Amaxa news #6 - lonza

amaxa
› amaxa news # 6
news
latest mission achievements
gene transfer begins here
# 6
amaxa’s mission 2005
› New Cell Line Nucleofector ® Solutions
and Cell Line Optimization Nucleofector ® Kit
› Mouse and rat hepatocyte nucleofection ®
› Mouse and human macrophage
nucleofection ®
upcoming
› amaxa's new
Nucleofector ® 96-well Shuttle System

96-well
transfection
Introducing:
amaxa's new Nucleofector ®
96-well Shuttle System
› applicable for cDNA and RNAi library
screening in primary cells and difficultto-transfect
cell lines
› up to 90% transfection efficiency with
DNA
› up to 99% transfection efficiency with
siRNA duplexes
Pre-register now: available Q 1, 2006!
amaxa Europe /Export
scientific-support@amaxa.com
+49 (0) 221 99199-400
amaxa Europe /Export
amaxa USA
scientific-support.US@amaxa.com
240-632-9110
Editorial
We have dedicated this issue of amaxa news to report on some of
our latest accomplishments, ranging from new protocols and kits
for additional cell types and applications, such as protein production,
to nucleofection in a 96-well format.
In the field of primary cell transfections, we have tackled two cell
types that have so far been impossible to efficiently transfect with
non-viral methods: macrophages (human and mouse, see pages
14-15) and hepatocytes (mouse and rat, see pages 6-7).
For cell line transfections you will find not only a steadily increasing
number of Optimized Protocols but also two new Cell Line Nucleofector
Kits. As shown on pages 4-5, transfection efficiencies of
60-80% are achieved in cell lines such as MDCK, NS0, BHK-21,
EL-4, WEHI-231 etc.
In 2006, we shall continue providing you with the latest cuttingedge
transfection technology. Our upcoming Nucleofector 96-well
Shuttle System (introduced on pages 12-13) is further proof of our
ongoing commitment to provide the best transfection solutions.
Soon you will be able to perform high-throughput transfections in
primary cells, such as T cells, or virtually any difficult-to-transfect
cell line. Like the Nucleofector, the new Nucleofector 96-well Shuttle
System delivers cDNA, siRNA as well as shRNA libraries into your
cell of interest with highest efficiency. Take a look at our new developments
to help you meet your personal research mission in 2006.
Rainer Christine
CEO

Editor
Wolfgang Kintzel
New products
New products
Technote
Product update
Hot topic
New products
New products
Hot topic
New products
amaxa insights
amaxa insights
amaxa insights
FAQ
amaxa insights
Editorial board
Martina Mohrs, Oliver Gresch,
Katrin Höck, Andrea Toell
Production coordinator
Petra Schmid
amaxa news is published by
amaxa GmbH
Nattermannallee 1
50829 Koeln
Germany
amaxa_news@amaxa.com
Tel: +49(0)221-99199-0
Fax: +49(0)221-99199-111
› Table of contents
› page 3 › www.amaxa.com
4 Nucleofection ® of cell lines further improved
6 Mouse and rat hepatocyte nucleofection ®
8 Use of Nucleofector ® technology for transient protein production
Markus Zumbansen, Stefanie Zahn, Oliver Gresch, Andreas Herrmann and Titus Kretzschmar
10 Is your Nucleofector ® Software up-to-date?
11 Understanding cell-matrix contacts with the help of nucleofection ®
Stefan Linder
12 High-throughput transfection
Introducing amaxa’s Nucleofector ® 96-well Shuttle System
14 Efficient non-viral transfection of human and
mouse macrophages
16 Essentials for the nucleofection ® of mouse T cells
17 amaxa’s Basic Nucleofector ® Kits
Broad-range nucleofection ® at its best
18 amaxa’s online information pool is growing
19 Representing amaxa in Canada – ESBE Scientific
20 Scientific Product Specialists: On the road all around the world
22 Frequently asked questions about achieving high viability
pre and post nucleofection ®
23 Meet amaxa in 2005/2006
Please note that to date amaxa’s Nucleofector ® technology is intended for research or investigational
use only.
amaxa's Nucleofector ® Process, Nucleofector ® Device and Nucleofector ® Solutions are covered by
PCT Applications PCT/EP01/07348, PCT/DE02/01489, PCT/DE02/01483, and other pending
patents, and domestic or foreign applications corresponding thereto. The Nucleofector ® 96-well
Shuttle System is covered by patent and/or patent-pending rights owned by amaxa.
amaxa, gene transfer begins here, Nucleofector, nucleofection, Nucleocuvette, 96-well Shuttle
and maxGFP are trademarks of amaxa GmbH. EndoFree is a trademark of Qiagen. Steady-Glo is a
trademark of Promega. Lipofectamine 2000 and Gibco are trademarks of Invitrogen. SpectraMax ®
is a trademark of Molecular Devices. Other product and company names mentioned herein are the
trademarks of their respective owners.
Design
vierviertel – Agentur für Kommunikationsdesign GmbH
info@vierviertel.com
© September/2005 amaxa. All rights reserved. Printed in Germany.

› New products
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new
Nucleofection ® of cell lines
further improved
amaxa’s dedication to help you succeed in your research by providing easy and reliable transfection
solutions has led to the creation of a wide range of Optimized Protocols for standard and difficult-totransfect
cell lines. To date amaxa’s Cell Database comprises information on the successful
transfection of more than 300 cell lines. Our commitment to provide the optimal approach for your
cell line transfection, has resulted in two additional new Cell Line Nucleofector Solutions.
Furthermore a redesign of our Cell Line Optimization Nucleofector Kit, makes your cell line optimization
even quicker, more convenient and economic than before.
A10 CCRF FDC-P1 MDCK WEHI-231 EL-4
Fig. 1: Average transfection efficiencies and viabilities for
cell lines transfected with the new Cell Line Nucleofector
Solution C and L. All cells were transfected with pmaxGFP.
24 h post nucleofection transfection efficiencies were determined
by flow cytometry. Viabilities are either given as % PI negative
cells (CCRF-CEM, FDC-P1, WEHI-231, EL-4) or compared to unnucleofected
controls (A10, MDCK). A10, FDC-P1, MDCK, WEHI-231
and EL-4 cells were transfected using Cell Line Nucleofector Kit
L, CCRF-CEM cells with Kit C.
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BHK-21
efficiency viability
efficiency viability
Fig. 2: Average transfection efficiencies and viabilities
of cell lines BHK-21 and NS0 cells, also used for protein
production. BHK-21 and NS0 cells were transfected with
pmaxGFP. 24 h post nucleofection transfection efficiencies and
viabilities were determined by flow cytometry (viability = % PI
negative cells). BHK-21 cells were transfected with Cell Line
Nucleofector Kit L, NS0 cells with Kit C.
› page 4 › amaxa news # 6
NS0
C and L: Two new Cell Line Nucleofector ® Solutions to
advance your cell line transfections
In addition to the existing Cell Line Nucleofector Solutions R, T
and V two new solutions are now available to further extend the
range of cell lines that can be successfully transfected. By screening
many new possible solutions, amaxa’s R&D team has identified
two new Cell Line Nucleofector Solutions, named C and L.
These have shown superior transfection results compared to the
existing solutions: over 60% for eight newly developed cell line
optimization protocols (Fig. 1 and 2). Transfection efficiencies
were thus frequently more than 20% higher than those achieved
with the existing solutions (data not shown).
Highly efficient transfection of optimization protocols cell
lines NS0 and BHK-21, also used for protein production
With the new Cell Line Nucleofector Solutions it is now possible to
transfect NS0 and BHK-21 cells, both of which are frequently used
for mammalian protein expression culture (also see pages 8-10 to
learn more about protein production in mammalian cells using
the Nucleofector technology). With Cell Line Nucleofector Solutions
C and L transfection efficiencies of over 80% can be achieved
for both NS0 and BHK-21 cells, with viabilities ranging from
50-90% (Fig. 2). The Optimized Protocol for NS0 cells enables
efficient transfection of NS0 cultured under serum-free conditions
(Fig. 3).

