INTRODUCTION
The enzyme-linked immunospot (ELISPOT) assay measures the secretion intensity of effector molecules released by immune cells inresponse to ex vivo antigenic stimulation, as well as the frequency of these responding cells. This assay is highly sensitive, quantitative, easy to use, and amenable to high-throughput screening. For these reasons, the ELISPOT assay is considered by many as a gold standard for monitoring cellular immune responses. Until recently, ELISPOT assays using chromophores to detect the T cell secretion of cytokines were limited to the characterization of a single effector molecule. Notably, studies evaluating the immune response to chronic viral infections often measured IFN-
secretion by ELISPOT because of the known antiviral effects of this cytokine as well as its correlation to the cytotoxic capacity of T cells. However, maintenance of both IFN- and IL-2 secretion by pathogen-specific T cells has been linked to a more favorable clinical outcome in human immunodeficiency virus (HIV) and Leishmania infections. Therefore, an ELISPOT assay able to simultaneously characterize T cell responses in terms of IL-2 and IFN- secretion is potentially relevant for the monitoring of immune responses to certain infectious agents. In this protocol, we describe an ELISPOT assay for the simultaneous detection of IL-2 and IFN- upon stimulation with viral peptides.
RELATED INFORMATION
Early uses of the ELISPOT assay for studies of immune cell secretionintensity and frequency are described in Currier et al. (2002) and Czerkinsky et al. (1988). The principles of the ELISPOT assay are reviewed in Letsch and Scheibenbogen (2003), and its use in high-throughput screening is described in Hernandez-Fuentes et al. (2003). The pathogen studies by Harari et al. (2005) illustrate the potential utility of simultaneous detection of IFN- and IL-2 secretion for monitoring immune response. Darrah et al. (2007) demonstrate that immune responses secreting IFN- and IL-2 and other cytokines play a protective role in a Leishmania major infection model. The assay presented in this protocol has also been described in Boulet et al. (2007).
MATERIALS
Reagents
AEC buffer (freshly prepared)
Immediately before use, activate AEC buffer using H2O2 (see Step 25).
Anti-CD3 monoclonal antibody (mAb) (Fisher Scientific)
Blocking buffer for ELISPOT assay
CEF peptide pool (available from the NIH AIDS Research and ReagentProgram and other commercial sources) (for use as control; see Step 13.iv)
Coating buffer (dual, IL-2, and IFN-)
Detection buffer for ELISPOT assay
Developing buffer for ELISPOT assay
Erythrosine B dye
H2O2 (30%; Sigma)
Methanol (MeOH; 90%) (American Chemicals)
Phosphate-buffered saline (PBS) (10X stock solution from Roche)
PBS-T buffer
Peptides from infectious agent (see Step 13.v)
Peripheral blood mononuclear cells (PBMCs)
R10 medium (prewarmed to 37°C)
R20 medium (prewarmed to 37°C)
RPMI medium 1640 (GIBCO)
Vector Blue solution
Wash medium for ELISPOT assay (prewarmed to 37°C)
Equipment
Centrifuge with swing-out bucket rotor for 15-mL polypropylene tubes
CTL-Immunospot Analyzer (Cellular Technology Ltd.)
Filters (0.45-µM)
Hemacytometer
Incubator (humidified atmosphere, preset to 37°C and 5% CO2)
Microscope
Multichannel pipette (Rainin)
MultiScreen IP white-walled sterile plates (Millipore)
Pipette tips
Pipettes (plastic transfer)
Pipettes (serological)
Plastic wrap
Trough (plastic, 50-mL)
Tubes (polypropylene [PP], 15-mL and 50-mL)
Vacuum plate washer (VPW) (Millipore)
Waterbath preset to 37°C
METHOD
All procedures on Days 1, 2, and 3 should be done under sterileconditions.
Day 1: Coating Plates and Thawing Cells
Coating Plates (30 min)
1. Permeabilize the MultiScreen IP white-walled ELISPOT plate by aliquoting 50 µL of 90% MeOH from a 50-mL plastic trough to each well using a multichannel pipette. Wait ~45 sec or until all the wells in the plate appear slightly translucent.
2. Immediately wash the plate with 1X PBS, adding 200 µL from a 50-mL plastic trough to each well using the multichannel pipette. Repeat four times.
