BioResearch

Amaxa™ 96-well Shuttle™ Protocolfor Stimulated Human T Cells
Cell Description

Product Description

Stimulated CD3+ human T cells (small, round suspension cells (lymphocyte)
are a subpopulation of human peripheral blood mononuclear cells(PBMCs). PBMCs purified from fresh human blood samples treated with
anticoagulant or from leucocyte rich buffy coat.

Recommended Kits

Example for 96-well Nucleofection™ of stimulated human T cells%

1008060
4020024 hours
Transfection efficiency

Viability

Transfection efficiency of stimulated human T cells 24 hours post Nucleofection™.
1×106 stimulated T cells were transfected with program 96-EO-115 (high functionality)
using 0.4 μg pmaxGFP™ Vector. Cells were analyzed 24 hours post Nucleofection™
using a FACSCalibur™ with HTS option (Becton Dickinson). Cell viability (CellTiterGlo™
Viability Assay, Promega Cat. No.: G7570) is approximately 60 % after 24 hours.

P3 Primary Cell 96-well Nucleofector™ Kits
Cat. No.
Size (reactions)
P3 Primary Cell 96-well Nucleofector™ Solution
SupplementpmaxGFP™ Vector (1 μg/μl in 10 mM Tris pH 8.0)
Nucleocuvette™ Plate(s)V4SP-3096
1×96
2.25 ml
0.5 ml50 μg1Cat. No.
Size (reactions)
P3 Primary Cell 96-well Nucleofector™ SolutionSupplementpmaxGFP™ Vector (1 μg/μl in 10 mM Tris pH 8.0)
Nucleocuvette™ Plate(s)V4SP-3960
10×96
22.5 ml
5 ml
50 μg
10Storage and Stability
Store Nucleofector™ Solution, Supplement and pmaxGFP™ Vector at 4°C. For
long term storage pmaxGFP™ Vector is ideally stored at -20°C. The expiry
date is printed on the solution box. Once the Nucleofector™ Supplement
is added to the Nucleofector™ Solution it is stable for three months at 4°C.

Note
96-well Nucleofector™ Solutions can only be used with conductive polymer
cuvettes, i.e. in the 96-well Shuttle™ Device and in the 4D-Nucleofector™
System. They are not compatible with the Nucleofector™ II/2b Device.

BioResearch
Amaxa™ 96-well Shuttle™ Protocolfor Stimulated Human T Cells

Required Material

1. Pre Nucleofection™

Note

Notes

Please make sure that the entire supplement is added tothe Nucleofector™ Solution.
– Nucleofector™ 96-well Shuttle System (Nucleofector™ Device, version IIS;
96-well Shuttle™ Device; laptop with 96-well Shuttle™ Software)– Supplemented 96-well Nucleofector™ Solution at room temperature– Supplied Nucleocuvette™ Plates– Supplied pmaxGFP™ Vector, stock solution 1 µg/µl– This protocol is designed for fresh unstimulated primary human T cells
from whole PBMCs. Depending on application T cells can be further
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 Nucleofection™ of unstimulated T cells, please refer to the Optimized Protocol for Unstimulated Human T Cells

Note

Coating of Culture Plates with Anti-CD3 and Anti-CD28 Antibodies

Volume of substrate solution added to each sample should not exceed
10 % of the total reaction volume (2 µl for 20 µl reactions). For positivecontrol using pmaxGFP™ Vector, please dilute the stock solution to reachthe appropriate working concentration.

