BioResearch

Amaxa™ 4D-Nucleofector™ Protocolfor Unstimulated Human T Cells
For 4D-Nucleofector™ X Unit
Unstimulated human T cells (small round lymphoblastoid cells) are a subpopulation of humanperipheral blood mononuclear cells (PBMC). PBMC should be purified from fresh human blood
samples treated with an anti-coagulant or from leukocyte-enriched buffy coat.

% 10090807060

Example for Nucleofection™ of human T cells

50

Transfection efficiency of fresh unstimulated human T cells 24 hours post Nucleofection™.1 x 106 enriched T cells weretransfected with program FI-115 (high efficiency) or EO-115 (high functionality) using 0.4 μg pmaxGFP™ Vector in
20 µl Nucleovette™ Strips. Cells were analyzed 48 hours post Nucleofection™ using a FACSCalibur™ [Becton Dickinson].
Cell viability (% PI negative T cells) is approximately 79% (for program EO-115) or 53% (for program FI-115) after
48 hours.Functionality (% of CD25 expression compared to non-transfected control) is usually 59% for EO-115.

40302010048 hours

FI-115
EO-115

Product Description
Recommended Kit(s)–P3 Primary Cell 4D-Nucleofector™ X Kit
Cat. No.

V4XP-3012

V4XP-3024

V4XP-3032

Transfection volume

100 µl100 µl20 µlSize [reaction]

2x624

2 x 16

Nucleofector™ Solution

2 x 0.675 ml

2.25 ml

0.675 ml

(0.492 ml + 27% overfill)(1.968 ml + 13% overfill)(0.525 ml + 22% overfill)2 x 0.15 ml

0.5 ml0.15 ml(0.108 ml + 27% overfill)(0.432 ml + 13% overfill)(0.115 ml + 22% overfill)pmaxGFP™ Vector (1 µg/µl in 10 mM Tris pH 8.0)

50 µg

50 µg

50 µg

Single Nucleocuvette™ (100 µl)

1224-

16-well Nucleocuvette™ Strips (20 µl)--2Supplement

Storage and stability

NoteStore Nucleofector™ Solution, Supplement and pmaxGFP™ Vector at 4°C.
For long-term storage, pmaxGFP™ Vector is ideally stored at -20°C. The
expiration date is printed on the solution box. Once the Nucleofector™
Supplement is added to the Nucleofector™ Solution, it is stable for threemonths at 4°C.

4D-Nucleofector™ Solutions can only be used with conductive polymer
Nucleovettes™, i.e. in the 4D-Nucleofector™ and the 96-well Shuttle™
System. They are not compatible with the Nucleofector™ II Device.

Research Solution
Amaxa™ 4D-Nucleofector® Protocolfor Unstimulated Human T Cells

Required Material

1. Pre Nucleofection™

NoteNotes

Please make sure that the entire supplement is added to the Nucleofector™
Solution. The ratio of Nucleofector™ Solution to supplement is 4.5 : 1 (seetable 1).

1.

2.
3.
–– 4D-Nucleofector™ System (4D-Nucleofector™ Core Unit and
4D-Nucleofector™ X Unit)–– Supplemented 4D-Nucleofector™ Solution at room temperature–– Supplied 100 µl single Nucleocuvette™ or 20 µl 16-well Nucleocuvette™
Strips–– Compatible tips for 20 µl Nucleocuvette™ Strips: epT.I.P.S. [US/CDN:
Eppendorf North America, Cat. No. 2491.431, Rest of World: Eppendorf
AG, Cat. No. 0030073.266], Matrix TallTips® [Matrix Technologies Corp.,
Cat. No. 7281] or LTS Tips [Rainin Instruments, LLC, Cat. No. SR-L10F,
SR/SS-L250S, SR/SS-L300S]. Before using other types of pipette tips,
please ensure they reach the bottom of the Nucleocuvette™ Wells
without getting stuck–– Supplied pmaxGFP™ Vector, stock solution 1 µg/µl

4.

5.