[k]
d
› New products
A
B
Fig. 3: Example of the transfection of NS0 with
pmaxGFP cultured under serum-free conditions.
› page 5 › www.amaxa.com
A new version of our Cell Line Optimization Nucleofector Kit –
making cell line transfection in your lab even quicker and more
convenient than before
For transfection of your cell line of interest you can rely on either
detailed Optimized Protocols for cell lines optimized by amaxa or
on our online Cell Database which provides information from
other users. For any other cell line, where there are no nucleofection
details available, the Cell Line Optimization Nucleofector Kit
is the method of choice. With the use of this comprehensive
approach you can transfect virtually any cell line. In addition we
have improved our current Cell Line Optimization Nucleofector
Kit (Fig. 4). The result is an even quicker and more convenient
tool to successfully transfect your cell line of choice.
The new Cell Line Optimization Nucleofector Kit is:
quick › only 18 transfections (including controls) required for the first
optimization round
convenient › now with just two Nucleofector Solutions for easier handling
economic › fewer cells required for optimization
1 Solution V L
program 1 A-020 A-020
program 2 T- 020 T- 020
program 3 T- 0 30 T- 0 30
program 4 X-001 X-001
program 5 X-005 X-005
program 6 L- 0 2 9 L- 0 2 9
program 7 D-023 D-023
Fig. 4: The new Cell Line Optimization Nucleofector Kit with just 18 reactions. The kit contains Cell Line Nucleofector Solutions V and L. In
combination with a new range of Nucleofector Programs you can achieve superior transfection results for virtually any cell line. This new cell line
optimization approach reduces the number of transfections that need to be done by almost 50%. For further optimization, our expert Scientific
Support Team is, of course, available to provide you with advice on how to improve your transfection results even further.
Ordering information
Cell Line Nucleofector ® Solution R Cat. No.: VCA-1001 Cell Line Nucleofector ® Solution C Cat. No.: VCA-1004
Cell Line Nucleofector ® Solution T Cat. No.: VCA-1002 Cell Line Nucleofector ® Solution L Cat. No.: VCA-1005
Cell Line Nucleofector ® Solution V Cat. No.: VCA-1003 Cell Line Nucleofector ® Optimization Kit Cat. No.: VCO-1001*
amaxa web information www.amaxa.com/celldatabase
2 3
> > > +
> > >
Step 1 The cell line of interest
is transfected with the
Nucleofector solutions V
and L in combination with
seven different Nucleofector
programs.
Step 2 The Nucleofector
solution and program which
result in highest transfection
efficiency and viability
are selected.
*available Nov 1 st , 2005
Step 3 Further optimization
of the nucleofection
conditions can be performed
with the help of our
Scientific Support Team.

› New products
new
Benefit from:
Mouse and Rat Hepatocyte
nucleofection ®
Our R&D team has set out to conquer yet another untransfectable cell type: primary hepatocytes.
This quest has resulted in specific Nucleofector Kits for mouse and rat hepatocytes.
First non-viral transfection of hepatocytes › Ability to use primary hepatocytes for toxicologic studies
Reliable performance › Up to 55% efficiency in mouse and rat hepatocytes
Functionality › albumin synthesis rates of nucleofected cells comparable to
transfection efficiency
› page 6 › amaxa news # 6
% transfected cells
those of untransfected control
Flexibility › suitable for DNA and siRNA
Due to their low ability to proliferate efficient
transfection of primary hepatocytes has so far
been restricted to virus-based protocols. amaxa
is now introducing Nucleofector Kits for mouse
and rat hepatocytes. A protocol for human
hepatocytes will follow soon. With these new
kits, efficient and reproducible transfection of
primary hepatocytes is possible for the first
time. Efficiencies of up to 55% can be achieved
while cells remain functional. Once again the
Nucleofector technology offers the only efficient
and reproducible non-viral transfection
method for these cells. Fig. 1 summarizes transfection
efficiencies for primary hepatocytes.
Furthermore, the siRNA Test Kit has successfully
been used. Fig. 2 shows downregulation of
maxGFP expression in mouse hepatocytes.
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mouse
rat
viability
Fig. 1: Average transfection efficiencies and viabilities of mouse and rat
hepatocytes 24 hours post nucleofection with pmaxGFP TM .
% living cells (trypan blue negative)
A B
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C D
mouse
Fig. 2: Efficient gene silencing in mouse hepatocytes. Hepatocytes were nucleofected
with 2 μg of pmaxGFP (A,B) or cotransfected with 2 μg pmaxGFP and
1.5 μg siRNA directed against maxGFP (C,D). 24 hours post nucleofection cells were
analyzed by light and fluorescence microscopy for maxGFP expression.
rat

