166 kD); the 1/10X MW

contaminant, Cytochrom C (12.4 kD); and the

1/100X MW contaminant, Vitamin B12 (1.35 kD).

All 3 membrane MWCO’s (20,

50 and 100 kD) retained nearly

all of the target protein, IgG over

24 hours. The 20 kD MWCO

eliminated most of Vitamin B12

(87,6%) but only a small percentage

of Cytochrome C (12%). The 50 kD MWCO

removed almost all of Vitamin B12 (97,5%)

but only 24% of Cytochrom C. The 100 kD

MWCO eliminated essentially all of Vitamin B12

(99,4%) and also removed the majority (72%) of

Cytochrom C.

Conclusion

The overall relative MWCO effectiveness for IgG

purification is: 100 kD >50 kD >20 kD; 100 kD

being the optimal MWCO for IgG purification.

Refering to the MWCO retention profile curves,

the 50 kD and 100 kD MWCO membranes

effectively eliminate Vitamin B12 (the 1/100X

low MW contaminant) in 24 hours; while the

20 KD MWCO would require approximately

36 hours. Only the 100 kD MWCO effectively

eliminates Cytochrome C (the 1/10X MW

contaminant), however it requires 2 - 3 days

for completion. The 20 and 50 kD MWCO’s are

simply ineffective in eliminating Cytochrome C.

General Rules for Protein

Purification by Dialysis:

1. Dialysis requires that the target protein or

macromolecule is at least 10X larger, ideally

100X larger, than the contaminants to be

removed.

2. The most effective MWCO for protein

purification is the highest MWCO just below

the size of the protein to be retained.

3. To achieve purification in 1 – 2 days, the

membrane MWCO should be at least 100X

larger than the contaminant MW.

4. To achieve, purification in 2 - 3, the membrane

MWCO should be at least 10X larger than the

contaminant MW.

Solute

IgG, 166 kD

(Target)

Cyt.C., 12.4 kD

(Target)

Vit. B12, 1.35 kD

(Target)

(1/100x)

Retention in membrane MWCO’s

Hours 20 kD 50 kD 100 kD

3 99.0 % 99.0 % 99.0 %

8 99.0 % 98.8 % 98.8 %

24 99.0 % 98.2 % 97.5 %

3 96.0 % 93.5 % 90.0 %

8 92.0 % 85.0 % 72.5 %

24 88.0 % 76.0 % 28.0 %

3 76.1 % 62.4 % 42.7 %

8 52.5 % 36.5 % 18.8 %

24 12.4 % 2.5 % 0.6 %

Table 1

VWR International I VWRbioMarke Issue 27 I September 2011 I

21


the market source for life science

Pall Life Sciences - centrifugal devices

Exclusive

to VWR

Pall Life Sciences centrifugal

devices simplify many common

nucleic acid and protein sample

preparation procedures. These

devices provide efficient

concentration and salt removal of

samples from 50 μl to 60 ml in just

minutes.

Ultra-Filtration (UF) is a membrane

separation technique based

on selection by molecular size,

although other factors, such as

molecule shape and charge, can

also play a role. Molecules larger

than the membrane pores in the

UF membrane will be retained

at the surface of the membrane

while solvent and smaller solute

molecules will freely pass. This

molecular exclusion at the UF

membrane surface leads to

concentration of the protein

solute in the retained fraction

(termed the retentate) and can

be recovered from above the

membrane.

There are 3 generic applications

for Ultra-Filtration:

1. Concentration.

Ultra-Filtration is a very convenient

method for the concentration of dilute

protein or DNA/RNA samples. It is gentle

(does not shear DNA as large as 100

Kb or cause loss of enzymatic activity

in proteins) and very efficient (typically

>90% recovery).

2. Desalting and buffer exchange

(diafiltration).

Ultra-Filtration provides a convenient and

efficient way to remove or exchange salts,

remove detergents, separate free from

bound molecules, remove low molecular

weight components, or rapidly change the

ionic or pH environment.

3. Fractionation.

Ultra-Filtration will not accomplish a

sharp separation of two molecules with

similar molecular weights. The molecules

to be separated should differ by at least

one order of magnitude (10X) in size for

effective separation.

Fractionation is effective in applications,

such as the preparation of protein-free

filtrates, the separation of unbound or

unincorporated label from DNA and

protein samples, and the purification of

PCR products from synthesis reactions.

Centrifugal devices can replace

traditional separation techniques,

such as column chromatography,

preparative electrophoresis, alcohol or

salt precipitation, dialysis, and gradient

centrifugation, when performing the

following:

• Protein or nucleic acid concentration

• Desalting

• Buffer exchange

• Deproteination of biological samples

• Fractionation of protein mixtures

• Separation of primers from PCR

products

• Separation of labelled nucleic acids

or proteins from unincorporated

nucleotides

• Virus concentration or removal

• Clarification of cell lysates and tissue

homogenates

Nanosep ® and Nanosep ® MF

centrifugal devices

Simple, reliable concentrating and

desalting of 50 to 500 μl samples

• Ensures rapid processing of samples.

• Typical recoveries are >90% - available

with low protein binding

• Omega , Bio-Inert ® , and GHP

membranes.

• A wide range of MWCOs, colour coded

for easy identification

• Constructed of low binding

polypropylene.

• Ultrasonically welded seals prevent

bypass or seal failure

• Fits standard centrifuge rotors that

accept 1.5 ml tubes

Pall Life Sciences offer a complete range

of Ultra-Filtration devices suitable for

various sample volumes. These centrifugal

devices are specifically engineered to

provide faster flow rates and easier

handling. Each device is available with

either Omega Ultra-Filtraion membrane

or 0,2 and 0,45 m Supor ® membrane for

microfiltration applications. Devices have

a colour coded label to provide quick,

easy identification of molecular weight

cut-off and pore size.

22

I VWR International I VWRbioMarke Issue 27 I September 2011


Proteomics

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

Microsep Advance

centrifugal devices

Precise, quick recovery of microlitre

volumes of concentrate from starting

volumes up to 5.0 ml

• High recovery - achieves 50X

concentration and >90% recovery in just

minutes

• Features deadstop to prevent samples

from spinning to dryness

• Versatile Omega membrane is

available in a variety of MWCOs

• Colour coded and laser etched for easy

identification.

See BioMarkeShop

for special offer!

Macrosep Advance

centrifugal devices

Quickly concentrates up to 20 ml of

biological sample without valuable sample

loss

• Rapidly concentrates 20 ml sample

volumes to 0.5 ml

• Provides high recoveries, typically >90%.

• Low protein-binding Omega membrane

and polypropylene housing minimises

loss due to non specific binding

• Versatile Omega membrane is available

in a variety of MWCOs

• Built-in deadstop prevents spinning to

dryness

• Colour coded for easy identification

Jumbosep

centrifugal devices

Convenient and reliable concentration,

purification, and diafiltration of 15 to 60

ml biological samples

• Concentrates 60 ml sample volumes to

5 ml in 30 minutes

• Provides high recoveries, typically >90 %.

• Low protein-binding Omega

membrane and polysulphone housing

minimise losses due to non specific

binding

• Versatile Omega membrane is available

in a variety of MWCOs

• Colour coded for easy identification

• Built-in deadstop prevents spinning to

dryness

• Unique sealing mechanism prevents

retentate leakage and filtrate

contamination

• Economical - sample reservoir and filtrate

Nanosep ® centrifugal devices with Omega membrane

Description Pk Cat. No.

10K, blue

24 516-8490

100 516-8491

30K, red

24 516-8501

100 516-8502

100K, clear

24 516-8519

100 516-8520

Macrosep Advance centrifugal devices with Omega membrane

Description Pk Cat. No.

10K, blue 6 516-0357

24 516-0358

30K, red 6 516-0360

24 516-0361

100K, clear 6 516-0363

24 516-0364

Microsep Advance centrifugal devices with Omega membrane

Description Pk Cat. No.

10K, blue

24 516-0372

100 516-0373

30K, red

24 516-0374

100 516-0375

100K, clear

24 516-0376

100 516-0377

Jumbosep centrifugal device starter kits

Description Pk Cat. No.

10K starter kit, blue 4 516-8159

30K starter kit, red 4 516-8160

100K starter kit, clear 4 516-8161

For more information about Pall’s complete range of Ultra-filtration devices,

including complete product portfolio and application notes, please visit

www.pall.com/lab or speak to your local VWR sales office.

VWR International I VWRbioMarke Issue 27 I September 2011 I

23


the market source for life science

OligoClear enables you to remove

oligonucleotide contaminations from

your molecular diagnostic application

Nowadays sensitivity of DNA amplification technology is so high that the slightest

contamination with oligonucleotide sequences can disrupt your production work

flow. This leads to costly incoming goods inspections, a need for increased stock

and possible difficulties during scale-up at a later stage of the process. While GMP

oligonucleotides are manufactured to be safe in human application they are not

necessarily designed to avoid the slightest cross contamination that may interfere

with PCR amplification and the cost and delivery time could be hardly bigger.

Since its foundation in 1995 Thermo

Fisher Scientific’s Custom Biopolymer site

is well known for the synthesis of finest

oligonucleotides, each one is delivered purified

by true reverse phase HPLC as standard.

Proactively preventing contaminations always

results in a smoother and more cost effective

production. Through close collaboration

with our customers in the field of molecular

diagnostics we developed highly customised

services to meet specific requirements in terms

of purity, reliability and cost.

Understanding that every customers demands

are different, we are now offering an

OligoClear “à la carte menu” of services to

help you removing contamination threats from

your production process. The service always

includes specifically trained employees, synthesis

on dedicated equipment, chemicals batch

tracking, analysis by mass spectrometry and

comprehensive documentation.

Optional services can be added to the basic

service to meet your requirements. For

example, dedicated HLPC columns, pipettes

or syringes for your project or for each oligo,

specific decontamination procedures, sterile

filtration, desalting, solubilisation, high precision

concentration measurement etc…

To discuss your needs and design your “a

la carte” OligoClear menu, contact our

experienced scientists at

services.biopolymers@thermo.com.

