In vitro and ex vivo IFN-γ ELISPOT Assays

Immune responses were evaluated with in vitro and ex vivo IFN-γ ELISPOT assays. For in vitro ELISPOT assays, PBMCs were thawed and stimulated with the target epitope. The next day, PBMCs were stimulated with IL-2 (120 U/mL) and incubated for 12-14 days. The PBMCs were then counted and plated in ELISPOT wells. Cells were restimulated with or without the target peptide. All conditions were performed in triplicates. For ex vivo IFN-γ ELISPOT assays, cells were thawed, rested, then plated on ELISPOT plates. Cells were stimulated with or without the target epitope for 24-48 h to ensure antigen presentation.
In vitro ELISPOT assays were performed with a cell density of 2.5 ×10⁵ cells/well. Ex vivo ELISPOTs were performed with cell densities of 9 ×10⁵ cells/well, for PBMCs, and 6.8×10⁵ cells/well, for bone marrow mononuclear cells. Plates were analyzed with the ImmunoSpot Series 2.0 Analyzer (CTL, Shaker Heights, Ohio). Results were generated by subtracting the background obtained with negative controls. A detailed description of our setup was described previously (32 (link)). Statistical significance of ELISPOT responses was analyzed by the DFR method (33 (link)). For non-triplicate samples, responses were evaluated empirically and defined as true if the number of spots observed in the peptide stimulated wells were at least double of the spot counts in the control wells.