Materials and Reagents

Nitrile gloves
Sterile 15 ml and 50 ml polypropylene tubes
Disposable sterile pipettes: 2 ml, 5 ml, 10 ml, 25 ml
Filters for syringes: 0.45 μM and/or 0.22 μM
Multiscreen _HTS-IP plates (PVDF membrane) (Merck, catalog number: MSIPS4510 )
Mouse (BioLegend, catalog number: 575402 ) or human (Thermo Fisher Scientific, Gibco^TM, catalog number: PHC0026 ) recombinant IL-2
Microbeads (Miltenyi Biotec)
Note: Catalog numbers depend on the cell type to be tested.
Dynabeads magnetic beads (Thermo Fisher Scientific, USA, https://www.thermofisher.com/kr/en/home/brands/product-brand/dynal.html)
Note: Catalog numbers depend on the cell type tested.
Ethanol
Peptides or antigen from the protein of choice to stimulate antigen-specific IFN-γ
Rat anti-mouse IFNγ mAb, clone AN18, purified (capture) (50 μg, Thermo Fisher Scientific, eBioscience^TM, catalog number: 14-7313-81 or 500 μg, Thermo Fisher Scientific, eBioscience^TM, catalog number: 14-7313-85 ) mouse anti-human IFNγ mAb, p clone NIB42, purified (capture) (50 μg, BioLegend, catalog number: 502403 or 500 μg, BioLegend, catalog number: 502404 )
Note: Either titrate or use as recommended by manufacturer. Prepare immediately before coating wells.
Rat anti-mouse IFNγ mAb, clone R4-6A2, biotinylated (detection) (50 μg, Thermo Fisher Scientific, eBioscience^TM, catalog number: 13-7312-81 or 500 μg, Thermo Fisher Scientific, eBioscience^TM, catalog number: 13-7312-85 ) or mouse anti-human IFNγ mAb, clone 4S.B3, biotinylated (detection) (50 μg, BioLegend, catalog number: 502503 or 500 μg, BioLegend, catalog number: 502504 )
Note: Either titrate or use as recommended by manufacturer.
0.5% BSA
Streptavidin-horseradish peroxidase (BioLegend, catalog number: 405210 )
3-amino-9-ethylcarbazole (AEC) substrate/chromogen (BD, catalog number: 551951 )
Milli-Q water
Sodium chloride (NaCl)
Potassium chloride (KCl)
Sodium phosphate dibasic (Na₂HPO₄)
Potassium phosphate monobasic (KH₂PO₄)
Hydrochloric acid (HCl)
Sodium bicarbonate (NaHCO₃)
Sodium phosphate (Na₂CO₃)
IMDM-1640
Fetal calf serum (FCS)
Note: Any brand that is suitable for cell culture and is heat inactivated at 56 °C for 30 min.
Penicillin-streptomycin (Tissue culture grade; Life technologies)
β-mercaptoethanol
Tween 20 (store at room temperature)
IMDM culture medium
L-glutamine (Tissue culture grade; Life technologies)
Phytohemagglutinin-L (PHA, positive control) stock (Roche Diagnostics, catalog number: 11249738001 ). Refer to Recipes section for details
Sterile phosphate buffered saline (1x PBS) (see Recipes)
Coating buffer working solution (see Recipes)
Blocking solution (see Recipes)
Anti-IFN-γ capture working solution (see Recipes)
Biotin-anti-IFN-γ working solution (see Recipes)
Streptavidin-horseradish peroxidase working solution (see Recipes)
Tween 20 working solution (see Recipes)
10% fetal calf serum in IMDM culture medium (see Recipes)
PHA stock solution (see Recipes)

Equipment

Note: ELISPOTS on the plate can be manually counted under a dissection microscope, or stereomicroscope (For example, Greenough, high-performance zoom stereomicroscope, SMZ 168-series). Alternatively, there are several specialist automated systems for high throughput screening (AELVIS, Autoimmun Diagnostika, Bio-Sys, Cellular Technology and the Zeiss reader) and there pros and cons discussed elsewhere and beyond the scope of this protocol (Janetzki et al., 2015).