OligoClear

optional services can include:

- Proprietary, reserved HPLC columns

- Reserved utility objects (syringes, bulbs,

spatulas)

- Special decontamination procedures

- Desalting by size exclusion column

- Special OD measurement for increased

precision

- Sterile filtration of final product

See BioMarkeShop

for special offer!

24

I VWR International I VWRbioMarke Issue 27 I September 2011


Proteomics

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

Quantitative Western blotting utilising new horizontal design

Amersham ECL Gel system and Amersham ECL Prime

Maria Winkvist, Susanne Grimsby and Karin Söderquist

GE Healthcare Bio-sciences AB, Björkgatan, 30, SE-751 84 Uppsala, Sweden.

GE Healthcare pioneered the ECL

approach to Western blotting

under the Amersham brand.

Traditional Western blotting with

chemiluminescence is a very well

established technique used to

study proteins from a wide variety

of sources.

See BioMarkeShop

for special offer!

The technique is used throughout life

sciences from basic research to medical

diagnostic applications. For some Western

blotting applications, a “yes or no”

answer is enough, but the addition of

quantitative data will for many applications

be beneficial. To obtain good quantitative

Western blotting data there is a demand

on broad linear dynamic range, high

signal-to-noise ratio, stability of signals

and normalisation of the total protein

amount that was loaded on the respective

gel lane. Moreover, high sensitivity will

have an impact on the linear dynamic

range as well as the possibility to detect

low abundant proteins, which is critical

for many Western blotting applications.

Here we demonstrate the use of a new

ECL reagent, Amersham ECL Prime

and new Amersham ECL precast gels,

in a number of typical Western blotting

applications. The results demonstrate

that Amersham ECL Prime reaches

limits of detection enabling analysis of

low abundant proteins, that signals are

very stable over time and cover a broad

dynamic range. These features make

Amersham ECL Prime highly suitable for

accurate quantitative Western blotting.

New Amersham ECL precast gels ensure

reliable and reproducible results. The novel

horizontal design makes gel handling and

sample loading very easy, which enables

new users to successfully perform

high quality protein

electrophoresis and

Western blotting.

Methods

Co-IP Western blotting

TAK1 was immunoprecipitated in

untreated and treated PC3U cell

lysates using anti-TAK1 mouse IgG1

monoclonal antibodies and Protein G Mag

Sepharose .

Unbound proteins were washed away and

TAK1 with its interacting proteins were

eluted from the beads. The eluate was

applied to Western blotting for detection

of the interacting protein TAB 1.

P38 phosphorylation assay

HEK 293T cells were exposed to TGF-ß.

SDS-PAGE of cell lysates on Amersham

ECL Gel was performed according to

standard procedure and was used to

evaluate levels of p38 and phosphorylated

p38 (pp38) by Western blotting. Relative

quantitation was performed after

normalisation by comparison to levels of

the housekeeping protein GAPDH.

Amersham ECL Prime Western

blotting

Western blotting was performed

according to the standard procedure and

protocol supplied with the reagent using

Hybond P membrane (PVDF), optimised

blocking solution and specific antibodies

at optimised concentrations.

Detection and imaging

Amersham ECL Prime detection

reagent was added to the membrane

(Amersham Hybond P (PVDF) and the

chemiluminescent signal was captured

using ImageQuant LAS 4000 mini

Biomolecular Imager.

Analysis

Images were analysed using ImageQuant

TL 7.0 software.

Signal stability

The signal duration was followed up to

three hours after addition of Amersham

ECL Prime. Signals were captured every

10 minutes using the same exposure time

at every time point (3 minutes). The signal

intensity for the band corresponding to

0,31 ng protein (transferrin) was analysed

and the remaining signal intensity was

compared to the initial signal intensity.

VWR International I VWRbioMarke Issue 27 I September 2011 I

25


the market source for life science

Results

High sensitivity of ECL Prime enables detection of low abundant proteins

With a limit of detection down to low

picogram levels, Amersham ECL

Prime demonstrates its usefulness for

applications ranging from confirmatory

protein detection with demand on high

sensitivity as well as detection of low

abundant proteins and post-translational

modifications.

A

B

Figure 1. Monitoring of a plasma purification process with a total protein

stain Deep Purple and confirmation of IgG content with Amersham

ECL Prime Western blotting (A). Western blotting detection of transferrin

in a twofold dilution series using Amersham ECL Prime (B).

Broad linear dynamic range is needed for reliable quantitation

A Western blotting system has a linear

dynamic range when the detected

signals are directly related to the amount

of protein on the blot. A broad linear

dynamic range allows the comparison

of weak and strong bands on the same

blot and requires that (1) high end

signals are not saturated and that (2)

the lower limit of detection is as low

as possible. A lack of linearity, caused

by saturation of strong signals, will

lead to underestimation of protein

abundances at the high end of the scale

(Figure 2). By using Amersham ECL

Prime and ImageQuant LAS 4000

mini system proteins can be detected

with a linear dynamic range of three

orders of magnitude starting on low

picogram levels as demonstrated here

using transferrin as an example. This

data demonstrates the usefulness of

the system when quantitation across a

wide level of protein concentrations is

required.

A

B

Figure 2. Western blotting detection of

transferrin in a twofold dilution series

using Amersham ECL Prime, image

obtained with ImageQuant LAS 4000

mini system (A). An illustration of broad

linear dynamic range and the consequence

of a saturated signal (B).

In-lane normalisation is a prerequisite for accurate quantitation

Normalisation with a housekeeping

protein can correct for uneven sample

loading, uneven transfer to membrane,

etc. This makes it a necessary prerequisite

A

for accurate quantitation. The best way

to do this is to relate the protein of

interest to an unregulated housekeeping

protein like actin (Figure 3).

C

In this way, it is possible to compare

levels of specific protein between lanes

even when the total protein loads are

not identical.

B

Figure 3. A schematic illustration of

how to relate your protein of interest

to a housekeeping protein (A). A Coimmunoprecipitation

of endogenous TAB1

with TAK1 in cell lysates from untreated

and TGF-ß treated PC3U cells. The Coimmunoprecipitation

was performed with

Protein G Mag Sepharose and detection

with Amersham ECL Prime and

ImageQuant LAS 4000 mini (B). Detection

of different levels of phosphorylated STAT3

(pSTAT3) in 5 different HeLa cell lysates.

Relative quantitation of pSTAT3 levels after

normalisation to actin for correction of

uneven sample amount (C).

26

I VWR International I VWRbioMarke Issue 27 I September 2011


Proteomics

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Stability of signal and efficient use of antibodies is convenient and cost saving

The stability of signal over time and the

ability to dilute the antibodies allows

flexibility, convenience and efficient use of

precious antibodies. (Figure 4)

Figure 4. Amersham ECL Prime signal duration

was followed 3 hours after the addition of

reagent by capturing images every 30 minutes

with the same exposure time (3 minutes). The

signal intensity for the band corresponding to

0.31 ng protein (transferrin) is analysed and the

remaining signal intensity was compared to the

initial signal intensity (A). Comparison of Western

blotting detection performance using highly

diluted antibodies. ß-catenin was detected in

twofold dilution series of NIH 3T3 whole cell

lysate using primary antibody dilutions ranging

from 1:3000 to 1:10 000 and secondary antibody

at 1:30 000 or 1:50 000 (B).

Quantitating post-translational modifications

p38 is a mitogen-activated protein

kinase involved in cell differentiation

and apoptosis and is regulated by

phosphorylation. Here, HEK 293T cells

were exposed to TGF-ß. After SDS-

PAGE of cell lysates on Amersham

ECL Gel, Western blotting was

performed to evaluate levels of p38 and

phosphorylated p38 (pp38) over time.

Relative quantitation was performed after

normalisation by comparison to levels

of the housekeeping protein GAPDH.

(Figure 5)

Figure 5. Quantitative Western blotting analysis

of p38 and pp38 following stimulation of

HEK 293T cells with TGF-ß. p38 responded to

stimulation with TGF-ß by phosphorylation

after 30 minutes. Note that pp38 signals

in isolation would indicate a peak in

phosphorylation levels after 120 minutes.

Normalisation by comparison with GAPDH

signals shows that this is not the case and that

phosphorylation of p38 is maximal after 30

minutes and then remains phosphorylated in

the presence of TGF-ß.

Figure 6. The new Amersham ECL Gel system,

consisting of the Gel Box electrophoresis unit

and a selection of ready-to-use pre-cast gels

(10%, 12%, 4 - 12%, 8 - 16%, and 4 - 20%

acrylamide, 10, 15 or 2 sample wells) has a

novel horizontal design. This simplifies handling,

especially the sample loading, reduces buffer

consumption (190 ml per run) and significantly

reduces the risk of leakage. It utilises Tris/Glycine

buffers. The gels can be run as denaturing gels

using the standard Laemmli system or as native

gels.

Conclusions

The high sensitivity of Amersham ECL

Prime combined with the broad linear

dynamic range of CCD-based imagers, such

as ImageQuant LAS 4000 series, enables

relative quantitation of target proteins

with confidence. This is demonstrated

with several typical Western blotting

applications, with a limit of detection

on the low picogram level and a broad

linear dynamic range. Moreover, stability

of the signal over time and possibilities to

dilute antibodies more allows convenient

handling, enables repeated exposures

and efficient use of precious antibodies.

Together with the new Amersham ECL

precast gels consistent separation with

high resolution was observed. The new

horizontal design enables easy operation

and sample loading.

Acknowledgements

Professor Marene Landström Umeå University,

Ludwig Institute for Cancer Research Uppsala is

acknowledged for kindly providing cell lysates

for p38 and Co-IP experiments and valuable

discussions.

Description Pk Cat. No.