[k]
coming soon
› New products
Nucleofected mouse and rat hepatocytes maintain their functionality
Hepatocytes are involved in numerous metabolic pathways and have important functions that include detoxification,
synthesis of proteins, lipids and bile acids as well as storage of glycogen and vitamins. Thus, primary hepatocytes are
frequently used tools to study mechanisms of cell growth and differentiation, drug pharmacokinetics as well as
biotransformation and toxicity of drugs. The Nucleofector technology will help you gain further insight into these and
other research topics. Figs. 3 and 4 demonstrate that transfected hepatocytes remain functional. Mouse hepatocytes
show albumin secretion rates comparable to those of untransfected control hepatocytes (Fig. 3). Fig. 4 proves that rat
hepatocytes show normal cell-cell interactions and formation of bile canaliculi post nucleofection.
albumin secretion [pg/cell/hour]
0.25
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untreated cells
nucleofected cells
Fig. 3: Nucleofected hepatocytes maintain functionality.
24 hours post plating cultured and nucleofected primary
mouse hepatocytes were analyzed for albumin secretion
(normalized to cell number) by ELISA.
Ordering information
Mouse Hepatocyte Nucleofector ® Kit Cat. No.: VPL-1002
Rat Hepatocyte Nucleofector ® Kit Cat. No.: VPL-1003
Human Hepatocyte Nucleofector ® Kit Cat. No.: VPL-1001
› Product update
Top
offer
A
Fig. 4: Nucleofected hepatocytes maintain their morphology and polarization.
Primary rat hepatocytes were nucleofected with pmaxGFP (A) or a plasmid containing
the cDNA sequence for a plasma membrane receptor-YFP fusion protein (B).
Hepatocytes were stained with antibodies against desmoplakin (A; blue) to visualize
cell boundaries, and against multidrug resistance protein 2 (MRP2; A+B; red) to show
the apical, canalicular membrane. maxGFP was located in the cytosol of transfected
cells (A). YFP-fusion protein was correctly targeted to both the basolateral and the
apical membrane domain as shown by colocalization with MRP2 (B).
(Data courtesy of V. Keitel, F. Schliess and D. Häussinger, Clinic for Gastroenterology,
Hepatology and Infectiology, Heinrich-Heine-University Düsseldorf, Germany)
q
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*
N = nuclei; * = bile canaliculi
N
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B
Supplementary information Gastroenterology Flyer
amaxa web information www.amaxa.com/gastroenterology.html
Trade in your Nucleofector ® I for
a Nucleofector ® II at low cost
and benefit from
› a two-year warranty extension
› virtually unlimited filing capacity for new Nucleofector programs
› compatibility with upcoming new applications such as our Nucleofector ® 96-well Shuttle System
› possibility to store your individually identified cell-type program
For more information contact your local sales representative or fill out the reply card in the center of this newsletter.
› page 7 › www.amaxa.com
*
N
*

› Technote
1 amaxa GmbH,
Köln, Germany
2 Celonic GmbH,
Jülich, Germany
Use of Nucleofector ® technology
for transient protein production
Markus Zumbansen 1 , Stefanie Zahn 2 , Oliver Gresch 1 , Andreas Herrmann 2 , Titus Kretzschmar 1
Mammalian cells are often the only possible protein expression system when it comes to recombinant expression of
complex proteins such as antibodies. While generation of stably expressing cell clones is usually the method-of-choice,
transient protein production in mammalian cell cultures is becoming more popular as it offers a quick and affordable
alternative to establishing stable clones. Here we describe the successful use of the Nucleofector technology to
transiently express proteins in difficult-to-transfect cell lines such as suspension CHO cells. With up to 2 mg produced
of e.g. a human IgG1 antibody as shown here, nucleofection is the ideal tool to quickly generate small amounts of
complex proteins in a mammalian expression system.
Introduction
Cultured mammalian cells are frequently
preferred over prokaryotic systems
for recombinant protein production
because they provide proper protein folding
and assembly, as well as post-translational
modification. Although stable
expression systems are extremely useful
for production of recombinant proteins,
such systems can be time consuming
and expensive to establish when only
small amounts of protein are desired.
Milligram quantities of protein are sufficient
for a wide range of research applications,
including proof-of-concept studies,
target validation, pre-clinical
research, and characterization (e.g.
chromatography, crystallography). In
contrast to stable expression cultures,
transient expression cultures enable
production of proteins on a small scale
(up to approximately 10 mg) within a
few days. The principal drawback to
transient expression has been the inability
to achieve high transfection efficiencies
into cell lines, e.g. suspension
CHO cells (sCHO), which are suitable to
Table 1
be grown in high density and under
serum-free conditions. The ability of the
Nucleofector technology to efficiently
transfect even difficult-to-transfect cell
lines, such as suspension cells, has suggested
its use to establish a transient
protein expression culture. In this study,
we compared nucleofection to the market-leading
lipofection reagent in the
establishment of transient expression
culture using suspension CHO cells.
Materials and Methods
Cell lines and cell culture
Two sCHO cell clones from different
sources were used in the transfection
experiments. sCHO-clone 1 was purchased
from the European Collection of Cell
Cultures (ECACC; Salisbury, UK), and
was cultured in CHO Protein-Free
Medium (SIGMA, Munich, Germany)
supplemented with 2 mM L-glutamine
(Invitrogen; Carlsbad, CA, USA). sCHOclone
2 was purchased from Invitrogen,
and was cultured in CD-CHO Medium
with 10 ml/L HT Supplement and 8 mM
L-glutamine (all from Invitrogen). All
Cell line Transfection Transfection DNA Cell number
method conditions [pDRIVE-hSEAP]
sCHO-clone 1
(ECACC)
sCHO-clone 2
(Invitrogen)
› page 8 › amaxa news # 6
Transfection conditions for transient expression of hSEAP
cells were grown in spinner culture
systems at 37°C and 5% CO 2.
Transfection
Each of the two sCHO lines was transfected
using either Lipofectamine 2000
(Invitrogen) or nucleofection (amaxa,
Cologne, Germany), after optimizing the
transfection conditions. The first set of
experiments involved transfection with
a plasmid containing the DNA sequence
encoding human secreted alkaline
phosphatase (hSEAP), using conditions
described in Table 1.
In the second set of experiments, 1 x 10 8
sCHO-clone 2 cells (= 10 samples) were
co-transfected with plasmids containing
the DNA sequence for either the light
chain or the heavy chain of human IgG1
antibody (total of 50 μg DNA) using
nucleofection (Cell Line Kit V in combination
with program U-024). After transfection,
sCHO cells were cultivated as
batches in spinner flasks containing
50 ml culture medium for 5 days under
non-regulated conditions.
Lipofectamine 290 μl 58 μg 5 x 10 7
2000
Nucleofector ®
Cell Line Kit V 25 μg 5 x 10 7
technology Program U-024 (= 5 samples)
Lipofectamine 580 μl 116 μg 5 x 10 7
2000
Nucleofector ®
Cell Line Kit V 25 μg 5 x 10 7
technology Program U-024 (= 5 samples)

