BioResearch

Amaxa™ 4D-Nucleofector™ Protocolfor stimulated Human T Cells
For 4D-Nucleofector™ X Unit
Stimulated CD3+ human T cells (small, round suspension cells (lymphocyte)) are a subpopulationof human peripheral blood mononuclear cells (PBMCs). PBMCs purified from fresh human blood
samples treated with anticoagulant or from leucocyte rich buffy coat.

% 100
908070605040

Example for Nucleofection™ of stimulated human T cells

30Transfection efficiency of stimulated human T cells 24 hours post Nucleofection™. 1 x 106 stimulated T cells weretransfected with program EO-115 (high functionality) using 0.4 μg pmaxGFP™ Vector in 20 μl Nucleocuvette™ Strips.
Cells were analyzed 24 hours post Nucleofection™ using a FACSCalibur™ [Becton Dickinson]. Cell viability (CellTiterGlo®
Viability Assay, Promega Cat. No.: G7570) is approximately 60 % after 24 hours

2010024 hours

Transfection efficiency
Viability

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

V4XP-3012

V4XP-3024

V4XP-3032

Transfection volume100 µl

100 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 Nucleovettes™
(conductive polymer cuvettes), i.e. in the 4D-Nucleofector™ Systemand the 96-well Shuttle™ Device. They are not compatible with theNucleofector™ II/2b Device.

BioResearch
Amaxa™ 4D-Nucleofector™ Protocol
for stimulated 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.

This protocol is designed for fresh stimulated primary human T cells
from whole PBMCs. Depending on application T cells can be further
enriched before stimulation (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 Nucleofection™ of unstimulated T cells, please refer to the
Optimized Protocol for Unstimulated Human T Cells.

–– 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 1µg/µl

1.2

Note

Blood samples

For positive control using pmaxGFP™, dilute the stock solution to an
appropriate 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).

1.3

4.

Coating of culture plates with anti-CD3 and anti-CD28 antibodies
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 MABat 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)at
37 °C/5 % CO2 for 5 hours
Wash the wells carefully three times with PBS/BSA

Fresh human blood treated with an anticoagulant (e.g. heparin,citrate, ACD-A) or alternatively, leukocyte-enriched buffy coat notolder than 8 hours

Preparation of PBMC
1.41.5–– Substrate of interest, highly purified, preferably by using endotoxinfree kits; 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 (for 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]; controlantibody [purified mIgG(K); BD-Pharmingen, Cat. No. 554 721];
antibodies should be diluted in carbonate 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)

1.6

1.7
1.8
1.9
1.10

Pipet 15 ml Ficoll-Paque™ Plus in a 50 ml conical tube
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
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
Add PBS/BSA to 50 ml mark, mix and centrifuge at 350xg for 10minutes at 4 °C. Remove the supernatant carefully
Resuspend the cell pellet in 25 ml of PBS/BSA and centrifuge at
160xg for 15 minutes at 4 °C. Remove the supernatant carefully
Resuspend the cell pellet in 25 ml PBS/BSA and centrifuge at
300xg for 10 minutes at 4 °C. Remove the supernatant carefully
Resuspend cell pellet in 5 ml PBS/BSA and count the cells

Note
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

2Primary human T cells can be further enriched by using Pan T CellIsolation Kit II [Miltenyi] or RosetteSep™ Isolation Kit for human
T cells [StemCell Technologies] according to the manufacturer’s
protocolBioResearch
Amaxa™ 4D-Nucleofector™ Protocol
for stimulated Human T Cells

Stimulation
1.12

2.16

Stimulate the isolated human T cells for 2–3 days priorNucleofection™ e.g. in plates coated with anti-CD3 antibody
and anti-CD28 antibody (please see 1.1–1.2). Seed cells at
5 x 106 cells per ml

2.17

2. Nucleofection™

2.18

For Nucleofection™ Sample contents and recommended Nucleofector™
Program, please refer to Table 3.
2.1
2.2
2.3
2.4

2.5
2.6
2.7
2.8
2.9
2.10
2.11
2.12

3. Post Nucleofection™

Please make sure that the entire supplement is added to theNucleofector™ Solution
Start 4D-Nucleofector™ System and create or upload experimental
parameter file (for details see device manual)
Select/Check for the appropriate Nucleofector™ Program(see table 3)
Prepare cell culture plates by filling appropriate number ofwells with desired volume of recommended culture media (seetable 4) and pre-incubate/equilibrate plates in a humidified
37 °C/5 % CO2 incubator
Pre-warm an aliquot of culture medium to 37 °C (see table 4)
Prepare plasmid DNA or pmaxGFP™ Vector or siRNA (see table 3)
Count an aliquot of the cells and determine cell density
Centrifuge the required number of cells (see table 3) at 200xg for
10 minutes at room temperature. Remove supernatant completelyResuspend the cell pellet carefully in room temperature 4DNucleofector™ Solution (see table 3)
Prepare mastermixes by dividing cell suspension according to
number of substrates
Add required amount of substrates to each aliquot (max. 10 % offinal sample volume)
Transfer mastermixes into the Nucleocuvette™ Vessels

3.1

3.2

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.
2.13
2.14

2.15

After run completion, carefully remove the Nucleocuvette™ Vessel
from the retainer
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 thesample
Plate desired amount of cells in culture system of your choice (forrecommended volumes see table 5)

Gently tap the Nucleocuvette™ Vessels to make sure the sample
covers the bottom of the cuvette
Place Nucleocuvette™ Vessel with closed lid into the retainer ofthe 4D-Nucleofector™ X Unit. Check for proper orientation of theNucleocuvette™ Vessel
Start Nucleofection™ Process by pressing the “Start” on the displayof the 4D-Nucleofector™ Core Unit (for details, please refer to thedevice manual)3

Incubate the cells in a humidified 37 °C/5 % CO2 incubator until
analysis. Gene expression or down regulation, respectively, isoften detectable after only 4–8 hours. If this is not the case, theincubation period may be prolonged to 24 or 48 hours
Culture stimulated T cells post Nucleofection™ in plates coatedwith anti-CD3 antibody and anti-CD28 antibody (see chapter 1)

BioResearch
Amaxa™ 4D-Nucleofector™ Protocolfor stimulated 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

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, Nucleocuvette, maxGFP, LGM-3,
Clonetics and Biowhitakker are registered trademarks of the Lonza Group or its affiliates.
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-3007_2011-03

Lonza Cologne GmbH50829 Cologne, Germany

BioResearch
Amaxa™ 4D-Nucleofector™ Protocol
for stimulated 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

240 µlCell number per Nucleofection™ Sample

1–5 x 106 cells (Lower or higher cell numbers may
influence transfection results)

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

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

100 µl Single Nucleocuvette™

20 µl Nucleocuvette™ Strip

1–5 x 101 x 1062 µg

0.4 µg

6Substrate*

pmaxGFP™ Vectororplasmid DNA (in H2O or TE)

1–5 µg0.4–0.8 µg

orsiRNA

30–300nM siRNA(3–30 pmol/sample)

30–300nM siRNA(0.6–6 pmol/sample)

P3 Primary Cell 4D-Nucleofector™ Solution

100 µl20 µl

Program

EO-115

EO-115* Volume of substrate should comprise maximum 10 % of total reaction volume

Table 4: Culture 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 µl

Culture medium to be added to the sample post
Nucleofection™

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