Amersham ECL Gel 10%, 10 wells 10 gels GEHE28-9898-04

Amersham ECL Gel 10%, 15 wells 10 gels GEHE28-9901-55

Amersham ECL Gel 12%, 10 wells 10 gels GEHE28-9898-05

Amersham ECL Gel 12%, 15 wells 10 gels GEHE28-9901-56

Amersham ECL Gel 4 - 12%, 10 wells 10 gels GEHE28-9898-06

Amersham ECL Gel 4 - 12%, 15 wells 10 gels GEHE28-9901-57

Amersham ECL Gel 8 - 16%, 10 wells 10 gels GEHE28-9898-07

Amersham ECL Gel 8 - 16%, 15 wells 10 gels GEHE28-9901-58

Amersham ECL Gel 4 - 20%, 10 wells 10 gels GEHE28-9901-54

Amersham ECL Gel 4 - 20%, 15 wells 10 gels GEHE28-9901-59

Amersham ECL Gel Box 1 GEHE28-9906-08

VWR International I VWRbioMarke Issue 27 I September 2011 I

27


the market source for life science

Clean, simple and rapid purification of antibodies

Purify IgG from serum in under 15 minutes with Pearl antibody purification resin

G-Biosciences’ new

Pearl IgG purification

resin functional groups

bind to the majority

of proteins present in

serum, ascites and tissue

culture supernatants,

allowing the IgG to

rapidly pass through and

be collected in the flowthrough

fraction. The

main advantage of Pearl

IgG purification resin is

that IgG antibodies are

not purified by a bind

and release mechanism,

which is rapid and

extremely gently on

the IgG molecules. This

results in highly pure

and active IgG antibody

molecules.

Another advantage is that it is compatible

with a wider variety of different species and

subclasses of antibodies, compared to the

expensive, bind and release, Protein A and

Protein G resins (Table 1).

Pearl IgG purification resin enables

antibodies purified from the resin are ready

for use in downstream applications, including

immunoassays or subsequent conjugation/

labelling reactions. The mild elution

conditions mean that further purification

is not required to neutralise or desalt the

sample, a common requirement with bind

and release resins, such as Protein A or

Protein G.

Products

In addition to Pearl IgG purification resin,

G-Biosciences offers four complete kits that

utilise the Pearl IgG purification resin.

Pearl IgG purification

(spin format):

For the purification of up to 25 mg antibody in


Proteomics

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

Results

Figure 1: IgG molecules were successfully purified

from rabbit serum with the spin format Pearl IgG

purification kit. Diluted samples of starting serum

and the purified IgG were resolved by SDS_PAGE

under reducing and non reducing conditions. The

IgG molecules are indicated.

the purified IgG. The original rabbit serum

and flow through was diluted 1:10 and 5 µl

were compared on a SDS polyacrylamide gel

under reducing and non reducing conditions.

The proteins were visualised with Labsafe GEL

Blue protein stain.

Conclusion

The IgG was rapidly purified in 90% recovery and

~80% purity, which is comparable or better than

published data for Protein A and Protein G resins.

Description Size Cat. No.

Pearl IgG purification (spin format) kit For 25 mg IgG GENO786-798

Pearl IgG purification kit For 200 mg IgG GENO786-799

Pearl IgG purification resin 3 ml resin GENO786-800

Pearl IgG purification resin 25 ml resin GENO786-801

Pearl Monoclonal IgG purification kit 1 l serum/0.2 l ascites GENO786-802

Pearl Antibody Clean Up kit 10 GENO786-803

Tube-O-DIALYZER, Micro 1K MWCO 20 GENO786-610

Tube-O-DIALYZER, Micro 4K MWCO 20 GENO786-611

Tube-O-DIALYZER, Micro 8K MWCO 20 GENO786-612

Tube-O-DIALYZER, Micro 15K MWCO 20 GENO786-613

Tube-O-DIALYZER, Micro 50K MWCO 20 GENO786-614

LabSafe GEL Blue 1 litre GENO786-35

LabSafe GEL Blue 1 gallon GENO786-35G

VWR International I VWRbioMarke Issue 27 I September 2011 I

29


the market source for life science

Batch and fed batch cultivation of different mammalian

cell lines in the BIOSTAT ® Aplus bioreactor

D. Lampe, B. Pütz, D. Lütkemeyer , Frank Gudermann, Apparative Biotechnology,

Department of Engineering Sciences and Mathematics, Bielefeld University of Applied Sciences, Bielefeld, Germany

Sabrina Armgart, Ute Noack, Sartorius Stedim Biotech GmbH, Goettingen, Germany

BIOSTAT ® Aplus is a

compact laboratory

bioreactor with an

autoclavable vessel

featuring a stirred tank

design. The system is

available in a choice

of culture 1, 2 and 5

litre volumes and preconfigured

versions

for microbial and cell

cultivation.

This wide selection makes it easy for users

to choose the bioreactor model that best

matches their specific needs. Thanks to the

intuitive operating design of this bioreactor

via notebook PC, it is especially suitable for

educational purposes or first time bioreactor

users. Typical applications range from small

scale protein expression to up-scale experiments

from uncontrolled shake or T-flasks to controlled

cultivation conditions. All systems measure

and automatically control dissolved oxygen

(DO), pH, temperature, foam and level. In

particular, DO control can be set-up as in larger

scale bioreactors normally used in process

development, while keeping operation easy.

Stirrer speed, gas mixing and substrate addition

can be selected for the DO cascade. Automatic

pH control can be accomplished by selecting

acid and base addition or CO 2

and base addition

to suit the needs of a specific application.

Three internal peristaltic pumps can be used

for corrective agents or substrate. A fourth

external pump can also be connected. Different

impeller and sparger designs are available to

suit organisms with a high demand for oxygen

as well as sheer sensitive cells. The BIOSTAT ®

Aplus also includes an installation video and

cultivation recipes for common cell lines to

facilitate installation and first time use of the

bioreactor.

The following application data was compiled at

the University of Applied Sciences in Bielefeld,

Germany.

At Bielefeld University the BIOSTAT ® Aplus is

used for various applications during students’

practical lab course work in cell cultivation.

1st application:

CHO cultivation in fed batch mode

For fed batch cultivations in a 2 litre BIOSTAT ®

Aplus system, a CHO cell line was used which

secretes a recombinant monoclonal IgG

antibody. The protein-free, chemically defined

medium, MAM-PF 2 from BioConcept Amimed,

was used as the culture medium.

The concentration of living and dead cells was

measured using the Cedex from Roche Applied

Sciences. The concentration of glucose, lactate,

glutamine, ammonium and IgG was determined

using the Cubian XC from Optocell. (Figure 1)

The bioreactor was equipped with a 3 blade

segment impeller with pull down mixing

direction in the upper section and a 6 blade

disc (Rushton) impeller approx. 2 cm above

the sparger. At an agitation speed of 200 rpm,

this configuration — in combination with the

microsparger used for the submerse gassing

— ensures a small gas bubble diameter as well

as good mixing conditions. The residence time

of the small gas bubbles in the medium is also

30 I VWR International I VWRbioMarke Issue 27 I September 2011


Cell biology

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

Figure 1: Schematic diagram of the BIOSTAT ® Aplus for fed batch cultivation

increased, as they are retained for some time in

the lower section of the bioreactor due to the

flow profile of the disc impeller.

The regulation of the pH at 7,1 using CO 2

was

performed exclusively via the headspace. A

constant flow of air (200 ccm) through the

headspace was mixed with the corresponding

proportion of CO 2

via the control of the Aplus.

Using sodium carbonate (1 M), the pH could, if

necessary, be maintained in the alkaline range

during the second phase of the cultivation.

The required amount of dissolved oxygen was

transfered into the suspension by submerse gassing

using the microsparger. Pure oxygen was used to

minimise the gas flow through the liquid. In case

of dissolved oxygen concentrations below the

target value of 40%, a short injection of oxygen

was used to generate a succession of bubbles,

which were held in the liquid for some time. In this

way, a continuous gas flow through the sparger

was avoided and pulsed gassing of the culture was

achieved. As a result foaming on the surface of

the liquid was significantly reduced, minimising the

need for anti-foam agents (Antifoam C, Sigma).

(Figure 2)

The Figure shows a fed batch cultivation

with the addition of separate 25 ml doses

of “CHO Feed Bioreactor Supplement”

from SAFC Biosciences. The feeding was

performed every 48 hours. Additionally the

glutamine and glucose concentration of each

sample were determined with the Cubian XC,

Optocell. The substrate need for the next 48

hours was calculated based on the results.

Furthermore, the specific growth rate µ as well

as the specific uptake rate qGLC and qGLN

were approximately determined from the

concentration of the last two samples.

The addition times of the substrate mix are

marked in the graph by grey bars. With this

strategy, a maximum viable cell density of

125 - 10 5 cells/ml with a viability of 90% was

achieved on the seventh day.

In a second fed batch cultivation using the

same cell line, the viable cell density was

additionally determined online by continuous

measurement of the capacity of the cell

suspension using the i-Biomass 465 from

Fogale. In this second scenario, the aim was

to keep the cell culture at a constant cell

concentration for as long as possible, as the

cell line only produces noteworthy amounts

of antibodies in the stationary phase. In this

process, 50 ml of amino acid concentrate

and 50 ml of glucose/glutamine mix — the

composition was adapted to the requirements

of the culture in advance — were added

at three separate times. The times of these

additions are once again marked by grey bars

in the following two graphs. (Figure 3)

Figure 2: Development of cell concentration and

viability over time

Figure 3: Variation of cell and product

concentration over time with feed times

VWR International I VWRbioMarke Issue 27 I September 2011 I

31


the market source for life science

The viable cell concentration reached 56 ∙ 10 5

cells/ml with a viability of 98%. The specific

growth rate was 1.06 d-1. At the end of the

process, the product concentration was 35 mg/l

(referred to as 100% in Figure 3). (Figure 4)

In order to continuously determine the

concentration of viable cells, the capacity of

the cell suspension was measured using the

i-Biomass 465. Figure 4 shows that, in the first

5 days of the cultivation, the capacity correlates

very well with the concentration of living cells

determined offline. After the third addition of

the concentrate and a slight reduction in the

concentration of viable cells the results of the

two measuring processes start to differ from

one another. (Figure 5)

The substrate concentrations of the glucose

and glutamine were kept between 2 g/l – 6

g/l (glucose) and 1.5 mM – 10 mM (glutamine)

during the cultivation by the addition of

concentrates. On the eighth day, the lactate

concentration rose to 7 g/l.

