Fluorescence activated cell sorting aria 2

For intracellular cytokine staining, cells were restimulated with phorbol myristate acetate and ionomycin in the presence of brefeldin A (all from Sigma-Aldrich) for the final 4 hours of culture. Cells were fixed and permeabilized with FIX and PERM (Thermo Fisher Scientific). Cells were stained with a Live/Dead fixable violet dead cell stain kit (Thermo Fisher Scientific) and fluorescent antibodies. Cells were acquired in a Fluorescence-Activated Cell Sorting Canto II (BD Biosciences) and analyzed with BD Fluorescence-Activated Cell Sorting Diva Software v6.1.1.
Cytokine secretion by PBMCs was measured by an enzymelinked immunosorbent assay or using the multiplex cytokine assay Milliplex Human Th17 magnetic bead (EMD Millipore Corporation, Billerica, MA).
Cell Sorting of Antigen-Specific CD4 þ T Cells and RNA Isolation CFSE-labeled PBMCs were cultured in the presence of TT, FlaX, A4-fla2, or YidX antigens. Recombinant interleukin (IL)2 (20 IU/mL) was added to the culture on day 7. Viable CFSE - CD4 þ cells were sorted on day 14 of culture in a Fluorescence-Activated Cell Sorting Aria II (BD Biosciences) and restimulated with antigen in the presence of autologous irradiated PBMCs. Ten days later RNA was extracted.