1.1

1.2
– Substrate of interest, highly purified, preferably by using endotoxin
free Kits; A260 : A280 ratio should be at least 1 : 8– Nucleocuvette™ compatible tips: 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 Instrument, 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– Anti-CD3/anti-CD28 coated 96-well and 6-well culture plates (seebelow) or coated culture plates of your choice– Culture medium: Clonetics™ Lymphocyte Growth Media-3 LGM-3™ for
serum-free culture (Lonza, Cat.No. CC-3211) or BioWhittaker™ IMDM
media for addition of 10 % serum (Lonza, Cat.No. BE12-722F)– 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); control
antibody (purified mIgG(K); BD-Pharmingen, Cat. No. 554 721); antibodies should be diluted in carbonate buffer (32 mM Na2CO3/16 mMNaHCO3) from 100 ng/μl stock solutions directly before use; Immuno™
Plate C96 Maxi Sorp™ (Nunc, Cat. No.: 430 341)
– Prewarm appropriate volume of culture media at 37°C (240 μlper sample)– Appropriate number of cells (1×106 cells per sample; 5×105 cells canbe used with slightly reduced transfection efficiency and viability;
at even lower cell numbers transfection efficiency and viability are
significantly decreased)

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 at 37°C/5 % CO2 for 5 hours
Wash the wells carefully three times with PBS/BSABlood 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

Preparation of PBMC
1.4
1.5

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 750×g for 20 minutes at 20°C in a swinging-bucket rotor withoutbrake
Remove the upper layer leaving the mononuclear cell layer undisturbed at the interphase. Carefully transfer the interphase cells(lymphocytes and monocytes) to a new 50 ml conical tube
Add PBS/BSA to 50 ml mark, mix and centrifuge at 350×g for 10minutes at 4°C. Remove the supernatant carefully
Resuspend the cell pellet in 25 ml of PBS/BSA and centrifuge at
160×g for 15 minutes at 4°C. Remove the supernatant carefully
Resuspend the cell pellet in 25 ml PBS/BSA and centrifuge at
300×g 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

2

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 (Stem Cell Technologies) according to the manufacturer’s
protocolBioResearch
Amaxa™ 96-well Shuttle™ Protocolfor Stimulated Human T Cells

Stimulation

Note

1.12

It is advisable to pre-dispense each cell suspension into a sterile roundbottom 96-well plate or to pipet from a pipetting reservoir for multi-channel
pipettes. Use a multi-channel or single-channel pipette with suitablepipette tips. As leaving cells in 96-well Nucleofector™ Solution for extended
periods of time may lead to reduced transfection efficiency and viability it is important to work as quickly as possible. Avoid air bubbles while
pipetting.

Stimulate the isolated human T cells for 2–3 days prior Nucleofection™
e.g. in plates coated with anti-CD3 antibody and anti-CD28 antibody (please see 1.1–1.2). Seed cells at 5×106 cells per ml2. Nucleofection™
One Nucleofection™ Sample Contains– 1×106 cells– 0.4–0.8 μg plasmid DNA (in 1–2 μl H2O or TE) or 0.4 μg pmaxGFP™
Vector or 30–300 nM siRNA (0.6–6 pmol/sample)– 20 μl P3 Primary Cell 96-well Nucleofector™ Solution

2.10
2.11
2.12

2.1
2.2

2.3
2.4

2.5
2.6

2.7
2.8
2.9

Please make sure that the entire supplement is added to theNucleofector™ Solution
Start Nucleofector™ 96-well Shuttle™ Software, verify device
connection and upload experimental parameter file (for detailssee Manual “Nucleofector™ 96-well Shuttle™ System”)
Select the appropriate Nucleofector™ Program 96-EO-115
Prepare cell culture plates by filling appropriate number of wellswith desired volume of recommended culture media, e.g. 160 μl
for one well of a 96-well plate and pre-incubate/equilibrate plates
in a humidified 37°C/5 % CO2 incubator
Pre-warm an aliquot of culture media to 37°C (80 μl per sample*
see comments at the end of this chapter)
Prepare 0.4–0.8 μg plasmid DNA or 0.4 μg pmaxGFP™ Vector.
For siRNA experiments we recommend to start using 30–300 nM
siRNA (0.6–6 pmol/sample)
Count the cells and determine cell density
Centrifuge the required number of cells (1×106 cells per sample)
at 200×g for 10 minutes at room temperature
Resuspend the cell pellet carefully in 20 μl room temperature 96well Nucleofector™ Solution per sample

