Materials and Reagents

Human blood or peripheral blood mononuclear cells (PBMCs), collected using heparin (1 ml per 60 ml of blood as an anti-coagulant)
Heparin (Sigma-Aldrich, catalog number: H3393 )
Ficoll-Hypaque (GE Life Sciences, catalog number: 17-1440-02 )
Human MACS Naive CD4⁺ T cell II Kit (Miltenyi Biotec, catalog number: 130-094-131 )
BSA (Thermo Fisher Scientific, catalog number: SH30574 )
PBS (Life Technologies, Invitrogen^TM, catalog number: 10010 )
RPMI medium (Life Technologies, Invitrogen^TM, catalog number: 11875093 )
Heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals or other supplier)
Hank's balanced salt solution without calcium or magnesium (HBSS) (Life Technologies, Invitrogen^TM, catalog number: 14170161 )
PHA (Sigma-Aldrich, catalog number: 61764 )
Neon Transfection System 100 μl kit (Life Technologies, Invitrogen^TM, catalog number: MPK10096 or MPK10025 )
Aqua Amine Live/Dead discriminator (Life Technologies, Invitrogen^TM, catalog number: L34957 )
7AAD (BD Biosciences, catalog number: 559925 )
Anti-CD3⁺ anti-CD28^- coated Dynal beads (Life Technologies, Invitrogen^TM, catalog number: 11131D )
Ionomycin (Sigma-Aldrich, catalog number: I3909 )
Phorbol myristate acetate (PMA) (Sigma-Aldrich, catalog number: P8139 )
One Glo luciferase reagent (Promega Corporation, catalog numbers: E6110 , E6120 , or E6130 )
AP-1-luciferase reporter (as described in Vaysberg et al., 2008)
Note: This plasmid consists of a pGL3 backbone (Promega Corporation, catalog number: E1751) with 5 copies of the AP-1 binding site from the metallothionein promoter inserted into the human IL2 minimal promoter.
pEGFP-N1 (Clonetech) or equivalent GFP-expressing plasmid
Beta-actin (b-actin)-driven luciferase reporter plasmid, or other highly expressing luciferase reporter
Note: Use of a renilla luciferase-expressing plasmid is not recommended in this assay, as we found it interferes with the firefly luciferase signal.
CD3-Alexa700 (eBiosceince, catalog number: 56-0037-42 )
CD4-PE-Cy7 (BD Biosciences, catalog number: 557852 )
CD4-PE (Life Technologies, Invitrogen^TM, catalog number: MHCD0404 )
CD45RA–PacificBlue (Life Technologies, Invitrogen^TM, catalog number: MHCD45RA28 )
CD45RO-PerCp-Cy5.5 (BD Biosciences, catalog number: 560607 )
CD4RA-APC (Life Technologies, Invitrogen^TM, catalog number: MHCD45RA05 ) or Alexa647 (BD Biosciences, catalog number: 562763 )
CD25-APC (Life Technologies, Invitrogen^TM, catalog number: MHCD2505 )
CD40L-FITC (BD Biosciences, catalog number: 555699 )
CD62L-PE (BD Biosciences, catalog number: 555544 )
CD69-PE-Cy5 (Life Technologies, Invitrogen^TM, catalog number: MHCD6906 )
MACS buffer (see Recipes)

Equipment

96-well round-bottom sterile tissue culture plates (BD Biosciences, catalog number: 353077 or equivalent)
96-well white-wall, flat-bottom plates (BD Biosciences, catalog number: 353296 or equivalent)
MACS LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
Neon Transfection System (Life Technologies, Invitrogen^TM, model: MPK5000 )
Dynal magnet (Life Technologies, Invitrogen^TM, model: 12321D )
Luminometer with 96-well plate capability (any manufacturer)
MACS midi magnets (Miltenyi Biotec, model: 130-042-302 )
Flow cytometer (BD LSR II or similar)

Procedure

Note: All centrifugation steps in 15 or 50 ml conical tubes should be performed at 450 x g for 10 minutes in a general-purpose centrifuge. All centrifugation steps in eppendorf tubes should be performed at 800 x g for 5 min in a microfuge.

Subject blood to standard Ficoll-Hypaque density gradient centrifugation; harvest interface layer and wash twice in HBSS, and purify with MACS Hunan Naive CD4⁺ II Kit and LS columns. Collect the flow-through; this is the naive CD4⁺ T cell fraction. Do not add EDTA to MACS buffer; use only 0.5% BSA in sterile PBS. Count cells. Resuspend in RPMI and centrifuge 540 x g for 10 minutes in general purpose centrifuge. You will need at least 1 x 10⁶ cells for each plasmid to be transfected. To allow for extra volume, stimulate a minimum 1.2 x 10⁷ cells. See Table 1 for a breakdown of cell number requirements.