The goal of washing with ~200 µL of fluid is to remove specific reagents after each step. As such, the volume used for the washes does not have to be precisely 200 µL.
3. Add 100 µL of coating buffer (dual, IL-2, and IFN-) to the appropriate wells.
4. Seal plate in plastic wrap and incubate overnight at 4°C.
Thawing Cells (30 min)
5. Remove frozen PBMCs from the liquid nitrogen tank and thaw in a 37°C waterbath, shaking gently until the frozen aliquot is reduced to a small ice pellet.
6. Transfer cells into a 15-mL PP tube using a plastic transfer pipette and add ~10 mL of warm (37°C) wash medium drop by drop while gently mixing the contents of the tube. Centrifuge at 1500 rpm for 7 min.
7. Discard the supernatant by decanting, and resuspend the pellet in 10 mL of warm (37°C) wash medium. Centrifuge at 1500 rpm for 7 min.
8. Repeat Step 7, but remove a 30-µL aliquot prior to centrifugation in order to count cells under the microscope. To count cells, dilute in Erythrosine B dye to check viability and use a hemacytometer.
9. After the last centrifugation, discard the supernatant and resuspend PBMCs at 2 x 106 cells/mL in warm (37°C) R10 medium. Aliquot amaximum 107 (or 5 mL) cells per 50-mL PP conical tube and incubateovernight in a humidified atmosphere incubator preset to 37°C and 5% CO2.
Day 2: Blocking Plates, Cell Plating
Blocking Plates (90 min)
10. Retrieve coated plates from the refrigerator and remove coating buffers by washing plates three times with 1X PBS, adding 200 µL of 1X PBS to each well with a multichannel pipette.
See Troubleshooting.
11. Add 200 µL of blocking buffer and incubate for at least 1 h at room temperature.
12. Wash plates five times with 200 µL/well of 1X PBS. Leave 200 µL per well of 1X PBS until ready to proceed to Step 13.
Cell Plating (2 h)
13. Remove 1X PBS from ELISPOT plate and add stimuli in RPMImedium 1640 to appropriate wells in a volume of 50 µL (or 100 µL for the medium control). During this step, do not allow wells to dry. Once done, place plate in a humidified atmosphere incubator preset to 37°C and 5% CO2. Stimuli are usually plated in triplicates and may include the following:
i. Medium control: 100 µL R10 medium with no stimulus added. No cells are added to these wells in Step 15.
No spots should be detected in Step 28. This controls for mediumquality.
ii. Negative control: 50 µL R10 medium with no stimulus added.
Unstimulated cells should give no or few spots in Step 28.
iii. Positive control: Anti-CD3 mAb at a final well concentration of 0.3 µg/mL.
iv. CEF control: Most individuals respond to this pool of peptidesisolated from cytomegalovirus (C), Epstein-Barr virus (E), and influenza virus (F).
v. Peptides from infectious agent: Optimal peptides (8- to 11-mers) or 15-mers are usually recommended. Pools of peptides may be used. Final concentration of the individual peptides or each peptide within a pool should be ~4 µg/mL.
14. Retrieve cells from the incubator and pool into a single 50-mL PP tube. Add warm (37°C) wash medium to fill the tube and remove a 30-µL aliquot to count PBMCs.
15. Centrifuge cells at 1500 rpm for 7 min. Discard the supernatantand resuspend the pellet in warm (37°C) R20 medium at 4 x 106 cells per mL. Retrieve plate from the incubator and add 50 µL of cells to appropriate wells.
Do not add cells to the "medium control" wells (Step 13.i).
16. Incubate plate for 28 h in a humidified atmosphere incubatorpreset to 37°C and 5% CO2.
Day 3: Detection (30 min)
17. Wash ELISPOT plate:
i. Three times with 1X PBS, 200 µL per well.
ii. Three times with PBS-T buffer, 200 µL per well.
18. Add 100 µL per well of detection buffer.
19. Seal plate in plastic wrap and refrigerate overnight at 4°C.
Day 4: Spot Color Development (3.5 h)
20. Wash the ELISPOT plate:
i. Three times with PBS-T buffer using 200 µL per well. Between each wash, empty wash fluid into a container.
ii. Three times with PBS-T buffer using 200 µL per well and the VPW.
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