2.13
2.14

2.15

Gently tap the Nucleocuvette™ Plate to make sure the sample
covers the bottom of the well
Place 96-well Nucleocuvette™ Plate with closed lid into the retainerof the 96-well Shuttle. Well “A1” must be in upper left position
Start 96-well Nucleofection™ Process by either pressing “Uploadand start” in the 96-well Shuttle™ Software or pressing “Upload”
in the 96-well Shuttle™ Software and then the “Start” button at the96-well Shuttle™ (for both options please refer to the respectiveManual)
After run completion, open retainer and carefully remove the96-well Nucleocuvette™ Plate from the retainer
Resuspend cells with desired volume of pre-warmed media (maximum cuvette volume 200 μl). Mix cells by gently pipetting upand down two to three times. Recommendation for 96-well plates:
Resuspend cells in 80 μl of pre-warmed media*
Plate desired amount of cells in culture system of your choice.
Recommendation for 96-well plates: Transfer 40 μl of resuspended
cells to 160 μl pre-warmed media prepared in 96-well culture
plates** Note
The indicated cell numbers and volumes have been found to produce optimal 96-well Nucleofection™ Results in most cases, however, dependingon your specific needs you may wish to test an extended range of cellnumbers. Cell numbers and volumes can be adapted such that fewer cells
are transferred or duplicate plates can be seeded.

A: One or several substrates (DNAs or RNAs) in multiples– Prepare mastermixes by dividing cell suspension according
to number of substrates– Add required amount of substrates to each aliquot(max. 2 μl per sample)– Transfer 20 μl of mastermixes into the wells of the96-well Nucleocuvette™ Modules–
B: Multiple substrates (e.g. Library Transfection)– Pipette 20 μl of cell suspension into each well of a sterile
U- or V-bottom 96-well microtiter plate– Add 2 μl substrates (maximum) to each well– Transfer 20 μl of cells with substrates into the wells of the96-well Nucleocuvette™ Modules

3. Post Nucleofection™
3.1

3.2

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™ 96-well Shuttle™ Protocolfor Stimulated Human T Cells

Additional Information
Up-To-Date List of all Nucleofector™ References
www.lonza.com/nucleofection-citations

Technical Assistance and Scientific Support
USA/Canada
Tel 800 521 0390 (toll-free)
Fax 301 845 8338
scientific.support@lonza.com
Europe and Rest of World
Tel + 49 221 99199 400
Fax + 49 221 99199 499
scientific.support.eu@lonza.com

www.lonza.com
Lonza Cologne GmbH – 50829 Cologne, Germany
Please note that the Amaxa™ Nucleofector™ Technology is not intended to be used for diagnostic purposes or for testing ortreatment in humans. The Nucleofector™ Technology, comprising Nucleofection™ Process, Nucleofector™ Device, Nucleofector™ Solutions, Nucleofector™ 96-well Shuttle™ System and 96-well Nucleocuvette™ plates and modules is covered bypatent and/or patent-pending rights owned by Lonza Cologne GmbH. Amaxa, Nucleofector, Nucleofection, 96-well Shuttle,
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 their respective owners.
This kit contains a proprietary nucleic acid coding for a proprietary copepod fluorescent protein intended to be used asa positive control with this Lonza product only. Any use of the proprietary nucleic acid or protein other than as a positivecontrol with this Lonza product is strictly prohibited. USE IN ANY OTHER APPLICATION REQUIRES A LICENSE FROM EVROGEN.
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
a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242. The useof this product in conjunction with materials or methods of third parties may require a license by a third party. User shallbe fully responsible for determining whether and from which third party it requires such license and for the obtainmentof such license. No statement is intended or should be construed as a recommendation to infringe any existing patent.
© Copyright 2009, Lonza Cologne GmbH. All rights reserved – D4SP-3007 2011-01

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