› Technote
volumetric productivity hSEAP [U/ml]
45
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5
0
time [d] 1 2 3 4 5
Analysis of protein production
Production of hSEAP was assayed using
the substrate p-nitrophenole-phosphate
from Roche (Basel, CH); the amount of
secreted hSEAP was quantified based on
absorbance at 405 nm by spectrophotometry
(SpectraMax ® , Molecular Devices,
Union City CA, USA).
Production of human IgG1 antibody was
assayed by ELISA, using antibodies from
Jackson Immuno-Research (West Grove,
PA, USA) and spectrophotometry (SpectraMax
® ) to measure OD at 490 nm. The
integrity of IgG1 produced by the transfected
sCHO lines was established by
Western blotting using anti-human IgG1
antibody (Jackson Immuno-Research).
Results
The volumetric productivity of hSEAP for
the two sCHO clones transfected using
Lipofectamine 2000 (LF) and nucleofection
(NF) is illustrated in Fig. 1. As can
be seen, nucleofection of both, sCHOclone
1 and sCHO-clone 2, resulted in
far higher volumetric productivity than
transfection using Lipofectamine 2000.
The comparison is even more striking
when the production of hSEAP is normalized
based on cell number. Fig. 2
illustrates cell-specific productivity for
the different transfection technologies,
based on a seeding density of 10 6 cells/ml
following transfection.
The production of human IgG1, following
co-transfection with plasmids separately
encoding IgG1 light and heavy
chains, is illustrated in Fig. 3. As can be
seen, IgG1 levels rose steadily before
peaking at day 4 at a concentration of
39 μg/ml. In summary, the 50 ml culture
had produced over 1.7 mg of IgG1 anti-
› page 9 › www.amaxa.com
sCHO-clone 1, lipofection
sCHO-clone 1, nucleofection
sCHO-clone 2, lipofection
sCHO-clone 2, nucleofection
Fig. 1: Volumetric productivity of hSEAP in nucleofected sCHO is higher compared to lipofected sCHO. Volumetric
productivity (units hSEAP activity/ml of culture medium) following transfection of two different strains of sCHO cells is shown
using either nucleofection or Lipofectamine 2000 (Invitrogen), as described in the text.
specific productivity hSEAP [μU/c/d]
antibody concentration [μg/ml]
10
9
8
7
6
5
4
3
2
1
0
45
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sCHO-clone 1 sCHO-clone 2
time [d] 1 2 3 4 5
Fig. 2: Cell specific hSEAP productivity
is higher in nucleofected cells compared
to lipofected cells. Specific percell
productivity of hSEAP (based on a
seeding density of 10 6 cells/ml) following
transfection of two different strains of
sCHO cells using either nucleofection or
Lipofectamine 2000 (Invitrogen), as
described in the text.
lipofection
nucleofection
Fig. 3: Production of up to 2 mg IgG1
antibody after nucleofection of sCHOclone
2. 1 x 10 8 cells of sCHO-clone 2
(10 samples) were co-nucleofected as described
in the text with plasmids encoding
IgG1 heavy and light chain. Post nucleofection
cells were cultivated for 5 days in
50 ml serum-free medium. Cells reached
a specific productivity of 5.5 pg/c/d and a
volumetric productivity of 39 μg/ml.
Within 5 days 1.7 mg of the antibody could
be produced (as determined by ELISA).
The integrity of the produced antibody
was verified by Western blotting.
body within 5 days. The integrity of the
produced antibody was verified by
Western blotting (data not shown).
Conclusions
These experiments demonstrate the
possibility to apply nucleofection for
transient protein expression in sCHO
cells grown under serum-free culture
conditions. In the hSEAP experiments,
cells transfected using nucleofection
demonstrated productivity more than an
order of magnitude greater than those
transfected using the market-leading
lipofection reagent whether assessed
on the basis of volumetric or per-cell
productivity.
Using nucleofection, transient expression
of human IgG1 was possible by cotransfecting
plasmids encoding IgG1
light and heavy chain into sCHO cells.

› Technote
› Product update
transfection efficiency/viability (%)
100
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sCHO-clone 1 sCHO-clone 2
Fig. 4: Transfection efficiencies and viabilities obtained after nucleofection for different suspension cell lines used for
protein production. sCHO cells and sHEK-293 (both with two different clones 1 and 2), BHK-21 and NS0 cells were transfected
with 2 μg pmaxGFP. Transfection efficiency and viability were analyzed after 24h using flow cytometry.
At day 4 post-transfection, the total IgG1
yield of a 50 ml culture was nearly 2 mg,
and the integrity of the secreted antibody
was verified by Western blotting.
It is important to note that nucleofection
has also demonstrated the ability to reliably
transfect other cell lines frequently
used for expression in suspension culture.
Fig. 4 illustrates transfection efficien-
cies and viabilities following nucleofection
with a plasmid encoding green fluorescent
protein (pmaxGFP) into different
suspension cell lines which are
frequently used for protein production,
e.g. sCHO, sHEK-293, BHK-21 and NS0.
In conclusion, nucleofection is a useful
method for generating transient expression
cultures using suspension cells
› page 10 › amaxa news # 6
efficiency viability
sHEK-293-clone 1 sHEK-293-clone 2 BHK-21 NS0
adapted for growth in serum-free media.
Nucleofection provides both high transfection
efficiencies and high cell viability
following transfection. The resulting
transient expression culture produces
milligram quantities of proteins which
can be used for proof-of-concept studies,
characterization, pre-clinical research or
target validation.
Is your Nucleofector ® Software
up-to-date?
Many recently developed Nucleofector Kits and protocols require that your Nucleofector is updated with the latest
software version so that the device is able to execute these new Nucleofector programs. For example, the new
Nucleofector Kits for mouse and human macrophages as well as many of the recently optimized cell lines need
Nucleofector programs which are only included in the latest software version.
Therefore check your Nucleofector device to see if you have the latest software version! An update is easily done
using a chip card provided by amaxa. In case your device does not have the latest version, order your complimentary
chip card today.
How to check for your software version:
Nucleofector I:
Simply switch on your device and the software version
will show up on the display.
Most up-to-date software version: V2.4
[k] d
Ordering instructions
Order your free chip card today either via our website or simply
use the reply card in the center of this newsletter.
Nucleofector II:
Turn on the device, and press the menu button repeatedly up
to the menu item “Software version”.
Most up-to-date software version: S3-5
amaxa web information
www.amaxa.com/nucleofectorprogram.html

› Hot topic
Understanding cell-matrix contacts
with the help of nucleofection ®
Stefan Linder, Institute for Cardiovascular Diseases, Ludwig-Maximilians-University, Munich, Germany
Podosomes are cell-matrix contact structures
which have recently started to attract widespread
attention. In contrast to other cell-matrix contacts
which are mainly involved in migration and
adherence, podosomes seem to be involved in
matrix degradation. They are typically formed in
cells that have to cross tissue boundaries including
monocytes/macrophages, immature dendritic
cells and some types of cancer cells. Podosomes
have a dot-like appearance and consist of a core
of F-actin and actin-associated proteins surrounded
by a ring structure consisting of plaque
proteins such as talin or vinculin. The role of
these and other proteins in podosome dynamics
and function are not entirely clear yet.
Nucleofection of YFP-vinculin into primary
human macrophages showed that vinculin is
localized to the ring structure of podosomes,
close to the matrix (Fig. 1). As shown in Fig. 2A
by nucleofection of a GFP-actin fusion construct
into human macrophages, larger precursor
podosomes are located at the cell periphery or
at the leading lamella of migrating cells. Live-cell
imaging of nucleofected macrophages showed
that these precursor clusters undergo constant
fusion and fission (Fig. 2B-D). Experiments such
as these will help understand the podosomes’
structure and fuction.
Reference
S. Linder and P. Kopp (2005).
Podosomes at a glance. J. Cell Sci. 118 (10): 2079-2082.
› page 11 › www.amaxa.com
vinculin TIRF Overlay
Fig. 1: Vinculin forms ring-like structures in macrophages.
Human macrophages were nucleofected with 2 μg of a pEYFP-C3 fusion construct to
the chicken vinculin sequence. Image was taken using total internal reflection fluorescence
(TIRF) microscopy, thus only parts less than 120 nm away from the substrate
are shown. The overlay shows that podosomes form close contact to substratum.
(Photographs courtesy of Dr. Stefan Linder, Ludwig-Maximilians-University, Institute
for Cardiovascular Diseases, Munich, Germany).
trailing edge
A B C D
leading edge
➔
primary human macrophage expressing GFP-actin
Fig. 2: Location and dynamics of podosomes in primary macrophages.
Human macrophages were nucleofected with 2 μg of a pEGFP-C1 fusion construct to
the human beta-actin sequence. The dotted structures are the actin-rich core
domains of podosomes (A). These structures are highly dynamic (B-D); dissolution
(C; ) or fission (D; ) can be observed within just a few minutes.
(Photographs courtesy of Dr. Stefan Linder, Ludwig-Maximilians-University, Institute
for Cardiovascular Diseases, Munich, Germany).
➔
➔

