ELISpot assays were performed as previously described [39]. A single-cell suspension was created by grinding a whole spleen through a 70 μm cell strainer with the rubber end of a 3 mL syringe plunger and rinsing with 15 ml of RPMI with 10% FBS and 1% PSG (RPMI complete). Cells were pelleted at 650 x g for 10 minutes and red blood cells were lysed with 1x Red Blood Cell Lysis Buffer (Biolegend) for 3 minutes, after which 10 ml of complete RPMI was added and cells were pelleted. Cells were resuspended in 3 ml RPMI complete medium and counted. Splenocytes, 2.5x105 cells per well, were added to Mouse IFN-γ ELISpot plates (MabTech) in addition to 20 μl of peptide (2 μg/well), 2 μl of DMSO, or 2 μl of a phorbol 12-myristate 13-acetate/ionomycin stock at a 1:300 dilution as a positive control. 18mer peptides corresponding to predicted H2b epitopes present in the MAYV structural polypeptide were ordered from Thermo Scientific. Plates were incubated for 48 h, washed and incubated with anti-mouse IFN-γ biotin antibody for 2 h. Plates were again washed and incubated with streptavidin-ALP antibody for 1 h following the manufacturers protocol. Spots were visualized using BCIP/NPT-plus substrate, after which the plates were washed and dried prior to counting with the aid of the AID EliSpot Reader Classic.

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