Table 1. Plasmids and number of replicates
Plasmid
Number of Replicates
AP-1 luciferase reporter
5
b-actin luciferase reporter
1
pEGFP reporter
1
No plasmid (mock)
3
Dilute PHA to 2.5 μg/ml in RPMI/10% FBS (no PSG). Concentration should be optimized for each PHA source in terms of transfection efficiency and low expression of activation markers. Resuspend cells in PHA/RPMI at 1 x 10⁶ cells/100 μl. Leave 1 x 10⁶ cells unstimulated for FACS analysis of activation markers.
Activate cells for 19.5-20 h at 37 °C in a humidified 5% CO₂ containing incubator. Set concurrent timers for 19.5 and 20 h. If you are doing more than 10 transfections, stagger the start of the stimulations by at least half an hour to allow enough time to finish transfections by 20 h. The samples must be transfected between 19.5 and 20 h after PHA stimulation.
Before 19.5 h is up, add DNA plasmids to Eppendorf tubes (2.0 μg/1 x 10⁶ cells), as shown in Table 2. Aliquot 1.0 ml RPMI/10% FBS (no antibiotics) to wells of a 24 well plate (one well for each transfection reaction), and keep at 37 °C until ready to transfect. Add 3.0 ml Neon buffer E (included in Neon kit) to transfection chamber.

Table 2. Volumes for transfections
Transfection
DNA
Final volume
Total number of
reactions

buffer T
cells necessary
1
3.0 μg
150 μl
1.5 x 10⁶
2
5.0 μg
250 μl
2.5 x 10⁶
3
7.0 μg
350 μl
3.5 x 10⁶
n
2 n + 1 μg
100 n + 50 μl
n x 10⁶ + 5 x 10⁵
At 19.5 h, begin washing cells in PBS by centrifuging for 800 x g for 5 min in microfuge. Resuspend cells in Neon buffer T (10⁶ cells/100 μl, see Table 2). Prepare at least 50 μl extra volume for each tube. Not allowing extra volume will introduce air bubbles into the Neon pipette, which will disrupt the transfection.
Make sure to finish transfections by 20 h. Set the Neon for 2,400 V, 2 pulses, 12 ms. For other human T cell subsets, these parameters should be optimized as described in the Neon manual.
Electroporate 100 μl at a time. Check tip to make sure there are no bubbles in the Neon tip, and watch for sparks. If a sample sparks, omit it and do not reuse that tip. Use tips no more than twice. Add each electroporation/transfection reaction (100 μl) to 1 well of 24 well plate. Repeat electroporation until finished. Work quickly to maintain consistency. To assess viability, you may electroporate one sample in the absence of plasmid.
Incubate cells for 24 h in 37 °C/5% CO₂ incubator.
After 24 or 48 h of activation, stain cells for activation markers. Stain one unstimulated sample and one PHA-stimulated sample. Include aqua amine live/dead discriminator. To stain, pellet 1 x 10⁶ cells in an eppendorf tube (800 x g, 5 min in microfuge), wash with PBS and follow manufacturer's instructions for aqua amine staining. Wash cells in MACS buffer and resuspend in a total volume of 100 μl MACS buffer with antibodies as listed below. Incubate for 10 minutes at room temperature, wash with 1.0 ml of MACS buffer, and resuspend in 1% paraformaldehyde in MACS buffer. Suggested antibodies and fluorophores are listed in the Table 3. This staining combination may be adapted according to the capability of the available flow cytometer.

Table 3. Suggested staining for activation markers
Epitope
Fluorophore
Volume/10⁶ cells
CD3
Alexa700
2.5 μl
CD4
PE-Cy7
10 μl
CD45RA
PacificBlue
2.5 μl
CD45RO
PerCp-Cy5.5
10 μl
CD25
APC
2.5 μ
CD69
PE-Cy5
2.5 μl
CD40L
FITC
10 μl
CD62L
PE
10 μl

Gate on lymphocytes, aqua amine- cells, singlets. To assess purity, gate on CD3⁺ CD4⁺then CD45RA⁺ CD45RO^- cells. To identify the percentage of cells that are not activated in the PHA-stimulated sample, use the unstimulated sample to set gates using a histogram plot for CD25^-, CD69^-, CD40L^-, and CD62L⁺. See Figure 1 for gating strategy.

Figure 1. Analysis of sample purity and activation by flow cytometry. Top row: unstimulated sample stained for purity. Bottom row: Overlays of unstimulated (gray) and PHA-stimulated samples (black) stained for activation markers as indicated, and gated on naive CD4⁺ T cells as indicated.
At 24 h after transfection, activate cells for measurement of AP-1 activity. Wash anti-CD3+anti-CD28-coated Dynal beads with 1 ml RPMI/10% FBS. Leave tube containing beads on magnet for 1 minute before aspirating medium, while leaving tube containing beads on magnet. Remove from magnet, add 1 ml medium, and repeat for a total of 3 washes. Prepare 25 μl beads per 10⁶ cells, plus enough for 1 extra sample. Resuspend in 4x original volume (e.g. 100 μl RPMI/10% FBS per 25 μl beads originally used).
Dilute and combine PMA and ionomycin to working concentrations of 50 ng/ml PMA and 250 nM ionomycin. Make a total volume of 1 ml.
Transfer transfected cells to eppendorf tubes and pellet (800 x g, 5 min in microfuge). Leave one replicate of unstimulated cells. Do not combine replicates. Aspirate supernatants and resuspend in 550 μl RPMI/10% FBS. Aliquot 100 μl per well and transfer to 96 well round-bottom plate. Split transfection replicates across rows for 5 wells each replicate. Pellet cells by centrifuging at 450 x g for 10 min and aspirate supernatants.
Note: To avoid this step, you may resuspend cells in 225 μl RPMI/10% FBS and prepare 2x working stocks of all stimulation reagents described above. In this case, aliquot 50 μl of cells per well according to the diagram below and add 50 μl of medium containing stimulation reagents.

