BioResearch

Amaxa™ HT Nucleofector™ protocol
for 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 mononuclearcells (PBMCs). PBMCs purified from fresh human blood samples treatedwith anticoagulant or from leucocyte rich buffy coat.

Recommended kits –P3 Primary Cell HT Nucleofector™ kits

Example for Nucleofection™ of stimulated human T cells%100Cat. No.
Size (reactions)P3 primary cell HT Nucleofector™ solutionSupplementpmaxGFP™ vector (1.0 μg/μl in 10 mM Tris pH 8.0)
384-well Nucleocuvette™ plate(s)V5SP-3002
2×384
22.5 ml
5 ml
50 μg
2Cat. No.
Size (reactions)P3 primary cell HT Nucleofector™ solution
Supplement
pmaxGFP™ vector (1.0 μg/μl in 10 mM Tris pH 8.0)
384-well Nucleocuvette™ plate(s)V5SP-3010
10×38490 ml
20 ml
150 μg
1080
60
40
200

24 hours
Transfection efficiency

Viability

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

Storage 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.

NoteHT Nucleofector™ solutions can only be used with conductive polymer
cuvettes, i.e. in the HT Nucleofector™, the 96-well Shuttle™ device andin the 4D-Nucleofector™ system. They are not compatible with theNucleofector™ II/2b device.

BioResearch
Amaxa™ HT Nucleofector™ protocol
for stimulated human T cell

Required material

1. Pre Nucleofection™

Note

Note

Please make sure that the entire supplement is added to the Nucleofector™
solution.

This protocol is designed for fresh primary human T cells from wholePBMCs. Depending on application T cells can be further enriched (seebelow).

–HT Nucleofector™
–Supplemented HT Nucleofector™ solution at room temperature–Supplied 384-well Nucleocuvette™ plates–Supplied pmaxGFP™ vector, stock solution 1 μg/μl–– 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

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

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;–– Substrate of interest, highly purified, preferably by using endotoxin
free Kits; A260 : A280 ratio should be at least 1 : 8–– 384-well Nucleocuvette™ plates are best pipetted with automatedliquid handling systems. If manual pipetting cannot be avoided,
please use compatible tips such as: 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). Please ensure the chosen pipette tips reachthe bottom of the 384-well Nucleocuvette™ wells without getting stuck.
–– Anti-CD3/anti-CD28 coated HT and 6-well culture plates (see below)
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
mM NaHCO3) 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 (>95 μl persample)–– 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)

1.2

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 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
1.6
1.7
1.8
1.9
1.10

at 750×g 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 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.

2BioResearch
Amaxa™ HT Nucleofector™ protocol
for stimulated human T cell

Enrichment of T cells (optional)
1.11 Primary human T cells can be further enriched by using Pan T Cell–– Transfer 20 μl of mastermixes into the wells of the 384-wellNucleocuvette™ plate

Isolation Kit II (Miltenyi) or RosetteSep™ Isolation Kit for human
T cells (StemCell Technologies) according to the manufacturer’s
protocol

B: Multiple substrates (e.g. library transfection)–– Pipette 20 μl of cell suspension into each well of a sterile U-bottom
384-well microtiter plate–– Add 2 μl substrates (maximum) to each well–– Transfer 20 μl of cells with substrates into the wells of the384-Nucleocuvette™ plates

Stimulation
1.12 Stimulate the isolated human T cells for 2–3 days priorNucleofection™ e.g. in plates coated with anti-CD3 antibody andanti-CD28 antibody (please see 1.1–1.2). Seed cells at 5×106
cells per mlNote
It is advisable to pre-dispense each cell suspension into a sterile roundbottom 384-well plate or to pipet from a pipetting reservoir for multichannel pipettes. Use a liquid handling system or manual pipette withsuitable pipette tips. As leaving cells in HT Nucleofector™ solution forextended periods of time may lead to reduced transfection efficiency andviability it is important to work as quickly as possible. Avoid air bubbles
while pipetting.

2. 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 HT Nucleofector™ solution

2.1
2.2
2.3
2.4

2.5
2.6
2.7
2.8
2.9

2.10 Briefly shake 384-well Nucleocuvette™ plate with an appropriate
microtiter plate shaker to make sure the sample covers the bottom
and sides of the well with no air bubbles. Alternatively, thoroughly
tap the 384-well Nucleocuvette™ plate2.11 Place 384-well Nucleocuvette™ plate with closed lid onto thecarousel of the Plate Handler. Well “A1” must be in upper leftposition
2.12 Start HT Nucleofection™ process by clicking “Start” in theHT Nucleofector™ Software
2.13 After run completion, carefully remove the 384-well Nucleocuvette™
plate from the carousel
2.14 Resuspend cells with desired volume of pre-warmed media(maximum cuvette volume 60 μl). Mix cells by gently pipetting
up and down two to three times. Recommendation for 384-wellNucleocuvette™ plates: Resuspend cells in 40 μl of pre-warmed
media*
2.15 Plate desired amount of cells in culture system of your choice.
Recommendation for 384-well cell culture plates: Transfer 6 μlof resuspended cells to 54 μl pre-warmed media prepared in384-well cell culture plates*

Please make sure that the entire supplement is added to theNucleofector™ solution
Start HT Nucleofector™ Software, verify device connection andupload experimental parameter file (for details please refer to theHT Nucleofector™ manuals)
Select the appropriate HT Nucleofector™ program EO-115-AA
Prepare cell culture plates by filling appropriate number of wellswith desired volume of recommended culture media, e.g. 54 μl forone well of a 384-well microtiter plate and pre-incubate/equilibrate
plates in a humidified 37°C/5 % CO2 incubator
Pre-warm an aliquot of culture media to 37°C (40 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
Nucleofector™ solution per sample* Note
The indicated cell numbers and volumes have been found to produce
optimal Nucleofection™ results in most cases, however, depending on yourspecific needs you may wish to test an extended range of cell numbers.
cell numbers and volumes can be adapted such that fewer cells aretransferred or duplicate plates can be seeded.

A: One or several substrates (DNAs or RNAs) in multiples–– Prepare mastermixes by dividing cell suspension according to numberof substrates–– Add required amount of substrates to each aliquot (max. 2 μl persample)

3

BioResearch
Amaxa™ HT Nucleofector™ protocol
for stimulated human T cell

3. Post Nucleofection™

Additional information

3.1

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 testingor treatment in humans.
The Nucleofector™ technology, comprising Nucleofection™ process, Nucleofector™ device, 4D Nucleofector™ device,
HT Nucleofector™ device, Nucleofector™ 96-well Shuttle™ system, Nucleofector™ solutions and 96-well Nucleocuvette™ platesand modules is covered by patent and/or patent-pending rights owned by Lonza Cologne GmbH.
Amaxa, Nucleofector, Nucleofection, maxGFP, Reagent Pack, HCM and BulletKit are either registered trademarks ortrademarks of the Lonza Group or its affiliates.
TallTips are a registered trademark of Matrix Technologies Corporation.
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 positive
control 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 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 theobtainment of such license.
No statement is intended or should be construed as a recommendation to infringe any existing patent.
© Copyright 2011, Lonza Cologne GmbH. All rights reserved D5SP-3007_2011-10

4D5SP-3007_2011-10

3.2

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)