Waste container
Gilson pipette and tips: P-2, P-10, P-20, P200, P1000
Gilson multichannel pipette with matched tips
Beckman Allegra 12 refrigerated centrifuge (Beckman Coulter, model: Allegra X-12 )
Class II biohazard hood
Incubator
1 L bottles

Procedure

There are two ways to measure cell type-specific responses. Firstly, isolate total CD4⁺ and/or CD8⁺ T cells and culture with dendritic cells (DCs) and the peptide of choice (Karunarathne et al., 2016). T cells are generally enriched from experimental mice using bead based techniques such as Dynabeads magnetic beads (Untouched Mouse T cells kit; Life Technologies, US) or Miltenyi Biotec (CD90.2 T cell isolation; Germany). DCs are immuno-magnetically isolated from naive mice (for use as antigen-presenting cells; APC) using anti-mouse CD11c Microbeads (Miltenyi Biotec; Germany). DCs should not be isolated from experimental mice where DC function is known to be compromised. Both cell types (T cells and DCs) can also be isolated using flow cytometry-based cell sorting. Approximately 2 x 10⁵ T cells, from individual mice, should be co-cultured with 2 x 10⁴ DC, in 4-8 replicate wells per sample with 20 μg/ml peptides, recombinant protein or no peptide, as previously described (Howland et al., 2013). Alternatively, if peptides are specific for CD4⁺ but not CD8⁺ T cells and vice versa, then total spleen cell with no additional DCs or total T cells with DCs may be used. These combined cells are then cultured with the specific peptide on ELISPOT plates coated with anti-IFN-γ antibody. The IFN-γ-specific antibody on an ELISPOT plate captures the IFN-γ immediately after secretion from the cells. For human T cells, total PBMCs containing APC and T cells are cultured with the peptide of choice. For the positive control, just add PHA to total cells or isolated T cells without DCs.

Day 1
1. Coat Multiscreen _HTS-IP plates with primary antibody diluted in coating buffer:
  Before coating the plate with antibody, humidify the membrane in each well with 15 μl of 70% ethanol (in Milli-Q water) for 1 min. Rinse with 150 μl sterile PBS three times before the ethanol evaporates. Then coat plates with 50 μl anti-IFNγ antibody (capture) in sterile coating buffer. Incubate overnight at 4 °C.
  Notes:
2. The following control wells should be incorporated into the assay:
  1. No antigen stimulation (DC and T cells cultured without peptide).
  2. Positive control with a lymphocyte mitogen (T cells without DC but with mitogen like PHA at 5-10 μg/ml).
  3. No cells; no primary antibody.
Day 2
1. Block membrane with blocking solution.
  1. Remove primary antibody solution.
  2. Wash off unbound antibody with 150 μl sterile PBS per well, at least twice.
  3. Block membrane with 150 μl per well of cell culture medium (see Recipes) for at least 2 h at 37 °C.
    Note: Keep changing media if it goes deep pink, until orange.
2. Prepare mouse splenocytes (MacPherson et al., 2001; Wykes et al., 2007) or isolate T cells/DCs, suspended in cell culture medium (see Recipes). Plan for each sample to have 4-8 replicate wells.
3. Cell stimulation
Day 3
1. Secondary antibody
  1. Remove culture medium with cells.
  2. Wash plate 3 times with PBS and 3 times with PBS/0.01% Tween 20.
    Note: Ensure you include Tween 20 in the wash buffer. Some cells will have started attaching after culture overnight. Tween 20 will help wash these off the membrane. Do not use a plate washer at this stage if available.
  3. Dilute biotinylated anti-IFNγ antibody (detection) in PBS/0.5% BSA. Pass the detection antibody mixture through a 0.45 μm or 0.22 μm filter to remove aggregates. Some of the mixture will be lost by filtration and original volume must be scaled up accordingly. Add 50 μl of detection antibody mixture to each well.
    Note: Failure to filter secondary antibody may results in non-specific spot formation due to protein aggregates.
  4. Incubate for 2 h at 37 °C, 5% CO₂, 95% humidity.
  5. Then incubate overnight at 4 °C, in a sealed box with tissues moistened with sterile water or PBS which provide humidity.
Day 4
1. Wash plate 6 times with PBS/0.01% Tween 20.
2. Enzyme conjugate and substrate development

Data analysis

Spots are counted under a dissection microscope or in an automated ELISPOT reader, and the frequency of secreting cells is calculated:
One cell = One spot
Number of spots = number of cells secreting cytokines
Intensity and size of spot = relative cytokine-secreting ability of cells
Background subtraction
For ELISPOT data analysis, the background value is subtracted from measured results. Each specimen group should have a single background spot mean/median value, calculated from the negative control (T cells and DCs without antigen). Then for each antigen group in the sample, the mean/median for the group is calculated, the background mean/median is then subtracted and the count is normalized by the number of cells per well.
Assay

Positive should be > 50 spots/10⁶ cells.
Negative control should ideally be 0 spots/10⁶ cells. If > 0 and < 50 spots/10⁶ cells, subtract the negative control reading from the test count to correct for background staining. If > 50 spots/10⁶ cells, repeat the assay.
Contamination:

If one of the replicate wells of one assay condition clearly appears contaminated, ignore that particular well and use the result from the other wells.
If multiple wells clearly appear to be contaminated, repeat the assay.

