Pe cy7 anti il 2

For intracellular cytokine staining, 2 × 10⁶ splenocytes were stimulated in the presence of protein transport inhibitor, GolgiStop^TM GolgiPlug^TM (BD Bioscience) with the same peptide pools as the ELISpots. Media alone and phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulations (BD Bioscience) were used as negative and positive controls respectively. To test for degranulation of cells, FITC anti-CD107a (Biolegend) antibody was also added during stimulation. After 6 h, cells were washed and stained with LIVE/DEAD violet. Surface staining was then added containing BV510 anti-CD4, APC-Cy7 anti-CD8, BV711 anti-CD62L, and AF700 anti-CD44 (Biolegend). After 30 min incubation, cells were spun, washed, and fixed using the CytoPerm CytoWash kit following the manufacturer’s protocol (BD Bioscience). Intracellular staining was then prepared using APC anti-IFNγ, BV650 anti-TNFα, PE-Cy7 anti-IL-2, and PE-Cy5 anti-CD3 (Biolegend). All data were collected on a modified LSRII flow cytometer (BD Bioscience) with FACS DIVA 6.0 GUI followed by analysis with FlowJo software 9.0 (BD Bioscience).