This protocol is designed for fresh unstimulated primary human
T cells from whole PBMCs. Depending on application T cells can befurther enriched (see below)
Transfection results may be donor-dependent
For preparation, do not perform protocols using hypo-osmolar
buffers. This may lead to high cell mortality after Nucleofection™
For freshly isolated cells no cultivation is required prior toNucleofection™. For cryopreserved cells we recommend incubating
the thawed cells for 1–2 hours at 37°C in culture medium beforeNucleofection™
For Nucleofection™ of stimulated T cells, please refer to the Optimized
Protocol for Stimulated Human T Cells

Coating of culture plates (optional for stimulation post
Nucleofection™)
1.1 Incubate each well with 1 ml (for 6-well) or 50 μl (for 96-well; Nunc
Immuno™ Plate C96 Maxi Sorp™) of a solution of Anti-Human CD3
MAB at a final concentration of 1 μg/ml and Anti-Human CD28 MAB
at a final concentration of 2 μg/ml (or with a solution of a control
antibody (purified mIgG(K)) at a final concentration 3 ug/ml)at37°C/5% CO2 for 5 hours
1.2 Wash the wells carefully three times with PBS/BSANote
For positive control using pmaxGFP™, dilute the stock solution to anappropriate working concentration. Further details are provided in table
3 of this Optimized Protocol. The volume of substrate solution added toeach sample should not exceed 10% of the total reaction volume (2 µl for20 µl reactions; 10 µl for 100 µl reactions).

Blood samples
1.3 Fresh human blood treated with an anticoagulant (e.g. heparin,citrate, ACD-A) or alternatively, leukocyte-enriched buffy coat notolder than 8 hours. The samples should be diluted with 2–4 volumesof PBS containing 0.5% BSA (PBS/BSA)–– Substrate of interest, highly purified, preferably by using endotoxin-freekits; A260:A280 ratio should be at least 1.8–– Cell culture plates of your choice–– For isolation: Ficoll-Paque™ Plus [GE Healthcare; Cat. No. 17-1440-03];
PBS containing 0.5% [w/v] BSA (PBS/BSA)–– For enrichment (optional): Pan T Cell Isolation Kit II [Miltenyi Biotec;
Cat. No. 130-091-156] or RosetteSep™ Isolation Kit for human T cells[StemCell Technologies, Cat. No 15021]–– For coating of plates (optional for post Nucleofection™ stimulation):
Anti-Human CD3 MAB [OKt 3; eBioscience, Cat. No. 14-0037-82]
and Anti-Human CD28 MAB [5E8; Research Diagnostics Inc., Cat.
No. 10R-CD28bHUμg/μl]; control antibody [purified mIgG(K); BDPharmingen, Cat. No. 554 721]; antibodies should be diluted incarbonate buffer (32 mM Na2CO3/16 mM NaHCO3) from 100 ng/μl stock
solutions directly before use; Immuno™ Plate C96 Maxi Sorp™ [Nunc,Cat. No.: 430 341]–– Culture medium: Clonetics™ Lymphocyte Growth Media-3 LGM-3® forserum-free culture [Lonza, Cat.No. CC-3211] or BioWhittaker® IMDM
media for addition of 10% serum [Lonza, Cat.No. BE12-722F]–– Prewarm appropriate volume of culture medium to 37°C (see table 2)–– Appropriate number of cells/sample (see table 2)

Preparation of PBMC
1.4 Pipet 15 ml Ficoll-Paque™ Plus in a 50 ml conical tube
1.5 Overlay Ficoll- Paque™ Plus with 35 ml blood sample and centrifuge
at 750xg for 20 minutes at 20°C in a swinging-bucket rotor without
brake
1.6 Remove the upper layer leaving the mononuclear cell layer
undisturbed at the interphase. Carefully transfer the interphasecells (lymphocytes and monocytes) to a new 50 ml conical tube
1.7 Add PBS/BSA to 50 ml mark, mix and centrifuge at 350xg for 10minutes at 4°C. Remove the supernatant carefully
1.8 Resuspend the cell pellet in 25 ml of PBS/BSA and centrifuge at160xg for 15 minutes at 4°C. Remove the supernatant carefully
1.9 Resuspend the cell pellet in 25 ml PBS/BSA and centrifuge at 300xg
for 10 minutes at 4°C. Remove the supernatant carefully
1.10 Resuspend cell pellet in 5 ml PBS/BSA and count the cells

2Research Solution
Amaxa™ 4D-Nucleofector® Protocol
for Unstimulated Human T Cells

Note

2.17 After run completion, carefully remove the Nucleocuvette™ Vessel
from the retainer
2.18 Resuspend cells with pre-warmed medium (for recommended
volumes see table 5). Mix cells by gently pipetting up and down
two to three times. When working with the 100 µl Nucleocuvette™ use
the supplied pipettes and avoid repeated aspiration of the sample
2.19 Plate desired amount of cells in culture system of your choice (forrecommended volumes see table 5)

Purified PBMC may be stored at 4°C overnight in PBS/BSA, but this cancause a significant loss of transfection efficiency.