Results
For each test antigen, results are reported as spot forming cells (SFC)/million for human PBMC or SFC/spleen for mouse spleen cells.

Notes

As horseradish peroxidase activity is completely stopped by sodium azide, please ensure it is never added to any buffer or reagents associated with this assay.
There are several brands of antibodies available that are suitable for this assay and can even be purchased as antibody pairs. However, each new antibody vial should be tested in a checkerboard titration to determine the optimal dilution (Asai, 2000 #14220).

Recipes

Sterile phosphate buffered saline (1x PBS)
Start with 800 ml of distilled water, to which add 8 g of NaCl; 0.2 g of KCl; 1.44 g of Na₂HPO₄ and 0.24 g of KH₂PO₄
Adjust the pH to 7.4 with HCl
Add distilled water to a total volume of 1 L
Stock is stored in 1 L bottles
Store at room temperature
Coating buffer working solution (0.5 M carbonate-bicarbonate buffer pH 9.6)
3.7 g NaHCO₃ and 0.6 g Na₂CO₃ (anhydrous) made up to 100 ml with distilled water
Make sure that the carbonate bicarbonate is dissolved by gently mixing it until there is no powder residue left and filter sterilized with 0.22 μM filter
Check pH which should be ~9.6 and occasionally needs to be adjusted
Store at room temperature in the dark
Blocking solution
IMDM culture medium
10% fetal calf serum
1% penicillin-streptomycin
0.1% β-mercaptoethanol
Anti-IFN-γ capture working solution
This is the capture antibody. Prepare immediately before coating wells. Need 50 μl per well, at a concentration according to manufacturer's instruction or by titration for each antibody stock
Biotin-anti-IFN-γ working solution
This is the detection antibody. Prepare as in PBS. Need 50 μl per well, at a concentration according to manufacturer's instruction or by titration for each antibody stock
Streptavidin-horseradish peroxidase working solution
Prepare a dilution of the stock in PBS. Need 50 μl per well, at a concentration according to manufacturer's instruction or by titration for each antibody stock
Tween 20 working solution
Add 0.5 ml Tween 20 stock to 1,000 ml 1x PBS
Store at room temperature
10% fetal calf serum in IMDM culture medium (store at 4 °C in the dark)

Fetal calf serum stock
Thaw and dispense into 50 ml aliquots. Store at -20 °C
IMDM culture medium (store at 4 °C in the dark)
L-glutamine stock
Supplied as 50 ml at 200 mM concentration. Store at -20 °C
Thaw and dispense into 1 or 5 ml aliquots depending on volume of culture medium to be prepared later. Store at -20 °C in the dark until needed
Penicillin-streptomycin (5,000 U/ml)
Supplied as 100 ml at 5,000 U/ml concentration. Store at -20 °C
Thaw and dispense into 1 or 5 ml aliquots depending on volume of culture medium to be prepared later. Store at -20 °C in the dark until needed
10% fetal calf serum in IMDM culture medium (complete medium)
For 100 ml: add 10 ml FCS (a), 1 ml L-glutamine (c), 1 ml penicillin-streptomycin (d), 100 μl β-mercaptoethanol, and to 88 ml IMDM (b). Store at 4 °C in dark

PHA stock solution
Add to 10 ml PBS to the PHA powder for a concentration of 500 μg/ml
Aliquot and freeze in volumes of 10 μl. Store at -80 °C
Notes:

Do not re-freeze thawed PHA.
PHA (Lectin, positive control) stock is supplied as 5 mg of purified, salt-free lyophilized powder.
In the assay, use at a final concentration of 5-10 μg/ml. Add 1-2 μl of stock solution to each well containing cells in a 100 μl volume

Acknowledgments

MNW was supported by ARC Future Fellowship and NHMRC. L.R. was supported by Singapore’s A*STAR and NRF Singapore (NRF2007NRF-RF001-226).The authors wish to thank Mr Josh Horne-Debets for the ELISPOT image.
The ELISPOT method was originally developed to detect antigen-specific antibodies from B cells (Czerkinsky et al., 1983) and has over the years been modified by several groups to detect cytokines produced by antigen-specific cells.

