Flow Cytometry and Intracellular Cytokine Staining

Single-cell suspensions of spleen, mLNs, and colon lamina propria cells were surface stained ex vivo with fluorescent antibodies to T-cell markers (CD3, CD4, CD62L, CD44, CD69, CD103, CD45.1, and CD45.2) and for PIRA/B (6C1). For cytokine staining, cells were ex vivo stimulated at 37°C for 4 hours with phorbol 12-myristate 13-acetate (PMA) (50 ng/mL), ionomycin (1 mg/mL), and Brefeldin A. Cells then were processed and stained using an intracellular cytokine staining kit (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer’s instructions with IFNγ and IL17a. For apoptosis staining, the dead cell apoptosis kit and live/dead viability assay (Thermo Fisher) was followed according to the manufacturer’s instructions. For activated caspase 3/7 staining, the caspase 3/7 kit (Thermo Fisher) was followed according to the manufacturer’s instructions. To assess cell cycling and entry into G1, Pyronin Y (Sigma Aldrich) staining was performed as previously described.82 To assess cell cycling and entry into S phase, the Edu flow cytometry kit (Sigma) was followed according to the manufacturer’s instructions. All flow cytometry samples were acquired on an Novocyte (ACEA Biosciences) and data were analyzed using FlowJo (Tree Star, San Carlos, CA) and Prism (GraphPad Software). Absolute cell numbers were calculated using Precision Count Beads (BioLegend) according to the manufacturer’s instructions.