› New products
The Nucleofector ® 96-well Shuttle System consists of three components:
Nucleofector ® II Device
High-throughput transfection
Introducing amaxa’s Nucleofector ® 96-well Shuttle System
To date, efficient and reproducible high-throughput transfection remains a challenge, especially when it comes to
difficult-to-transfect cell types. Based on the well-established Nucleofector technology, amaxa is currently developing
a 96-well system for highly efficient transfection of primary cells, such as human T cells and hard-to-transfect cell
lines (e.g. Jurkat, HL-60).
Searching for an efficient and reliable high-throughput transfection method for basic research and applications such as
target identification and target validation many researchers approached amaxa about a high-throughput nucleofection
system. Encouraged by this demand our R&D team set out to develop such a system based on our Nucleofector
technology.
Our new Nucleofector 96-well Shuttle System is an add-on to the Nucleofector II Device and, thus, offers the same
unique features as the existing single-cuvette based Nucleofector technology. In addition to difficult-to-transfect cell
lines even non-dividing primary cells, such as resting T cells, can be transfected efficiently in a 96-well format, as the
DNA is directly delivered into the nucleus.
Furthermore, as the same transfection parameters apply to any nucleic acid substrate used, the system offers high
flexibility in terms of applications: DNA vectors, e.g. expression plasmids or shRNA vectors, and RNA, e.g. siRNA
duplexes, can be transfected using the same transfection protocol. Efficiencies and viabilities achieved with the
Nucleofector 96-well Shuttle System are comparable to the single-cuvette Nucleofector, i.e. up to 99% transfection
efficiency with siRNA-duplexes and up to 90% efficiency with plasmid DNA.
+ +
Figure: The Nucleofector 96-well Shuttle System is based on the Nucleofector II Device which serves as the program delivery unit. The interaction
between Nucleofector II Device and Nucleofector 96-well Shuttle is controlled via the 96-well Shuttle Software. The system only works in conjunction
with cell-type specific 96-well Nucleofector Kits. The Kits contain the cell-type specific 96-well Nucleofector Solution, disposable, sterile
96-well Nucleocuvette plates (2 x 8) in a microtiter frame with lid, and a cell-type specific Optimized Protocol.
› page 12 › amaxa news # 6
96-well Shuttle Laptop with 96-well Shuttle Software

d
› New products
Benefit from:
Nucleofector ® technology › Transfect a wide variety of primary cells and
amaxa web information www.amaxa.com/96-wellnucleofection.html
› page 13 › www.amaxa.com
difficult-to-transfect cell lines using one technology
› Up to 90% transfection efficiency with DNA
› Up to 99% siRNA duplex transfer also in suspension cells
› High cell viability
› Easy and convenient handling
› Optimized protocols and kits for cell lines and primary cells
Flexible system › Modular system: add-on to Nucleofector II Device
› Suitable for DNA and siRNA applications
› Sterile and disposable 96-well Nucleocuvette plates (2 x 8)
for small and high throughput
› Up to 96 different programs applicable per plate
Optimal performance › Comparable transfection efficiencies to Nucleofector
› Fast processing of 96-well plate
› High-throughput optimization of cell lines with just one plate
› 20 μl transfection volume
Be among the first users of the system!
The Nucleofector 96-well Shuttle System will be tested by several selected laboratories in fall 2005, and is scheduled
to be available to other researchers in the first quarter of 2006.
If you want to be contacted as soon as the system is available, please register now either via our website form or just
fill out and return the reply card in the center of this newsletter.
For registration visit www.amaxa.com/96-well-form.html

› New products
new
Efficient non-viral transfection of
human and mouse macrophages
The inability to efficiently transfect primary macrophages has been a major obstacle in immunology,
cancer and cell biology research and has led our R&D team to challenge the transfection of macrophages
during amaxa’s mission in 2005. With transfection efficiencies of up to 60%, we think that
we have accomplished that mission well. What is more, nucleofected macrophages remain functional
and can be stimulated after nucleofection. So accelerate your research and employ our Nucleofector
Kits to finally address topics, such as gene regulation, signaling or differentiation in primary mouse
or human macrophages.
At a glance - most important facts about nucleofection of mouse and human macrophages:
Transfection efficiencies Human macrophages: up to 60%
› page 14 › amaxa news # 6
Mouse macrophages: up to 50%
Cell viabilities Human and mouse macrophages: up to 90% for both
Cell sources Human macrophages: differentiated from PBMC
100
90
80
70
60
50
40
30
20
10
0
Fig. 1: Average transfection efficiencies of human and mouse
macrophages (derived from C57BL/6 or Balb/c strains). Cells
were transfected with 2 μg pmaxGFP and analyzed 24 and 48 h
post nucleofection by flow cytometry.
Mouse macrophages: differentiated from mouse
bone marrow (evaluated for
C57BL/6 and Balb/c mice)
Macrophages are a key mediator in cellular immunity with multiple roles, e.g. in antigen processing
and presentation, phagocytosis and cytokine secretion. Efficient transfection of primary macrophages
has so far been restricted to viral methods. With specifically developed Nucleofector Kits for
mouse and human macrophages, efficient non-viral transfection of these cells is now possible
(see Figs. 1 and 2 for details).
% transfection efficiency 24h 48h 100
90
80
70
60
50
40
30
20
10
0
% living cells (7-AAD negative)
24h 48h
Human macrophages Mouse macrophages Mouse macrophages
Human macrophages Mouse macrophages Mouse macrophages
C57BL/6
Balb/c
C57BL/6
Balb/c
Fig. 2: Average viabilities of human and mouse macrophages
post nucleofection. Cells were transfected with 2 μg
pmaxGFP. Viability (as %7-AAD negative cells) was determined
24 and 48 h post nucleofection by flow cytometry.