References

Andersson, U., Hallden, G., Persson, U., Hed, J., Moller, G. and DeLey, M. (1988). Enumeration of IFN-γ-producing cells by flow cytometry. Comparison with fluorescence microscopy. J Immunol Methods 112(1): 139-142.
Bradley, L. M., Duncan, D. D., Tonkonogy, S. and Swain, S. L. (1991). Characterization of antigen-specific CD4⁺ effector T cells in vivo: immunization results in a transient population of MEL-14-, CD45RB- helper cells that secretes interleukin 2 (IL-2), IL-3, IL-4, and interferon gamma. J Exp Med 174(3): 547-559.
Carvalho, L. H., Hafalla, J. C. and Zavala, F. (2001). ELISPOT assay to measure antigen-specific murine CD8⁺ T cell responses. J Immunol Methods 252(1-2): 207-218.
Czerkinsky, C. C., Nilsson, L. A., Nygren, H., Ouchterlony, O. and Tarkowski, A. (1983). A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65(1-2): 109-121.
Gray, P. W. and Goeddel, D. V. (1982). Structure of the human immune interferon gene. Nature 298(5877): 859-863.
Horne-Debets, J. M., Faleiro, R., Karunarathne, D. S., Liu, X. Q., Lineburg, K. E., Poh, C. M., Grotenbreg, G. M., Hill, G. R., MacDonald, K. P., Good, M. F., Renia, L., Ahmed, R., Sharpe, A. H. and Wykes, M. N. (2013). PD-1 dependent exhaustion of CD8⁺ T cells drives chronic malaria. Cell Rep 5(5): 1204-1213.
Horne-Debets, J. M., Karunarathne, D. S., Faleiro, R. J., Poh, C. M., Renia, L. and Wykes, M. N. (2016). Mice lacking Programmed cell death-1 show a role for CD8⁺ T cells in long-term immunity against blood-stage malaria. Sci Rep 6: 26210.
Howland, S. W., Poh, C. M., Gun, S. Y., Claser, C., Malleret, B., Shastri, N., Ginhoux, F., Grotenbreg, G. M. and Renia, L. (2013). Brain microvessel cross-presentation is a hallmark of experimental cerebral malaria. EMBO Mol Med 5(7): 984-999.
Janetzki, S., Price, L., Schroeder, H., Britten, C. M., Welters, M. J. and Hoos, A. (2015). Guidelines for the automated evaluation of Elispot assays. Nat Protoc 10(7): 1098-1115.
Karunarathne, D. S., Horne-Debets, J. M., Huang, J. X., Faleiro, R., Leow, C. Y., Amante, F., Watkins, T. S., Miles, J. J., Dwyer, P. J., Stacey, K. J., Yarski, M., Poh, C. M., Lee, J. S., Cooper, M. A., Renia, L., Richard, D., McCarthy, J. S., Sharpe, A. H. and Wykes, M. N. (2016). Programmed death-1 ligand 2-mediated regulation of the PD-L1 to PD-1 axis is essential for establishing CD4⁺ T cell immunity. Immunity 45(2): 333-345.
MacPherson, G. G., Wykes, M., Huang, F. P. and Jenkins, C. D. (2001). Isolation of dendritic cells from rat intestinal lymph and spleen. In: Robinson, S. P. and Stagg, A. J. (Eds.). Dendritic Cells Protocols. Humana Press.
Schmittel, A., Keilholz, U., Bauer, S., Kuhne, U., Stevanovic, S., Thiel, E. and Scheibenbogen, C. (2001). Application of the IFN-γ ELISPOT assay to quantify T cell responses against proteins. J Immunol Methods 247(1-2): 17-24.
Schreiber, R. D. (2001). Measurement of mouse and human interferon gamma. Curr Protoc Immunol Chapter 6: Unit 6 8.
Schroder, K., Hertzog, P. J., Ravasi, T. and Hume, D. A. (2004). Interferon-γ: an overview of signals, mechanisms and functions. J Leukoc Biol 75(2): 163-189.
Wykes, M. N., Liu, X. Q., Jiang, S., Hirunpetcharat, C. and Good, M. F. (2007). Systemic tumor necrosis factor generated during lethal Plasmodium infections impairs dendritic cell function. J Immunol 179(6): 3982-3987.
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