Ctl immunospot s6 macro analyzer

IFNγ ELISpot assays were performed as described.^{41 (link)} Briefly, 96-well ELISpot plates (BD ELISPOT Mouse IFNγ ELISPOT Set, BD Biosciences, San Jose, CA) were coated with an IFNγ capture antibody (BD Biosciences) in PBS overnight (ON) at 4°C, and then plates were blocked with complete medium for 2 h at RT. Peptide or a synTac of known specificity (HPV E7 or IAV NP) was then added to wells, followed by the addition of 2.5 × 10⁵ splenocytes (whole splenocytes to detect antigen-specific CD8 T cells is standard for optimized ELISpot assays),^{42 (link)} 2×10⁴ tumor cells, or 2×10⁴ lung cells depending on the experiment. Positive control wells for all experiments contained 1X Cell Stimulation Cocktail (Invitrogen) prior to the addition of splenocytes or tumor cells, and negative control wells contained media only (Supplemental Figure 10). Plates were then washed and incubated with a biotinylated IFNγ detection antibody (BD Biosciences) for 2 h, followed by SA-HRP (BD Biosciences) for 1 h at RT. The plates were developed with 3-amino-9-ethyl-carbazole substrate (BD ELISPOT AEC Substrate Set) for 5 min and dried for at least 24 h. Spots were enumerated using the CTL ImmunoSpot S6 MACRO Analyzer.