2nd application:

BHK cultivation in batch mode

In a conventional batch process, the aim was

to achieve a maximum viable cell concentration

without additional substrate feeding. For this

purpose, a BHK cell line was cultivated in a

chemically defined medium, specially designed for

this task by Teutocell. The culture volume was 1 litre.

A 6 blade disc impeller was used for the mixing

and a ring sparger was used for gassing. In the

same way as in the cultivation described above,

the agitation speed was set at 200 rpm and the

regulation of the pH at the target value of 7.1

was achieved using CO 2

exclusively via the head

space. The DO set point was set at 40% air

saturation was achieved using pure oxygen by

submerse gassing using the ring sparger.

In this way, it was possible to achieve a

concentration of viable cells of 135 ∙ 10 5 cells/

ml. However, on the third day, the glutamine

concentration of the cultivation was 2.5 mM.

The grey bar in Figure 6 shows that, at this

point, the substrate concentration in the

bioreactor was increased to over 6 mM by the

addition of a glutamine concentrate. (Figure 6)

Summary

The results of the batch and fed-batch

cultivation of animal cells show that the

compact and easy-to-use BIOSTAT ® Aplus can

be used very effectively in teaching. The system

is not only outstandingly suitable for batch

operation, in which cell concentrations of over

13 million cells per ml are achieved, it can also

carry out fed-batch processes over a period

of more than a week. Using a corresponding

adapter (Sartorius Stedim Biotech), additional

electrodes besides pH, DO, PT100 and antifoam

can be installed for online measurements

via the top plate of the bioreactor. In this

example, the capacity of the cell suspension was

measured online and compared with the cell

concentrations determined offline.

Figure 4: Correlation between the

concentration of living cells and the

capacity of the suspension

Figure 5: Variation in the concentration of

glucose, lactate and glutamine over time

Figure 6: Variation in cell concentrations

over time

32 I VWR International I VWRbioMarke Issue 27 I September 2011


Cell biology

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

Drug screening using Corning ® Osteo Assay Surface

plates

Customer application note

Gazelle Crasto, Norbert Kartner and Morris F. Manolson

Faculty of Dentistry, University of

Toronto

Toronto ON M5G 1G6 Canada

A healthy skeleton is

a dynamic framework

of bone that is

constantly remodelled.

Without remodelling,

bone accumulates

microfractures and

loses its mechanical

strength. The process

of remodelling is

mediated primarily by

the concerted action

of two specialised cell

types, bone resorbing

osteoclasts and bone

depositing osteoblasts.

In healthy bone, a finely tuned balance between

resorption and deposition is achieved, but

menopause and disease can shift this balance in

favour of resorption. The result is pathological

bone loss, as is seen in osteoporosis,

inflammatory arthritis, periodontitis and some

metastatic tumors. These diseases erode the

quality of life in millions and result in tens of

billions of dollars in increased healthcare costs.

Consequently, there has been a remarkable

research interest in the pathophysiology and

molecular cell biology of bone remodelling,

and especially in the development of new

antiresorptive therapeutics. In vitro, osteoclast

resorption is studied by plating the cells on thin,

polished slices of dentin obtained from teeth or

tusks. Dentin slices are traditionally the “gold

standard” for assaying osteoclast resorption,

because of their close resemblance to bone

matrix, while offering a microscopically uniform

surface. The acquisition of dentin is limiting, its

preparation is extraordinarily labour intensive,

and sample handling is tedious. Therefore, for

high throughput screening applications, dentin

is not cost effective.

Corning Life Sciences has recently met the need

for a reliable, cost effective, bone-like mineral

surface, for both routine resorption assays

and high throughput screening, with its new

Corning ® Osteo Assay Surface multiple well

plates. The wells of these plates are coated with

a proprietary hydroxyapatite mineral surface

that is highly uniform and microscopically

fine grained. These plates offer an excellent

alternative to dentin for the study of both

osteoclast and osteoblast function. The surface

allows for easy quantification without affecting

cell differentiation and function.

We have used Corning ® Osteo Assay Surface

plates for secondary screening of potential

antiresorptives identified in a molecularinteraction

primary screen. This approach allows

rapid, cost effective elimination of compounds

that are toxic, affect osteoclast differentiation,

or do not inhibit resorption at a reasonable EC 50 ,

prior to committing to expensive animal testing.

This technical report will cover materials and

procedures that were used for in vitro screening

of osteoclast function, tartrate-resistant acid

phosphatase (TRAP) staining of osteoclasts,

von Kossa staining of the mineral surface for

increased contrast in pit identification, and

the use of NIH ImageJ for quantification of

resorption pit images.

Materials and procedures

Osteoclast cell culture for pit formation

RAW 264.7 cells, a mouse macrophage cell

line, were differentiated into osteoclasts in the

presence of RANKL (GST-soluble recombinant

murine RANKL fusion protein). These cells were

maintained in high glucose DMEM (Sigma-

Aldrich, Cat. No. D5796) in tissue culture

treated polystyrene 75 cm 2 flasks at 37 °C in

a humidified 5% CO 2

incubator. Typically, cells

of low passage and 50 to 70% confluence

were washed with DPBS after removing spent

DMEM, and were removed from the plastic

by scraping. Cells were centrifuged at 300

x g for 5 minutes and were resuspended in

4 ml of α-MEM (without ribonucleosides or

deoxyribonucleosides; Invitrogen, Cat. No.

12571-063), containing fetal bovine serum

(FBS) and antibiotics, which was pre-warmed to

37 °C. Cell density was determined by counting

with a hemacytometer or Coulter ® counter

calibrated for RAW 264.7

For 96 well plates, the number of cells seeded

was 5000 per well. Cells were diluted to 5 x

104/ml in the complete medium and 100 μl of

the cell suspension was added to each well.

Plates were incubated for 2 hours to allow

for cell attachment. The effects of various

antiresorptive lead candidates on the ability

of RAW 264.7 to differentiate, and then

resorb mineral, was measured. Typically, serial

dilutions of drug compounds were tested on

cells being differentiated in situ with RANKL.

For RAW 264.7 cells, media controls and serial

drug dilutions in medium all contained 100

ng/ml of RANKL and these conditions were

maintained to the end of the experiment.

VWR International I VWRbioMarke Issue 27 I September 2011 I

33


the market source for life science

Drug-free and vehicle controls were always

included. The total volume of medium per well

in the 96 well plates was 200 μl. Experimental

media were prepared as 2X concentrates with

respect to the drugs being tested. 100 μl

volume was added per well to the plates, after

the 2 hours of cell attachment incubation. The

final RANKL concentration was 50 ng/ml and

drug concentrations were 1X. The plates were

incubated for another 72 hours, then observed

under the microscope. Complete 1X media were

exchanged and the plates were incubated for

a further 48 hours. On day 5, all media were

aspirated and plates were stained for TRAP to

visualise osteoclasts (Figure 1). Alternatively,

plates were bleached and contrast enhanced

to assess resorbed surface area (Figure 2). If

storage was required, bleached plates were

stored with 200 μl of water per well (aspirated

for imaging) at 4 °C. It was important to store

the bleached plates in water to avoid cracking

of the dry mineral surface. TRAP stained plates

were stored in DPBS at 4 °C. If salts crystallised

and precipitated during storage, the plates

were rinsed with distilled water to dislodge and

dissolve the crystals, and the buffer was then

replaced.

Figure 1. Dose-dependent drug response as measured by TRAP staining of osteoclasts on

Corning Osteo Assay Surface.

Figure 2. Von Kossa staining for image enhancement on Corning Osteo Assay Surface.

Staining and image analysis

For TRAP staining, TRAP buffer (pH 5.0)

was prepared fresh for use by mixing 50 ml

acetate buffer (35.2 ml 0.2M sodium acetate

and 14.8 ml 0.2 M acetic acid), 10 ml 0.3M

sodium tartrate, 1 ml 10 mg/ml naphthol AS-

MX phosphate disodium salt (Sigma-Aldrich,

Cat. No. N-5000), 0.10 ml Triton ® X-100

and 38.9 ml distilled water. TRAP stain was

prepared fresh for use by dissolving 0.3 mg

Fast Red Violet LB salt (Sigma-Aldrich, Cat. No.

F-3381) per ml of TRAP buffer at 37 °C. For

TRAP staining, medium was aspirated from

cells and cells were washed with PBS. Cells

were fixed with 200 μl/well formalin (Sigma-

Aldrich; Cat. No. HT501128) for 15 minutes

at 37 °C, then washed three times with PBS

at 37 °C. Cells were incubated in TRAP stain

for 5 to 10 minutes at 37 °C. TRAP stain was

aspirated and cells were washed with Ca++/

Mg++-free PBS and were stored in the same

buffer at 4 °C. The modified von Kossa staining

protocol was used to improve the contrast for

resorbed pit image analysis and quantification.

For brightfield visualisation, von Kossa staining

greatly increases the image contrast, facilitating

imaging and quantification of resorption pits

(see Figure 2). For von Kossa staining, 100 μl

of 5% (w/v) aqueous silver nitrate solution was

added to each of the bleached wells of a 96

well plate. Plates were incubated for 30 minutes

at room temperature in the dark (covered with

foil). The silver nitrate solution was discarded

into a hazardous waste container and the

plates were then soaked in distilled water for

5 minutes. The water was discarded into a

hazardous waste container. The mineral surface

appears yellow after this step. The ionic silver

(I) was reduced to metallic silver, developing a

dark colour, by adding 100 μl of 5% sodium

carbonate (w/v in commercial buffered

formalin) and incubated for 4 minutes at room

temperature. The carbonate/formalin solution

was discarded into a hazardous waste container

and the plates were dried at 50 °C for 1 hour.