Enrichment of T cells (optional)
1.11 Primary human T cells can be further enriched by using Pan T Cell
Isolation Kit II [Miltenyi] or RosetteSep™ Isolation Kit for human
T cells [StemCell Technologies] according to the manufacturer’s
protocol

3. Post Nucleofection™

2. Nucleofection™

3.1 Incubate the cells in a humidified 37°C/5% CO2 incubator until
analysis. Gene expression or down regulation, respectively, is often
detectable after only 4–8 hours. If this is not the case, the incubation
period may be prolonged to 24 or 48 hours
3.2 Medium change 6 hours post Nucleofection™ leads to an increased
viability after transfection. Spin the culture dishes for 8 minutes at
140xg in a tissue culture centrifuge. Carefully remove the medium
and add fresh pre-equilibrated culture medium
3.3 Stimulation (optional): Please do not add stimuli immediately
after Nucleofection™ as this may lead to increased cell mortality.
For stimulation by antiCD3/antiCD28, transfer cells to an antibody
coated 6-well plate or Nunc MaxiSorp™ 96-well plate (see chapter1) containing fresh medium 5 hours post Nucleofection™, andincubate cells for another 43 hours. Alternatively you may add freshmedium containing IL-2 or other suitable stimuli 4 –12 hours post
Nucleofection™

2.1 For Nucleofection™ Sample contents and recommended Nucleofector™
Program, please refer to Table 3
2.2 Please make sure that the entire supplement is added to theNucleofector™ Solution
2.3 Start 4D-Nucleofector™ System and create or upload experimental
parameter file (for details see device manual)
2.4 Select/Check for the appropriate Nucleofector™ Program (see table 3)
2.5 Prepare cell culture plates by filling appropriate number of wellswith desired volume of recommended culture media (see table 4)
and pre-incubate/equilibrate plates in a humidified 37°C/5% CO2incubator
2.6 Pre-warm an aliquot of culture medium to 37°C (see table 4)
2.7 Prepare plasmid DNA or pmaxGFP™ Vector or siRNA (see table 3)
2.8 Count an aliquot of the cells and determine cell density
2.9 Centrifuge the required number of cells (see table 3) at 200xg for
10 minutes at room temperature. Remove supernatant completely
2.10 Resuspend the cell pellet carefully in room temperature 4DNucleofector™ Solution (see table 3)
2.11 Prepare mastermixes by dividing cell suspension according to
number of substrates
2.12 Add required amount of substrates to each aliquot (max. 10% of
final sample volume)
2.13 Transfer mastermixes into the Nucleocuvette™ Vessels

NoteAs leaving cells in Nucleofector™ Solution for extended periods of time maylead to reduced transfection efficiency and viability it is important to work
as quickly as possible. Avoid air bubbles while pipetting.
2.14 Gently tap the Nucleocuvette™ Vessels to make sure the sample
covers the bottom of the cuvette2.15 Place Nucleocuvette™ Vessel with closed lid into the retainer ofthe 4D-Nucleofector™ X Unit. Check for proper orientation of the
Nucleocuvette™ Vessel
2.16 Start Nucleofection™ Process by pressing the “Start” on the displayof the 4D-Nucleofector™ Core Unit (for details, please refer to thedevice manual)
3Research Solution
Amaxa™ 4D-Nucleofector® Protocolfor Unstimulated Human T Cells

Additional Information
For an up-to-date list of all Nucleofector™ References, please refer to:www.lonza.com/nucleofection-citations
For more technical assistance, contact our Scientific Support Team:USA /Canada
Phone: 800 521 0390 (toll-free)Fax:
301 845 8338
E-mail: scientific.support@lonza.com
Europe and Rest of World
Phone: +49 221 99199 400
Fax:+49 221 99199 499
E-mail: scientific.support.eu@lonza.com

References
1. Baumgrass R et al, J Biol Chem. 2003 Oct 28
2. Chun HJ et al, Nature. 2002;419(6905):395-9.
3. Cron RQ et al. Immunol Res. 2003;27(2-3):185-202.
4. Harriague J et al, Nat Immunol. 2002;3(11):1090-1096.
5. Kunzmann S et al, FASEB J. 2003;17(2):194-202.
6. Kunzmann S et al, FASEB J. 2003;17(9):1089-95.
7. Messi M et al, J Biol Chem. 2003;278(15):13003-7.
8. Nambiar MP et al, J Immunol. 03;170(6):2871-6.
9. Roy J et al, 2002;46(11):3447-55.
10. Salazar-Fontana LI et al, J Immunol. 2003;171(5):2225-32.
11. Salazar Murphy LL et al, J Immunol. 2002;169(7):3717-25.
12. Schmidt-Weber CB et al, Eur J Immunol. 2002;32(4):1196-204.
13. Smith JL et al, J Biol Chem. 2003;278(42):41034-46.
14. Tenbrock K et al, J Immunol. 2003;170(6):2971-6.
15. Vang T et al, J Biol Chem. 2003;278(20):17597-600.