[k]
› New products
Activation of nucleofected macrophages
A major pre-requisite for our development was
that macrophages had to remain functional after
nucleofection. Macrophages, as antigen presenting
cells, respond to uptake of foreign compounds,
such as bacterial membrane components
or DNA. In response, antigen processing
and presentation, as well as cytokine release are
initiated. Transfected DNA itself is a stimulus for
macrophages, which is reflected by a baseline
release of TNF- shortly after transfection with
a
DNA (an effect that is not seen when macrophages
are nucleofected without DNA). However,
this stimulation effect is transient and only
observed during the first hours after transfection.
Therefore, the culture medium should be
changed before nucleofected macrophages are
stimulated to remove all initially released cytokines.
For stimulation of human macrophages, the
culture medium should be changed 24 hours
post nucleofection; for stimulation of mouse
macrophages the medium can be exchanged as
soon as 6 hours after nucleofection. Nucleofected
human and mouse macrophages treated
in such a way, showed high TNF- release after
a
stimulation with LPS, which was comparable to
non-nucleofected control samples (Fig. 3).
Ordering information
Human Macrophage Nucleofector ® Kit Cat. No.: VPA-1008
Mouse Macrophage Nucleofector ® Kit Cat. No.: VPA-1009
d
› page 15 › www.amaxa.com
A
TNF- a pg/well 24h post activation
B
pg/ml TNF- a per 1000 cells
Fig. 3: Example for the stimulation of nucleofected human and mouse macrophages.
Primary human (A) and mouse (C57BL/6) macrophages (B) were transfected
with 2 μg pmaxGFP using the Human or Mouse Macrophage Nucleofector Kit.
24 hours (human macrophages) or 6 hours (mouse macrophages) post nucleofection,
the culture medium was exchanged and cells were stimulated with LPS. TNF- a
secretion was determined by ELISA after 48h for human monocytes or 24h for
mouse monocytes. For mouse macrophages values were normalized to the number
of living cells after transfection and are expressed as pg/ml TNF- a per 1000 cells. In
all samples stimulated with LPS, comparable TNF- a production could be detected. In
non-stimulated samples, no TNF- a was detected, indicating that no further TNF- a is
produced after medium change.
Tips and hints for human macrophage transfection
› For cultivation of nucleofected human macrophages we recommend using
Macrophage-SFM (M-SFM, Gibco ® /Invitrogen) supplemented with FCS. Compared
to those cultured in RPMI, macrophages cultured in M-SFM show an up to 20%
higher transfection efficiency post nucleofection.
› Transfected human macrophages can be activated 24h post nucleofection after
changing the culture medium. Cells are then ready for stimulation with stimuli,
such as LPS.
q Supplementary
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information
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amaxa web information www.amaxa.com/immunology
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-LPS +LPS
H

› Hot topic
H
Essentials for the nucleofection ®
of mouse T cells
Initial feedback from users of the recently introduced Mouse T cell Nucleofector Kits show that with the following
hints and tips you can obtain optimal transfection results.
Important points Comments
› Use lymphocytes from 6-12-week-old mice.
› Use lymphocytes freshly isolated from spleen.
› Media component B should always be added freshly to
an aliquot of Mouse T Cell Nucleofector Medium for each
experiment.
› Fully supplemented Mouse T Cell Nucleofector Medium
should be pre-equilibrated for at least 30 minutes
(37°C/5% CO2).
› Use 100 µm and then 70 µm cell strainer for isolation.
› An erythrocyte lysis step is not needed.
› Avoid centrifuging the cells higher than 90xg
and additional spins.
› Clean plasmid DNA is recommended.
› Handle cells very carefully when transferring the cells
from the cuvette to the 12-well plate.
› Incubating the nucleofected cells in fully supplemented
Mouse T Cell Nucleofector Medium, is recommended for
optimal results.
› page 16 › amaxa news # 6
›› T cells from mice younger than 6 weeks or older
than 12 weeks may lead to different nucleofection
results.
›› Mouse T cells isolated from lymph nodes or
thymus may yield different nucleofection results,
e.g. T cells from thymus (or thymocytes) show
higher mortality as many cells are apoptotic.
›› Media component B is essential for high transfection
efficiency. It is not stable in Mouse T Cell
Nucleofector Medium at 4°C.
›› If the medium is not pre-equilibrated cell
mortality after nucleofection may increase.
›› If only the 100 μm cell strainer is used, the
sample will contain higher amounts of fat, cell
debris, and aggregates which will decrease both
the transfection efficiency and cell viability.
›› Osmotic differences can cause apoptosis in
murine T cells. Erythrocyte lysis will decrease
cell viability post nucleofection.
›› Centrifugation speeds of over 90xg result in
increased cell damage and mortality.
›› Contamination with bacterial components may
result in lower transfection efficiency and
viability. Therefore avoid overloading the DNA
isolation column.
›› Gentle pipetting and handling of the cells after
nucleofection helps improve cell viability.
›› Using any other medium post nucleofection is
likely to result in lower cell viability and transfection
efficiency.

[k]
q
› Product news
new
Ordering information
amaxa’s Basic Nucleofector ® Kits
Broad-range nucleofection ® at its best
amaxa’s recent introduction of Basic Nucleofector Kits has significantly increased the range of
primary mammalian cells that can now be transfected non-virally. Offering great flexibility, Basic
Nucleofector Kits are available for fibroblasts, neurons, smooth muscle, epithelial and endothelial
cells. The kits allow you to determine the optimal nucleofection conditions of your cell, regardless of
mammalian species or tissue source, in just one simple experiment. By testing one Basic Nucleofector
Solution with five different Nucleofector programs you can work out the optimal program to obtain
highest transfection efficiency and viability for your cell.
See for yourself.
Results from Basic Nucleofector Kit users can be viewed on www.amaxa.com/celldatabase.
Fig. 1: Nucleofection of primary
porcine vascular smooth muscle cells.
Cells were nucleofected with the Basic
Nucleofector Kit for Primary Smooth
Muscle Cells, program A-33/A-033 and a
plasmid encoding the green fluorescent
protein, maxGFP. 48h post nucleofection
the cells were analyzed by fluorescence
microscopy. A transfection efficency of
around 80-90% was achieved.
(Data courtesy of Dr. Tao Wang and Dr.
Cathy M. Holt, University of Manchester,
United Kingdom.)
Examples of transfection efficiencies
obtained from Nucleofector users working
with Basic Nucleofector Kits.
Epithelial cells: pig tracheobronchial
epithelial cells; smooth muscle cells: rat
aortic smooth muscle cells; endothelial
cells: sheep lung endothelial cells; neurons:
cow neural precursor cells; fibroblasts:
human tunica albuginea. Viabilities
were in the range of 60% to 99%.
Basic Endothelial Cell Nucleofector ® Kit Cat. No.: VPI-1001
Basic Fibroblast Nucleofector ® Kit Cat. No.: VPI-1002
Basic Neuron Nucleofector ® Kit Cat. No.: VPI-1003
Supplementary information
Basic Nucleofector ® Kits Flyer
100
90
80
70
60
50
40
30
20
10
0
› page 17 › www.amaxa.com
% transfection efficiency
Epithelial cells Smooth muscle Endothelial
cells
cells
Neurons
Basic Smooth Muscle Cell Nucleofector ® Kit Cat. No.: VPI-1004
Basic Epithelial Cell Nucleofector ® Kit Cat. No.: VPI-1005
Fibroblasts