For microscopy, the multiple well plate was

oriented with the bottom of the wells closest

to the objective lens of an inverted microscope.

Digital images were captured using resident

image capture software. NIH ImageJ software

was used for image analysis (downloaded from

the NIH web site, http://rsbweb.nih.gov/ij/).

Table 1. Resorbed Surface Area Analysis on Corning

Osteo Assay Surface

Test compound (μM)

Resorbed surface

(percent area)

Control 46.36

0.60 33.95

1.25 27.04

2.50 26.60

5.00 17.95

10.00 3.83

34 I VWR International I VWRbioMarke Issue 27 I September 2011


Cell biology

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

tool. The uniformity of the mineral surface

of the Corning ® Osteo Assay Surface plates

is far superior to other artificial surfaces on

the market and tested to date, allowing

unprecedented precision in quantitative

resorption assays.

Conclusion

Results and discussion

TRAP staining was performed as described in

the methods above, to determine the effect

of an anti-resorptive lead compound on RAW

264.7 cell differentiation. Wells were observed

using brightfield microscopy, and TRAP positive

multi-nucleated osteoclasts were scored for a

drug concentrations range (0.6 μM to 10 μM).

In the example shown in Figure 1, no effect

on differentiation was seen at the EC 50 of the

test compound (1.25 μM), although some

inhibition was evident at higher concentrations.

Plates to be quantified for resorption were first

bleached with a 1:4 dilution of commercial

household bleach in distilled water. Von Kossa

staining was performed, as detailed above.

Results for brightfield microscopy are shown

here (Figure 2). The white areas of the image

are the pits resorbed by osteoclasts derived

from RAW 264.7 cells; the silver stained mineral

is black. Percentages of surface resorbed by

differentiated osteoclasts at various drug

concentrations were quantified and are shown

in Table 1. A serial dilution of test compound

shows an EC 50 of 1.2 μM. At this concentration,

growth and differentiation of RAW 264.7

number cells were unaffected, suggesting that

this compound is a potential lead candidate for

further drug development. The imaging and

image analysis can be automated, providing

an efficient, high throughput screening

The objective of this study was to screen

chemical libraries for potential novel

antiresorptive therapeutics, using defined

molecular targets. Lead candidates identified

using in vitro assays must at some point

be validated in vivo; however, if hundreds

of candidates are available, a simple, high

throughput intermediate or secondary

assay needs to be performed to choose

only the best possible candidates before

proceeding to costly animal studies. Prior to

the availability of the Corning ® Osteo Assay

Surface plates, alternative artificial mineral

coated plates and dentin slices were used.

The former lacked uniformity and were

prohibitively expensive, while the latter had

the serious procurement and handling issues

mentioned in the introduction. Neither

choice was suitable for high throughput

formats. The present results show that the

osteoclast model cell line, RAW264.7, could

differentiate without difficulty on Corning

Osteo Assay Surface plates, and effects on

differentiation and mineral resorption were

observed in the presence of test compounds.

In addition, pit formation was analysed with

an easily automated microscopic approach.

While studies on bone-like matrices such

as dentin will remain desirable as a final

analytical tool, and animal studies are a

requirement for drug development, using

Corning Osteo Assay Surface plates in an

intermediate cellular assay allows high

throughput secondary screening to isolate

the most valuable lead candidates. These

plates provide an ideal assay surface for

high throughput drug screening, or for basic

research in bone biology.

Further reading

Kartner, N., Y. Yao, K. Li, G.J. Crasto, A. Datti

and M.F. Manolson. Inhibition of Osteoclast

Resorption by Disrupting V-ATPase a3-B2

Subunit Interaction (2010) submitted.

Forgac, M. Vacuolar ATPases: rotary proton

pumps in physiology and pathophysiology.

(2007) Nature Rev Molec Cell Biol 8, 917-929.

Bradley, E.W. and M.J. Oursler. Osteoclast

culture and resorption assays. (2008) Met Molec

Biol 455, 19-35.

VWR International I VWRbioMarke Issue 27 I September 2011 I

35


the market source for life science

Thermo Scientific Nunclon Vita surface

Feeder cell and extracellular matrix-free cultivation of human pluripotent stem

cells using Thermo Scientific Nunclon Vita surface and Rho-Kinase inhibition

The promise of pluripotent stem cells lies

in their ability to form any cell or tissue in

the body. However, this promise requires

a stable and reproducible method to grow

the cells. Current methods rely on feeder

cells or extracellular matrix proteins to

cover the cultureware growth surface,

and either manual selection or enzymatic

dissociation in cell passaging and

harvesting. This technical note describes

a novel and simple method to grow

pluripotent stem cells without the use of

feeder cells or extracellular matrix proteins.

Methods

Human ESC cultivation

Cells.

Passage-49 human ESC (H1 line from

WiCELL, USA) were maintained in mouse

embryonic fibroblast (MEF)-conditioned

medium on Nunclon Delta surface

(Thermo Fisher Scientific, USA) coated

with a 1:30 dilution of growth factor

reduced Matrigel (Becton Dickinson,

USA). Cells were dissociated from the

surface for passage by treatment with 1

mg/ ml collagenase, and then seeded onto

Nunclon Vita surface with or without

Rho-kinase inhibitor in the medium, as

described below.

Cultivation without Rho-kinase

inhibition. H1 ESC were grown for 4

passages in MEF-conditioned medium

on Nunclon Vita surface. Cells were

dissociated from the surface for passage

by treatment with 1 mg/ ml collagenase.

Normal passage time for H1 ESC was 3 - 4

days on Matrigel . However, cells plated

on the Nunclon Vita surface took 7

days of culturing before they were ready

for passage, and a spontaneous decrease

in growth rate over the passages was

observed.

Cultivation with Rho-kinase

inhibition.

H1 ESC were grown in MEF-conditioned

medium supplemented with Rhokinase

inhibitor, Y-27632 (10 µM unless

otherwise indicated; Sigma-Aldrich, USA).

Cells were dissociated from the surface

for passage by treatment with 1 mg/ml

collagenase. Cells plated in medium with

10 µM Y-27632 on the Nunclon Vita

surface were ready for passage 4 days

after plating. Cells were grown for the

number of passages indicated.

Human ESC characterisation

Colony presence and morphology

were determined using phase contrast

microscopy, and by the naked eye after

staining colonies with 0.5% crystal violet.

Pluripotency was determined by the

presence of pluripotency markers through

the use of qRT-PCR for gene expression,

flow cytometry for cell surface marker

expression, and immunofluorescence for

cell surface and nuclear proteins.

Human ESC can be passaged a few times on the Nunclon Vita surface

without Rho-kinase inhibition before the growth rate spontaneously

declines

Karyotypic stability was determined by

cytogenetic analysis of 20 G-banded

metaphase cells, and by fluorescent in situ

hybridisdation (FISH) on 200 interphase

nuclei using probes for the ETV6 BAP (TEL)

gene located on chromosome 12 and for

chromosome 17 centromere.

Ability to form embryoid bodies was

determined by growing ESC in a low

binding plate for 10 days in DMEM/F12

containing 10% FBS.

The decline in growth rate of

human ESC on the Nunclon

Vita surface is not observed

if the culture medium is

supplemented with Rho-kinase

inhibitor Y-27632

Figure 2. Dose response effect of Rho-kinase

inhibitor on the attachment of human ESC

to the Nunclon Vita surface. Y-27632

was added to the cultures at a specified

concentration (0, 1, 2, 4, or 10 µM from upper

left well to lower middel well) at seeding. The

cells were then maintained from day 2 onward

in media containing 10 μM Y-27632 with daily

media changes for five days after which cells

were stained with crystal violet

Figure 1. Phase-contrast micrographs of human ESC passaged twice on 1:30 dilution of Matrigel

(A), a standard tissue culture-treated surface (B), or the Nunclon Vita surface (C).

Figure 3. Detachment of human ESC from the

Nunclon Vita surface upon withdrawal of

Rho-kinase inhibitor. Cells were seeded and

maintained for 4 days in medium containing 10

μM Y-27632 (left well) or Y-27632 was removed

from medium for 24 hours on the 3rd day (right

well). After 4 days in culture, the cells were

stained with crystal violet.

36 I VWR International I VWRbioMarke Issue 27 I September 2011


Cell biology

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Human ESC grown on the Nunclon Vita surface in the presence of Y-27632 have normal karyotype, express

pluripotency markers, and can be differentiated into embryoid bodies

Figure 4. Normal karyotype (A) and FISH pattern (B) for human ESC

after 11 passages on the Nunclon Vita surface.

Figure 5. Human ESC can form embryoid bodies after 11 passages

on the Nunclon Vita surface.

Figure 6 A.Expression of pluripotency markers

in human ESC as determined by qRT-PCR

after 1 passage (p1), 4 passages (p4), and 8

passages (p8) on the Nunclon Vita surface.

Figure 6 B. Expression of pluripotency

markers in human ESC as determined by

flow cytometry after 4 passages (p4) and 11

passages (p11) on the Nunclon Vita surface.

Figure 6 C. Expression of pluripotency

markers in human ESC as determined by

immunofluorescence staining after 11

passages on the Nunclon Vita surface.

Conclusions

The Nunclon Vita surface supported

feeder cell and extracellular matrix-free

attachment, colony formation and growth

of human ESC:

- For a few passages in medium

conditioned by mouse embryonic

fibroblasts

- For several passages in medium

conditioned by mouse embryonic

fibroblasts and supplemented with Rhokinase

inhibitor Y-27632

Human ESC grown 11 passages on the

Nunclon Vita surface in medium with

Y-27632 had normal karyotype, expressed

pluripotency markers, and could be

differentiated into embryoid bodies.