Please note that the Amaxa™ Nucleofector™ Technology is not intended to be used for
diagnostic purposes or for testing or treatment in humans.
The Nucleofector™ Technology, comprising Nucleofection™ Process, Nucleofector™
Device, Nucleofector™ Solutions, Nucleofector™ 96-well Shuttle™ System and 96-wellNucleocuvette™ plates and modules is covered by patent and/or patent-pending rights
owned by Lonza Cologne GmbH.
Amaxa, Nucleofector, Nucleofection, 4D-Nucleofector, 96-well Shuttle, Nucleocuvetteand maxGFP are either registered trademarks or trademarks of the Lonza Cologne GmbHin Germany and/or U.S. and/or other countries.
Other product and company names mentioned herein are the trademarks of theirrespective owners.
This kit contains a proprietary nucleic acid coding for a proprietary copepod fluorescentprotein intended to be used as a positive control with this Lonza product only. Any useof the proprietary nucleic acid or protein other than as a positive control with this Lonza
product is strictly prohibited. USE IN ANY OTHER APPLICATION REQUIRES A LICENSE FROMEVROGEN. To obtain such a license, please contact Evrogen at license@evrogen.com.
The CMV promoter is covered under U.S. Patents 5,168,062 and 5,385,839 and its use
is permitted for research purposes only. Any other use of the CMV promoter requires alicense from the University of Iowa Research Foundation, 214 Technology Innovation
Center, Iowa City, IA 52242.
The use of this product in conjunction with materials or methods of third parties may
require a license by a third party. User shall be fully responsible for determining whether
and from which third party it requires such license and for the obtainment of suchlicense.
No statement is intended or should be construed as a recommendation to infringe any
existing patent.
© Copyright 2010, Lonza Cologne GmbH. All rights reserved.
4D4XP-3002_2011-03

Lonza Cologne GmbH50829 Cologne, Germany

Research Solution
Amaxa™ 4D-Nucleofector® Protocol
for Unstimulated Human T Cells

Table 1: Volumes required for a single reaction
100 µl Single Nucleocuvette™

20 µl Nucleocuvette™ Strip

Volume of Nucleofector™ Solution

82 µl

16.4 µl

Volume of Supplement

18 µl

3.6 µlTable 2: Required amounts of cells and media for Nucleofection™
100 µl Single Nucleocuvette™

20 µl Nucleocuvette™ Strip

Culture medium per sample post Nucleofection™ (fortransfer and culture)

2 ml

230 µlCell number per Nucleofection™ Sample

5–10 x 106 cells(Minimal cell number: 7 x 105 cells, a lower cell number may lead to a major decrease of cell viability;
maximal cell number: 2 x 107 cells)

1 x 106 cells(5 x 105 cells can be used with slightly reduced
transfection efficiency and viability; at even lower
cell numbers transfection efficiency and viability
are significantly decreased)

Table 3: Contents of one Nucleofection™ Sample and recommended program
Cells

100 µl Single Nucleocuvette™

20 µl Nucleocuvette™ Strip

5–10 x 106 cells

1 x 106 cells

2 µg

0.4 µgor plasmid DNA (in H2O or TE)

1–5 µg0.2–1 µg

or siRNA30–300nM siRNA(3–30 pmol/sample)

30–300nM siRNA(0.6–6 pmol/sample)

P3 Primary Cell 4D-Nucleofector™ Solution

100 µl20 µl

Program

FI-115 (for high efficiency) or
EO-115 (for high functionality)

FI-115 (for high efficiency) or
EO-115 (for high functionality)

Substrate* pmaxGFP™ Vector

Table 4: Culture media volumes required for post Nucleofection™ Steps
100 µl Single Nucleocuvette™

20 µl Nucleocuvette™ Strip*

12-well culture plate

1.5 ml-96-well culture plate-160 µlCulture medium to be added to the sample postNucleofection™

500 µl

80 µl* Maximum cuvette volume 200 µl

Table 5: Recommended volumes for sample transfer into culture plate
100 µl Single Nucleocuvette™

20 µl Nucleocuvette™ Strip*

Culture medium to be added to the sample post
Nucleofection™

500 µl80 µlVolume of sample transferred to culture platecomplete sample (use supplied pipettes)

40 µl* Maximum cuvette volume 200 µl5