d
› amaxa insights
amaxa’s online information
pool is growing
We are constantly gaining information from you
and other users on your experiences with our
Nucleofector technology. We feel, sharing this
information in the form of our online cell, citation
and FAQ databases can greatly support you in
your day-to-day work.
In amaxa’s cell database you will find data on nucleofection ® conditions for more than 400 primary
cells and cell lines. These data include detailed information on every listed cell and on the experimental
set-up for their nucleofection.
Whenever you are planning to transfect a new cell type with amaxa's Nucleofector ® , just check the
cell database. Chances are high that you will find programs and solutions that will produce satisfactory
results with that specific cell type. To help you find the cells you are looking for more easily, we
have implemented advanced search functions.
amaxa‘s citation database is expanding fast. More than 600 publications using amaxa's Nucleofector
technology are available to date with new papers being published on a daily basis. Here, as with the
other databases, sophisticated search functions help you find the publications relevant for your work
quickly. You can search for author, title, journal, applications and various other topics – and for any
combination of these. For each publication, information on the cells and substrates used, relevant
applications and topics, as well as the link to the PubMed publication details page are available.
To complete our service the FAQ database offers a collection of questions and answers relevant to
existing and prospective Nucleofector users. The questions cover not only the use of the Nucleofector
technology, but also related topics, such as cell culturing, cell preparation and handling, vector
information and siRNA recommendations. Our aim is to provide you with helpful tips on all issues
influencing successful transfection.
All of these databases are frequently being updated, so be sure to check with www.amaxa.com
regularly or subscribe to our RSS feeds or e-mail news to be notified of new entries in the cell and
citation databases.
amaxa web information
www.amaxa.com/celldatabase www.amaxa.com/faq
www.amaxa.com/citations www.amaxa.com/news www.amaxa.com/rss
› page 18 › amaxa news # 6

ESBE Scientific Team
› amaxa insights
Representing amaxa in Canada
ESBE Scientific – Long experience meets absolute commitment
Founded in 1967 as a supplier
of disposables to the
clinical laboratory, ESBE Scientific has become
one of the largest wholly Canadian-owned
companies serving the life sciences and hospital
markets. The company has experienced steady
growth and stature in these laboratories by offering
knowledgeable support, quality products and
exceptional value. Forming exclusive partnerships
with manufacturers of scientific products has
allowed ESBE Scientific to constantly improve its
support to the scientific community. ESBE representatives
based in all major centers across the
6 million sq miles of Canada provide government,
industry, university and hospital research laboratories
with state-of-the-art diagnostic and research
technology such as the amaxa Nucleofector
technology. One example for the successful application
of nucleofection is the Samuel Lunenfeld
Research Institute (SLRI) at Mount Sinai Hospital,
established in 1985 in Toronto, Ontario.
Using the Nucleofector technology to understand
the dynamic role of TGF-b‚ pathway components
The primary focus of Dr. Jeff Wrana’s lab at the
SLRI is in defining the signal transduction pathways
for the TGF-b superfamily of proteins.
These growth factors regulate a number of bio-
› page 19 › www.amaxa.com
Lab members: Dr. Carrie Causing, graduate student
Valbona Luga and Dr. Christine LeRoy
logical processes that include cell growth, differentiation, embryonic
pattern formation and cancer progression.
Studies from the Wrana lab have been crucial in defining the key
steps in the TGF-b signaling pathway. Important discoveries include
mutations of the SMAD2 gene in some colorectal cancers, the
role of Smurf1, a novel ubiquitin ligase that degrades proteins in
the pathway, and the association of Par6 to the TGF-b receptor to
regulate cellular transformation.
To further investigate the network of TGF-b receptor–interacting
proteins and their role in the TGF-b signal transduction pathway in
mammalian cells, the Wrana lab is currently using nucleofection
to express various protein components of this pathway. With the
Nucleofector technology the team has successfully transfected
various cell lines and primary cells such as: MDCKs (canine kidney),
MDAs (human mammary), NMuMGs (mouse mammary),
Mv1Lu (mink lung), and primary cortical neurons.
Mouse cortical neurons MDAMB231 MDCK
MuLv NMUMG
Fig. 1: Nucleofection of different cell lines and primary neurons with a GFPencoding
plasmid.

› amaxa insights
Scientific Product Specialists:
On the road all around the world
Supporting Nucleofector ® users at the bench
It’s one thing to read about a new technique in a research paper,
or to review a sales brochure on a new product system. But when
you are ready to bring a new technology like nucleofection into
your laboratory, there is no substitute for watching an expert
demonstrate it live in an interactive format.
Helping Nucleofector users to successfully apply the technology
and drive their research is one of the principal missions of amaxa’s
Scientific Product Specialists, according to Kevin Grady, Director
of International Scientific Support at amaxa. To that end, these
specialists spend 70-80% of their time actively working with
Nucleofector users in their labs.
› page 20 › amaxa news # 6
USA Dr. Greg Alberts / Dr. Hartmut Tintrup
Peer-to-Peer Communications
As one would expect, the six Scientific Product
Specialists (two in Europe, two in the United
States and two in our Export region) are thoroughly
experienced in the optimal use of
Nucleofector technology. Moreover, all have
earned Ph.D. degrees in the biological sciences,
enabling them to provide true support to researchers
about using the Nucleofector technology
to tackle specific scientific problems and challenges.
Greg Alberts, Ph.D., Scientific Product
Specialist, explains, “As scientists, we speak the
same language as our customers; as a result, we
have the opportunity to work closely with them
as they integrate amaxa technology into their
research programs. It is tremendously rewarding
to provide the expertise that helps a research
team solve a challenging problem.”