Human ESC could be passaged without

the use of enzymes or manual selection by

withdrawing the Rho-kinase inhibitor from

the culture in order to lift the cells from

the Nunclon Vita surface, followed by

re-plating cells in the presence of the Rhokinase

inhibitor.

Non enzymatic passaging of human ESC by Y-27632 withdrawal

Grow ESC in

the presence of

Y-27632

Remove

Y-27632 and

ESC detaches

Figure 7. Human ESC grown in the presence of Rho-kinase inhibitor can be dissociated from the

Nunclon Vita surface by incubating the plate with fresh growth medium without Rho-kinase

inhibitor for 15 - 30 min. Cells can then be mechanically dissociated from the plate by gentle

pipetting or scraping, briefly centrifuged, and resuspended in growth medium containing Rho-kinase

inhibitor. The ESC can finally be mechanically dissociated by gentle trituration and re-seeded.

For research use only

Important information about patents:

Attachment, cultivation and detachment of cells

using methods described herein are covered by

patent applications WO 2009/105570 and US

12/388,930. A license to use these methods with

Nunclon Vita Surface cultureware solely in

connection with research is granted with the

purchase of Nunclon Vita cultureware.

Enquiries for a license to use these methods for

commercial purposes, except for those purposes

relating to amelioration of diabetes mellitus, should

be sent to: Thermo Fisher Scientific, 81 Wyman

Street, Waltham, MA 02451, Attn: Legal Dept.

Enquiries for a license to use these methods

directly or indirectly in the amelioration of diabetes

mellitus should be sent to: Att. Vice President of

Harvest ESC

Re-seed with

Y-27632 and

ESC attaches

BetaLogics Centocor Research & Development, Inc,

145 King of Prussia Road, Radnor, PA 19087, USA.

Particular types of cells, as well as methods for

manipulating cells, may be covered by one or more

patents held by others. Use of Nunclon Vita

cultureware is recommended only for applications

which do not violate proprietary rights of others or

for which the user has a license or other permission

under such proprietary rights.

Legal

www.thermoscientific.com - © 2011 Thermo

Fisher Scientific Inc. All rights reserved. Matrigel

is a trademark of Becton Dickinson and Company.

All other trademarks are the property of Thermo

Fisher Scientific Inc. and its subsidiaries.

VWR International I VWRbioMarke Issue 27 I September 2011 I

37


the market source for life science

ChillProtec ® : New protective medium for cold

storage of cells

Even primary cells remain intact after cold storage for

longer periods of time

When kept in the new protective ChillProtec ® medium, adherent cells, cell

suspensions or small tissue pieces are able to remain intact after cold storage.

Furthermore, cell functions are retained better than in alternative solutions. The

new medium is ready for use, sterile, free of animal components, and completely

chemically defined.

ChillProtec ® is suitable for the cold storage of

all cell types, including primary cells. Primary

human hepatocytes, for example, remained

intact at 2 to 8 °C for several days. In addition,

ChillProtec ® qualifies for the temporary storage

of fresh clinical specimens until cell isolation.

It is also suited for the short-term storage

of remaining or isolated cells, as well as for

the transport of cells and tissue. We offer

two variations of ChillProtec ® : ChillProtec ®

and ChillProtec ® plus. The macromolecular

substance that ChillProtec ® plus contains has

an additional protective effect on different cell

types. You should therefore test cells using

both versions.

Overview

ChillProtec ® is a medium for the cold storage of

adherent cells, cell suspensions or small tissue

preparations. When kept in this protective

medium, cells are able to remain intact after

cold storage without any loss of functionality.

Cold (hypothermia) is a widely used protection

principle for the storage and transport of cells.

It slows down cell metabolism and reduces

damaging processes caused by lack of oxygen

or substrate. It is this cold, however, that causes

damages within the cells. ChillProtec ® prevents

such damage, allowing for significantly longer

periods of cold cell storage than seen with

normally used liquids, such as cell culture media,

physiological salt solutions or organ protection

solutions.

General applications

ChillProtec ® is a sterile and ready-to-use

medium for the protection against cold.

In order to avoid frequent temperature

fluctuations during cell storage, it is advisable to

use a less frequently used refrigerator or a cold

storage room.

During the transport of cells and tissue on ice,

ice water should be used in order to prevent

frost damages. If ice packs are used during

transport, you should ensure that the ice packs

do not have any direct contact with the cell

culture flask or the vessel containing the tissue.

During transport, cell culture flasks should be

completely filled with ChillProtec ® .

cell culture medium

37 °C

storage in ChillProtec ®

15-20 °C à 2-8 °C

cell culture medium

2-8 °C à 37 °C

Figure 1: Application principle

38

I VWR International I VWRbioMarke Issue 27 I September 2011


Cell biology

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Figure 2: Porcine aortic endothelial cells that were stored in culture medium (left fFgure) and in ChillProtec ® (right

Figure) at 4 °C for seven days and then warmed at 37 °C for three hours

In case of longer transportation periods, check

that the temperature is kept at 2 to 8 °C (range

of tolerance: 0 to 10 °C), by means of a data

logger, for example.

Example: Application to adherent cells

When storing adherent cells, ChillProtec ® should

be warmed up to 15 to 20 °C immediately

before application.

If possible, the cells should be in their

logarithmic growth phase. Approximately 24

hours before cold storage, a complete medium

change should be performed. For longer periods

of cold storage, cell culture flasks are preferable.

1. Slightly warm up the protective medium at

room temperature (RT) to reach 15 to 20 °C.

2. Prior to cold storage, wash the cells under a

laminar flow with (warm) HBSS or PBS (two or

three times); exhaust HBSS or PBS respectively.

3. Put the protective medium (15 to 20 °C) at the

cells (use the same volume that is normally used

when using cell culture medium; e.g. 5 ml for a

25 cm 2 flask, 15 - 20 ml for a 75 cm 2 flask, 2 ml

per well of a 6 well plate). When finished, close

the cell culture flasks. Cover gas permeable caps

with Parafilm®. Store cell culture vessels in a

refrigerator or a cold storage room (2 to 8 °C).

4. When using wells or cell culture dishes, use

a cap and mask the margins with Parafilm®

or adhesive tape (one strip along the entire

opening or above all four lateral edges

respectively). Store wells or cell culture dishes

in a refrigerator or a cold storage room (2 to

8 °C).

5. At the end of the required period, take the cells

out of the refrigerator/cold storage room and

use the laminar flow to exhaust the protective

medium.

6. Take normal cell culture medium (complete

medium with serum and further additives

normally required for the respective cell type)

directly out of the refrigerator (approx. 2 to

8 °C) and apply it to the cells. Store the cells in

an incubator.

7. Change the cell culture medium the following

day; in case a larger number of cells have

detached, replace the medium after 4 to 6

hours after warming up.

8. Split the cell culture 48 hours after warming up

if possible, but not earlier than 24 hours

Cell suspensions and small tissue preparations,

as well as adherent cells may be stored in

ChillProtec ® . The respective instruction can be

found and downloaded at www.biochrom.de.

VWR International I VWRbioMarke Issue 27 I September 2011 I

39


the market source for life science

Suggestions on how to store cells

in ChillProtec ®

Compared to cell culture media, physiological

salt solutions and organ protection solutions,

ChillProtec ® causes significantly less cell damage

after cold storage of cells and tissue (cf. Table 1).

Cells and tissue tested with ChillProtec ®

ChillProtec ®

Aortic endothelial cells (primary, porcine)

Liver endothelial cells (cell line, rat)

Vero B4 (kidney epithelial cell line, monkey)

LLC-PK1 (kidney epithelial cell line, porcine)

Hepatocytes (primary, human), adherent

Hepatocytes (primary, human), suspension

Hepatocytes (primary, rat), adherent

Hepatocytes (primary, rat), suspension

Hepatocytes (primary, mouse)

Hepatocytes (primary, porcine)

HepG2 (hepatoma cell line, human)

A549 (lung epithelial cell line, human)

L929 (fibroblast cell line, mouse)

RIN-m5f (islet cell line, rat)

K-562 (myeloma cell line, human)

Muscle (diaphragm, mouse)

As there are the two different versions of

the protective medium, you should test both

media. At the moment, comparative studies of

ChillProtec ® and ChillProtec ® plus are available

for a broad range of cells, recommending

the use of the respective medium mentioned

(cf. Table 2).

Storage period

The possible length of cold storage highly

depends on the respective cell type and

cultivation or the use of the cells respectively,

varying between three days and more than two

weeks (up to five weeks). Users should test the

storage period for each cell type and each type

of application.

ChillProtec ® overview

Parameter ChillProtec ® ChillProtec ® plus

Cat. No. F 2285 F 2295

Pk 500 ml 500 ml

Storage (°C) +2 to +8

Raw material Chemically defined, proprietary formulation

Intended use Cold storage of cells

Note

For in vitro use only

Table 1

See bioMarkeShop

for an offer and

free samples of

ChillProtec ®

Cells tested with ChillProtec® and ChillProtec ® plus

ChillProtec ®

Aortic endothelial cells (primary, porcine)

Vero B4 (kidney epithelial cell line, monkey)

Hepatocytes (primary, human)

Liver endothelial cells (cell line, rat)

Hepatocytes (primary, mouse)

Hepatocytes (primary, porcine)

HepG2 (hepatoma cell line, human)

Table 2

ChillProtec ® plus

L929 (fibroblast cell line, mouse)

Hepatocytes (primary, rat)

A549 (lung epithelial cell line, human)

RIN-m5f (islet cell line, rat)

40

I VWR International I VWRbioMarke Issue 27 I September 2011


Cell biology

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Building a reliable foundation for stem

cell research

As the promise of

commercialised stem cell

therapy grows, so does

the need for reliable

tools for cell expansion.