Resuspend each well in 100 μl medium containing appropriate stimulation agents and transfer to a 96 well white-wall plate on ice. See Table 4 for layout and diagram of distribution of replicates.
Use RPMI/10% FBS for unstimulated samples, and for b-actin-luciferase-transfected sample. There is room for additional stimuli, such as anti-CD3 alone. If using soluble antibodies, use a secondary cross-linking antibody.

Table 4. Sample plate layout for stimulation

1

2

3

4

5

6

7

8

9

10

11

12

A

AP-1 (1)

medium

CD3+CD28 Dynal beads

Iono. + PMA

B

AP-1 (2)

medium

CD3+CD28 Dynal beads

Iono. + PMA

C

AP-1 (3)

medium

CD3+CD28 Dynal beads

Iono. + PMA

D

AP-1 (4)

medium

CD3+CD28 Dynal beads

Iono. + PMA

E

AP-1 (5)

medium

CD3+CD28 Dynal beads

Iono. + PMA

F

Untransfected

medium

medium

medium

medium

medium

G

b-actin-luc

medium

medium

medium

medium

medium

H

Once all stimulation reagents have been added, remove plate from ice and incubate for 4 h at 37 °C, 5% CO₂.
During stimulation incubation, assess transfection efficiency by staining one untransfected sample and the pEGFP-transfected sample with 2.5 μl of anti-CD4-PE and 2.5 μl of anti-CD4RA-APC or Alexa-647. After staining, add 5 μl 7AAD to each sample to assess viability. This is detected on the PE-Cy5 channel. Do not fix these samples. Transfection efficiency is expected to range between 5 and 40% of live (7AAD^-), CD4⁺ CD45RA⁺ cells. Set EGFP⁺ gate at top 1% of untransfected cells. See Figure 2.

Figure 2. Assessment of viability and transfection efficiency by flow cytometry. Top: mock-transfected sample gated on live cells (7AAD^-), then lymphocytes, and CD4⁺ CD45RA⁺ cells. Bottom: Measurement of transfection efficiency by %EGFP⁺ in population described in top row. EGFP⁺ gate is set at top 1% of signal from mock-transfected cells.
Prepare Promega One-Glo reagent according to the manufacturer's instructions, or thaw an aliquot covered in foil. You will need 100 μl per well. At 4 h after stimulation, remove plate from incubator and add 100 μl One-Glo reagent per well using a multi-channel pipette. Cover with foil and incubate for 3 min.
Note: There is no need to remove Dynal beads as these do not affect the luciferase reading (A. Palin, unpublished data). Read on a luminometer with a 1 sec read per well. One Glo reagent does not require the use of injectors. Include an empty well as a control for background from the plate.
To analyze data, subtract absorbance values for unstimulated samples, pairing the transfection replicates (i.e. subtract A1 value from A2 or A3; B1 from B2 or B3, etc.). Divide instead of subtract to calculate fold change. The untransfected control gives background fluorescence from the cells and the b-actin luciferase signal is a control for luciferase activity. This will be significantly higher than the stimulated cells. The anti-CD3⁺CD28^- stimulated cells give a measure of the upregulation of AP-1 in response to TCR engagement and co-stimulation, while the ionomycin + PMA serves as a measure of the capacity of the cell to induce AP-1. To compare between two sample types or two groups, use the average of the 5 stimulated replicates minus the unstimulated replicates and an unpaired student's t-test.

Recipes

MACS buffer
1x PBS
0.5% BSA
Sterilize by vacuum filtration.
Note: Do not include EDTA in this buffer, as it may interfere with T cell receptor signaling.

References

Cron, R. Q., Schubert, L. A., Lewis, D. B. and Hughes, C. C. (1997). Consistent transient transfection of DNA into non-transformed human and murine T-lymphocytes. J Immunol Methods 205(2): 145-150.
Palin, A. C., Ramachandran, V., Acharya, S. and Lewis, D. B. (2013). Human neonatal naive CD4+ T cells have enhanced activation-dependent signaling regulated by the microRNA miR-181a. J Immunol 190(6): 2682-2691.
Vaysberg, M., Hatton, O., Lambert, S. L., Snow, A. L., Wong, B., Krams, S. M. and Martinez, O. M. (2008). Tumor-derived variants of Epstein-Barr virus latent membrane protein 1 induce sustained Erk activation and c-Fos. J Biol Chem 283(52): 36573-36585.