› amaxa insights
Export region Dr. Klaus-Peter Rehorn / Dr. Bernd Neufeld
In addition to Greg Alberts, based in USA, the other Scientific
Product Specialists are Hartmut Tintrup, Ph.D. (USA), Christian
Bickel, Ph.D. (Europe), Bernd Neufeld, Ph.D. (Export), Klaus-Peter
Rehorn, Ph.D. (Export) and the newest member of the team,
Stefan Meinzinger, Ph.D. (Europe).
From Start-up to Established Science
Until recently, Kevin Grady notes, Scientific Product Specialists
spent most of their “field time” presenting seminars and product
demonstrations that introduced scientists to the Nucleofector
technology. However, as nucleofection has become accepted
as an important and useful technique, and as amaxa has
become a leader in the burgeoning RNAi arena, the focus has
broadened to provide more support for the existing community
of Nucleofector users.
Now, in addition to introductory seminars, Scientific Product Specialists
lead onsite user group meetings, sharing the latest news
and tips from amaxa’s R&D team, as well as other Nucleofector
› page 21 › www.amaxa.com
Europe Dr. Christian Bickel / Dr. Stefan Meinzinger
users. They also help the sales team troubleshoot
any difficulties encountered by users as
they put the Nucleofector technology to use.
“The Specialists are important ‘pipelines’ between
amaxa’s R&D team and the customer
base”, says Grady.
If you are interested in setting up a visit to your
laboratory or institution by an amaxa Scientific
Product Specialist, please contact your amaxa
sales representative or fill out and return the
reply card in the center of this news issue.

› FAQ
FAQ - frequently asked questions
about achieving high viability pre and post nucleofection ®
One of the things that is well known but frequently forgotten: Cells need to be treated with special care at all times.
Pre-warmed media, gentle centrifugation and cell handling, etc. are crucial for their viability.
› Are there any general recommendations to avoid high
mortality before nucleofection?
›› Gentle centrifugation - not too long, too fast or too many times.
Pay attention to the centrifugation speeds in the protocol, especially
if it says to spin at less than 200xg! If there is any doubt, or if you are
doing an unknown cell line, then just spin at 100xg. Most cells will
still pellet nicely, but will be less stressed by the lower speed spin.
Avoid multiple spins if possible.
› Are there any general recommendations to avoid high
mortality post nucleofection?
›› Immediately after nucleofection, the cells are very fragile and
must be handled very carefully to avoid shear stresses that can
damage or kill cells. You should slowly and gently add the media to
the cuvette (don't squirt the media onto the cells), and remove it
just as slowly and gently. The same is true for plating the cells or
placing them back into a 1.5 ml tube for the recovery step. Don't
pipette the cells up and down, as this, too, can introduce those shear
stresses and kill your cells.
› For cell lines grown in high-calcium media, such as DMEM,
the mortality post nucleofection may be elevated. What
do you recommend?
›› If you observe high mortality post nucleofection we often
recommend a recovery step with a calcium-free version of the
growth medium or a low-calcium medium, such as RPMI. Immediately
after nucleofecting your cells, you routinely add 500 μl of
pre-warmed “recovery medium” to the cuvette. Then gently remove
the entire contents of the cuvette, transfer into a 1.5 ml tube
and place at 37 C° for 5-10 minutes to allow the cells to recover.
After that time you can plate them in the appropriate growth
medium. There is no need to replace the “recovery medium”, just
add the cell suspension to the culture plate containing the appropriate
growth medium.
› page 22 › amaxa news # 6
› How can I avoid high mortality during
T cell nucleofection?
›› Try to keep the DNA amount for nucleofection
quite low (e.g. 1 μg plasmid DNA) since higher
DNA amounts might cause increasing toxicity
and mortality. It is also important to use highly
purified DNA (e.g. QIAGEN EndoFree ® Plasmid
Kits). According to various publications, the presence
of endotoxins may increase cell mortality
especially in primary cells such as blood cells.
The A260:A280 ratio of the DNA preparation
should be at least 1.8 for nucleofection.
› Is there a way to improve cell viability of
human mesenchymal stem cells or chondrocytes
post nucleofection?
›› As discussed before the biggest influence
on cell viability is the DNA preparation. In addition
you can also increase cell viability of these
two cell types by adding 20% FCS to your culture
medium post nucleofection.
› Is it possible to work with frozen samples
of primary PBMCs or CD34 + hematopoietic
stem cells? What about the viability
of the cells post nucleofection?
›› For cryoconserved PBMCs or enriched
CD34 + populations, we recommend culturing the
thawed cells for 1-2 hours in culture medium
before nucleofection. Any further enrichment
procedure after thawing is not recommended.
Viability of frozen nucleofected cells might be
lower than that of fresh ones. Efficiency is equal
to freshly isolated cells.

› amaxa insights
We will be pleased to welcome
you at our booth at any of
these meetings and shows!
* Talks will be held about amaxa‘s
new Nucleofector ® 96-well Shuttle
System during these meetings.
For details check our website.
Meet amaxa in 2005/2006
Oct 1 - 5 DGHO – German Society for Haematology and Oncology
Booth # 18, Hannover, Germany, www.dgho.de
*
Oct 18 – 20 Biotechnica
Hall 2, Booth # D24, Hannover, Germany, www.biotechnica.de
*
Oct 18 – 21 Dicovery on Target
Booth # 24, Boston, USA, www.healthtec.com
*
Nov 6 – 10 AAPS – American Association of Pharmaceutical Scientists
Booth # 2631, Nashville, USA, www.aapspharmaceutica.com
Nov 10 - 12 8th International Conference on Signal Transduction
Weimar, Germany, www.sigtrans.de
Nov 11 - 15 AASLD - 56th Annual Meeting of the American Association
for the Study of Liver Diseases
Booth # 424, San Francisco, USA, www.aasld.org
Nov 12 – 16 SFN – 35th *
Annual Meeting of the Society for Neuroscience
Booth # 1913, Washington, USA, www.sfn.org
Nov 15 – 18 SFI – French Society for Immunology
Toulouse, France, www.sfi-immunologie.com.fr
Dec 6 – 9 BSI – 11th Annual Congress of the British Society for Immunology
Harrogate, UK, www.immunology.org
Dec 8 – 9 DSI – Dutch Society for Immunology
Leeuwenhorst, NL, www.med.rug.nl/nvvi/
Dec 10 – 14 ASCB – 45th Annual Meeting of the American Society
for Cell Biology
Booth # 206, San Francisco, USA, www.ascb.org
*
Jan 25 - 27 Cell Signalling World 2006
2006 Luxembourg, www.transduction-meeting.lu
www.amaxa.com/meetings
› page 23 › www.amaxa.com

WAA-1001_06
b
amaxa GmbH
Nattermannallee 1
50829 Koeln
Germany
Scientific Support Scientific Support
Europe/World USA
phone +49(0)221-99199-400 phone (240) 632-9110
fax +49(0)221-99199-499 fax (240) 632-9112
e-mail scientific-support@amaxa.com e-mail scientific-support.US@amaxa.com
Help your friend and get two Nucleofector ®
Kits for free and the chance to win an iPod
Are you a convinced user of the Nucleofector technology?
Then why not tell your friends working in other labs or on other
projects about how effectively they can improve their transfection
efficiencies by using nucleofection?
You will receive a useful gift for your recommendation. Additionally,
if your friend buys a Nucleofector Device you will be rewarded with
two free Nucleofector Kits.
You will also be automatically entered in a lottery in which you can
win an iPod.
To participate visit www.amaxa.com/helpyourfriend
› www.amaxa.com
amaxa Inc.
205 Perry Parkway
Suite 7
Gaithersburg, MD 20877
USA