BD Biosciences is at the

forefront of development

of next generation,

integrated systems for

the expansion of hMSC

that provide researchers

and clinicians consistency,

performance, speed,

safety and lower costs in

use. BD Mosaic hMSC SF

Cell Culture Environment

is the first in a series of

integrated cell culture

reagent systems for

stem cell expansion. The

product is a complete

package that contains

the media, supplement

and surface required

for cell expansion, and

comes with the assurance

of reliability that only BD

Biosciences can offer.

Mesenchymal stem cells (MSCs) are commonly

isolated from the mononuclear fraction of the

bone marrow 1 , but are native to many resident

tissues, including bone marrow, umbilical cord

blood (placenta) 2 , adipose tissue (fat), muscle,

and dermis. MSCs represent ‘multipotent’ stem

cells that possess the ability to regenerate, and

can differentiate into a variety of cell types,

such as bone, cartilage and fat cells. Because

of their large capacity for self-renewal, while

maintaining their multi-potency and their

immunosuppressive nature, MSCs are a source

of research and clinical significance. More and

more interest is arising around the potential use

of MSCs in cell therapy applications. Therefore,

the demand for defined and efficient culture

and expansion systems is increasing.

Greater cell expansion over shorter

culture time with no medium

change required.

BD Mosaic provides for greater MSC

expansion in shorter time with no re-feeding

requirements between passages and low

minimum seeding density. When compared

to traditional 5% serum containing medium

and using two analysis methods, BD Mosaic

produced approximately 5 - 50 times the cell

output in 12 - 15 days (Figure 1 and Table 1).

Table 1 compares the key characteristics of the

cell culture, expansion and scale up conditions

of BD Mosaic medium versus traditional 5%

serum containing medium.

The BD Mosaic hMSC SF Cell Culture

Environment Advantage:

• Chemically defined and serum-free (SF)

expansion system

• Greater cell expansion in shorter culture time

• Higher cell doublings per passage

• No re-feeding required between passages

• Lower minimum seeding density

• Maintenance of immunophenotype,

multipotency and T Cell suppression

• Comprises medium, supplement, and surface

coating for culture vessel

• Reliably composed

Figure 1: Cumulative population doublings of BD Mosaic vs. traditional serum

MSCs were expanded with either BD Mosaic (red) or a traditional (5% serum) method (blue) and counted using

both imaging (brightfield, solid lines) and a standard automated cell counter (ViCell, dashed lines). Both culture

systems were administered according to recommended protocols. Results: By both measurement methods, BD

Mosaic produced at least ten MSC doublings (left vertical axis), or 1,024-fold expansion (right vertical axis), within

15 days. The traditional serum method produced only six doublings (64-fold expansion) in the same time period.

Standard errors were calculated for the brightfield method at time of passaging.

VWR International I VWRbioMarke Issue 27 I September 2011 I

41


the market source for life science

Table 1

Superior performance of BD Mosaic hMSC SF Cell Culture Environment compared to traditional serum-based medium.

Greater cell expansion over shorter culture time with no medium change required

BD Mosaic hMSC SF Cell Culture Environment

Traditional (5% serum-containing medium)

Seeding density 3 - 4000 cells/cm 2 5 - 6000 cells/cm 2

Harvest density 24 000 cells/cm 2 20 000 cells/cm 2

Passage frequency 3 days 7 days

Medium change frequency None between passage 3 - 4 days

Medium volume needed 0.2 ml/cm 2 0.2 – 0.4 ml/cm 2

Theoretical scale-up of 1x1 06 cells after 12 - 15 days in culture

BD Mosaic hMSC SF Cell Culture Environment

Traditional (5% serum-containing medium)

Medium required 10.5 l 41,0 l

Total surface area 52 610 cm 2 67 505 cm 2

Resulting cell number 1.0x1 09 0.02 – 0.2x1 09

Figure 2: Cultured MSCs exhibit normal

immunophenotype

MSCs were cultured in BD Mosaic MSC SF and

analysed by flow cytometry for MSC surface markers 3 .

Results:

Reliable maintenance of MSC

morphology, imunophenotype, and

multi-lineage potential.

A. Compared with an isotype control (blue) after a

representative passage, cultured MSCs (orange) expressed

the ISCT’s three positive markers (CD73, CD90, and CD105)

but did not express three negative markers (CD79a, CD19,

and CD14).

B. After two or four passages in BD Mosaic, virtually 100%

of cultured MSCs expressed the three positive markers

(CD73, CD90, and CD105) while almost none expressed

the five negative markers (CD14, CD34, CD45, CD79a, and

HLA-DR).

Our studies demonstrate that MSCs expanded

in BD Mosaic hMSC SF Medium maintain their

morphology (verified up to passage number

8, data not shown). After passage number 2

and 4, cells cultured in BD Mosaic hMSC

Medium showed the typical immunophenotype

of mesenchymal stem cells, as judged by the

staining of cells with antibodies to CD14, CD34,

CD45, CD73a, CD79a, CD 90, CD105 and HLA-

DR, and subsequent flow cytometric analysis

(Figure 2).

Cells previously cultured in

BD Mosaic hMSC SF Medium were

successfully induced to differentiate into

osteobalsts, adipocytes and chondrocytes

(Figure 3).

Numerous publications in recent years

have shown that MSCs can create an

immunosuppressive environment by secreting

cytokines, and thus influence the function of

dendritic cells and T-cells 4,5 . MSCs cultured in

BD Mosaic hMSC Medium at early and late

passages suppress T-cell activation equally to

cells grown in traditional 5% serum containing

medium (data not shown).

These experiments prove that mesenchymal

stem cells expanded in BD Mosaic hMSC Cell

Culture Environment maintain their full multipotency.

42 I VWR International I VWRbioMarke Issue 27 I September 2011


Cell biology

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send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

Figure 3: MSCs cultured in BD Mosaic hMSC SF Medium at early and late passages

suppress equally to cells grown in traditional 5% serum containing medium.

Proliferation measured using Bromodeoxyuridine (BrdU) incorporation and subsequent staining

with anti BrdU antibodies and FACS-analysis. Left, population doubling 6, right population

doubling 12.

Population Doublings (PD) Growth Medium % Suppression

6 BD Mosaic hMSC SF 76

5% Serum 82

12 BD Mosaic hMSC SF 78

5% Serum N/A

Extensive benchmarking studies have

demonstrated that BD Mosaic hMSC SF

Cell Culture Environment shows considerable

advantages over current hMSC media by

enabling researchers to obtain higher cell

numbers in shorter time, while using less media

as compared to conventional culture methods.

References:

1. Campagnoli C. et al. Identification of mesenchymal stem/

progenitor cells in human first-trimester fetal blood, liver,

and bone marrow. Blood. 98(8):2396. 2001

2. Erices A. et al. Mesenchymal progenitor cells in human

umbilical cord blood. Br J Haematol.109(1):235. 2000

3. Dominici, M. et al. Minimal criteria for defining

multipotent mesenchymal stromal cells. The International

Society for Cellular Therapy position statement.

Cytotherapy 8 (4):315. 2006

4. Bartholomew A. et al. Mesenchymal stem cells suppress

lymphocyte proliferation in vitro and prolong skin graft

survival in vivo. Exp Hematol. 30(1):42. 2002

5. Di Nicola M. et al. Human bone marrow stromal cells

suppress T-lymphocyte proliferation induced by cellular or

nonspecific mitogenic stimuli. Blood. 99(10):3838. 2002

Description Pk Cat. No.

BD Mosaic system

Complete kit: Medium, supplement and surface coating 1 kit 734-2458

Medium 500 ml 734-2459

Growth supplement 8.3 ml 734-2460

Additional products

BD Falcon 6 well plates, TC treated, flat bottom with lid 36 734-0054

BD Falcon 175 cm 2 tissue culture flask, TC treated, vented cap 40 734-0047

VWR International I VWRbioMarke Issue 27 I September 2011 I

43


the market source for life science

Petakas´ internal environment reflects

the O 2

concentration of tissues under

normal physiological conditions

In vitro culture of cells

and tissues requires a

controlled environment

of both temperature and

gas concentrations. In the

last 40 years, researchers

discovered that exposing

cells to variable oxygen

conditions affects their

physiology in dramatic

ways compared to

cells kept in traditional

hyperoxic (21% of the air)

culture conditions.

Cells cultured in devices exposed to normal

atmospheric concentration of oxygen are

traditionally used in basic research, in spite

of the artifacts consequent to that hyperoxic

environment, however, culturing the cells

at lower oxygen concentrations, such as 25

to 40% of the atmospheric concentration

(5 to 8% oxygen in air), reduces DNA

damage, does not alter cellular size/shape,

enhances chromosomal stability and an

increases proliferative potential 1 . This reduced

oxygen concentration better reflects the O 2

concentration (pO 2

) of tissues under normal

physiological conditions, since most tissues

are not exposed to external atmospheric

O 2

concentrations (Table 1). Actually, at the

cellular level, that excessive O 2

concentration

exposure can be significantly toxic 2 .

Tissues cells, in mammalians, are exposed to

a wide range of O 2

concentrations depending

on their blood supply position, capillary flow,

red blood cell concentration and hemoglobin

content 3 , even within a single organ. For

instance, oxygen tensions within the liver vary

from about 30 - 40 mm Hg in the liver lobule

periphery (portal domain, close to the hepatic

artery capillaries) and about 15 mm Hg in the

liver lobule centres (suprahepatic domain) 4 .

Moreover, It is established that cells in different

phases of the cycle consume different quantities

of oxygen, for example HeLa cells in S phase

consume about 240% more than the same cells in

G1, and in mitosis O 2

consumption is slightly lower

than in G1 6 . Therefore depending on the growing

state of the culture (proportion of cells in S phase)

the O 2

needs of a culture are different.

