Multiscreen hts ip 0.45 μm filter plates

ELISpot assays were carried out on Multiscreen HTS IP 0.45μm filter plates (Millipore Sigma). Briefly, the plates were coated with anti-IFNγ capture antibody (clone AN18, BioLegend) overnight at 4°C and blocked with RPMI-1640 supplemented with 20% FBS for 2 hours at 37°C. 500,000 splenocytes were then added, and the plates were incubated in the presence of 10 μg/ml of pooled peptides or 25 ng/ml of PMA and 0.5 μg/ml of Ionomycin in positive control wells, or 0.1% DMSO in negative control wells. In addition, for freeze-thawed splenocytes (Figure 7) 50 IU/ml of mouse IL-2 was added to the stimulation medium to enhance T cell survival. After incubating the plate for 44 hours at 37°C, the wells were washed to remove the cells. The wells were then incubated with biotinylated anti-IFNγ detection antibody (clone R4–6A2, BioLegend) for 2 hours at 37°C followed by streptavidin-HRP for 1 hour at room temperature. IFNγ spots were developed using 3-Amino-9-ethylcarbazole chromogen (Sigma) and images were captured by the ELISpot Reader (AID GmbH, Germany). Automatic spot counts were obtained using the AID ELISpot Reader software.