What today is still sometimes called mild

hypoxia in cell culture (5% O 2

in the incubator

atmosphere) only can be identified as absence

of typical hyperoxic classic cell cultures. It is

known that actual normoxia, to be an inducer

of large numbers of genes, not restricted only

to those directly involved in the physiological

response to hypoxia, such as those required

for haematopoiesis and angiogenesis 7,8 . It is

becoming progressively clearer that for the in vitro

culture of many cell types, consistent exposure

to O 2

concentrations well below normal 21%

atmospheric O 2

concentration is important.

For instance culturing fetal lung fibroblasts 9 ,

embryonic stem cells 1,10 , chondrocytes 11 ,

mesenchymal stem cells 12 , and haematopoietic

stem cells 13, 14 .

Cell cultures developed in classic cell culture

devices such as T flasks, Petri dishes and

derivatives, provide metabolic O 2

to the cells

through the water. Gases from the environment

dissolve first in the water, which transfer the

dissolved gas to the cells. Those devices all have

two common parts: (1) The gas phase in contact

with (2) the liquid phase. A large interface

guarantees the gas transfer in-between the two.

The gas phase, in regular culture applications, is

the open atmosphere with air containing up to

21% O 2

. Gas diffusion coefficient, temperature

and composition of the media force the formation

of an O 2

concentration gradient with a high

partial pressure (pO 2

= 140 - 150 mm Hg)

on the surface layer and a low concentration

layer (pO 2

= 70 - 100 mm Hg) at the deepest

level (2 - 4 mm), where the cells are anchored.

Therefore, to achieve the needed physiological

concentrations in the cell culture media, a

Table 1. Petaka is the one and only cell culture device autonomously providing Oxygen concentrations in the media at physiological levels, either for normal

tissues or tumor cells.

44

I VWR International I VWRbioMarke Issue 27 I September 2011


Cell biology

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environment. This means that following each

culture manoeuvre, for example, scheduled

sampling, or re-seeding of cell cultures in

fresh media, cells are exposed to undesirably

high (initially atmospheric) concentrations of

dissolved O 2

for a significant period, which may

introduce undesirable experimental alterations

and important physiological effects.

In order to overcome these problems, sealed

glove boxes for manipulation without exposure

to external atmospheric conditions are used 16 .

Cultures in “Normoxia”

Figure 1.- The Petaka auto-controlled gas

diffusion system (GDS), when exposed to

regular atmosphere with 21% O2, automatically

produces a “Progressive adaptive depletion of

O2” dropping the functional partial pressure of

dissolved oxygen from 140 mm Hg to 40 mm Hg,

which is equivalent to the tissue “normoxia”

reduction is required of the environmental O 2

concentration in the incubator to about 5%

(media O 2

partial pressure, ppO 2

= 38 mm Hg).

Today there are many cell culture incubators

which provide control of O 2

levels. However,

those instruments are cumbersome, costly

in maintenance, and limited in space and

use. Moreover, they have several important

functional setbacks: it takes approximately

30 minutes for an oxygen controlled incubator

to displace sufficient air to create a 5% O 2

environment every time it is opened. Within a

common 50 l incubator it takes a minimum of

180 minutes to create a 5% O 2

headspace in

T25 flasks and 420 minutes in T150 flasks 15 . The

deficiencies of these incubators are exacerbated

by a further issues: The rate of O 2

diffusion

into the cell culture media. During culture, cells

adhere to the bottom of the flask, 2 to 4 mm

below the liquid/gas interface between the

media and gas space above, which acts

effectively as a semi-permeable membrane.

The time required for excess of O 2

to diffuse

out of cell culture media at 37 OC. is minimally

80 minutes when cultured cells reach confluent

monolayer and maximally 180 minutes at low

cell densities [11]. It can therefore take more

than three hours for adjusting the desired O 2

concentration at the cellular peripheral micro

Petaka is a unique cell culture device with

auto-controlled in/out gas diffusion system

(GDS) 17 , which exposed to regular atmosphere

with 21 % O 2

automatically produces a

“Progressive adaptive normoxia” dropping the

functional partial pressure of dissolved oxygen

from 140 mm Hg to 40 to 53 mm Hg (Figure 1),

adjusted to the physiological environment of the

majority of mammalian cells (Table 1), in parallel

to the cell growth. DO concentration remains

stable, within an insignificant fluctuation

responding to the feedback between cell

doubling time and oxygen availability. When

cells grow over a certain threshold, the

controlled oxygen supply through the Petaka

GDS, holds the level of DO 2

, slowing the

doubling time and the average O 2

consumption

by the cell population, and vice versa. (Figure 1)

Initial oxygen concentration of the average

bottled media is about 6.8 ppm based on

regular sea level atmospheric pressure (760 mm

Hg) and standard culture temperature (310 ºK).

Beginning the cell growth the dissolved O 2

partial pressure (ppDO 2

) is dictated by the

normal media exposed to the atmospheric air.

Depending on the cell type (CHO cells as an

example), after a short period of growth, when

cell number exceeds 6 to 8 million, the rate of

oxygen consumption is balanced with the rate

of oxygen diffusing through Petaka GDS, and

the culture media ppDO 2

is maintained at the

tissue physiological limits, between 40 and

60 mm Hg (Figure 1). That auto-control normoxia

level remains balanced from the sea level up to

10 000 ft altitude (3000 m, 537 mm Hg).

The Petaka GDS special feature allows

culturing cells in physiological oxygen

concentration inside any regular incubator, with

regular open air environment (with or without

CO 2

enrichment), and allow manipulating the

cultures, with absolute freedom, without risk

VWR International I VWRbioMarke Issue 27 I September 2011 I

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the market source for life science

of breaking the levels of dissolved oxygen.

Conditions needed for culturing stem cells and

others sensitive to the hyperoxic traditional

systems. 18,19,20

Shelf media contains O 2

up to 150 mm Hg,

therefore media exchange represents a difficulty

for normoxia cell culture protocols, because

part of the fresh media O 2

, should be extracted

before use. Specific devices dedicated to

prepare media with the right O 2

content are

available today 21 . However, using Petaka as

media reservoir is an easy alternative method of

keeping hypoxic media ready to be used. Storing

Petakas, full of media, in a simple glass vacuum

chamber at 30 Torr for more than 24 hours

(degassing), provides enough hypoxic media to

substitute other Petaka media. The procedure

of withdrawing old media and transferring the

new one from the Petaka reservoir should be

no longer than 3 minutes.

Cultures in severe hypoxia

Many experiments of gene expression or cell

behaviour, under hypoxia conditions, demand

O 2

concentration in media below 20 mm Hg.

These are experiments in deep hypoxia. These

certainly require special incubators and glove

boxes to handle cultures in classic open flasks

and dishes. However, Petaka is designed

to work simply using standard cell culture

incubators without special gas flow controls.

Bagging each Petaka in a gas impermeable

bag, like those used for food preservation

(Mylar bags) in vacuum conditions (Figure 2),

will avoid gas transfer in/out of the bag, and

the vacuum produced in the bag will force

the release of gas from the media due to

decompression. (Figure 2)

Media sampling whilst maintaining

hypoxia conditions

The vacuum bag allows Petaka tips to

penetrate it, whilst avoiding vacuum rupture.

By doing so, samples of media, up to a total

of 7000 micro litres, can be extracted with a

syringe, avoiding contact with the atmospheric

air (Figure 4). More than 7000 micro litres could

be dangerous as it may result in rupturing the

Petaka walls.

Cell harvesting in hypoxia

conditions

Standard harvesting of cells kept in hypoxia,

force one to open the incubator to the open

atmosphere or to use glove boxes. This is

a critical moment in which very high levels

of oxygen can come into contact with the

cells introducing uncontrolled artifacts in

the experiments. Petaka avoids this critical

moment, firstly by allowing cell detachment

using a small paramagnetic vehicles driven by

magnets moving underneath (Figure 5). These

vehicles make linear displacements gliding very

close to the cells causing a Bernoulli effect,

that induces cell detachment without requiring

proteolysis enzymes or disrupting chemicals.

This process will generate levels of ppO 2

below 10 mm Hg after several hours of culture

(Figure 3A) starting with media stored at

normal 21% O 2

in atmosphere. If the media

is previously oxygen depleted, same levels of

hypoxia will be established in much less time

(Figure 3B), always depending on the cultured

cell type. (Figure 3)

Figure 4.- Severe hypoxia experiments in Petaka

allow periodical sampling of media and free cells

for measurements and other protocols, without

the hypoxia environment alteration.

Figure 2.- Cell culture in enforced severe hypoxia using Petaka TM . (1) Petaka TM containing growing cells in vacuum

sealed in a gas impermeable bag. (2) Petaka TM inside a closed vacuum bag. (3) Petaka TM in a vacuum bag incubated

in normal tissue culture incubators, alongside normal cultures. (4) Using the same transparent bags cells in culture

can be observed under the microscope during the experiment of deep hypoxia culture. Media alterations are

avoided.

46 I VWR International I VWRbioMarke Issue 27 I September 2011


Cell biology

For more information on these products contact your local VWR sales office,

send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com

Figure 3.- (A) The Petaka autocontrolled

gas diffusion system

(GDS), when enclosed into a gas

impermeable bag, automatically

produces a “Progressive adaptive

depletion of O 2

” dropping the

functional partial pressure of

dissolved oxygen from 140

mm Hg to 1.4 mm Hg, which is

equivalent to the tissue “severe

hypoxia”. (B) Previously O 2

depleted media up to 35 mm Hg

allows a rapid progression of the

progressive adaptive depletion

of O 2

, and the cultured cells are

exposed to severe hypoxia earlier.

When cells are detached, they can be collected

without any possible contact with the air in

the syringe when carefully penetrating the

bag. Cells can be transferred to other devices

common in biochemistry and molecular biology.

Figure 5.- Cell harvesting from severe hypoxia

cultures in Petaka does not alter the gas

environment. The surfers system (arrow) of cell

detachment allows cell release without media

alteration. Detached cells can be collected by

aspiration with a closed syringe.

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