Anti cd3 alexafluor700 ucht1

When sufficient cells were available, freshly isolated matched BAL lymphocytes and PBMCs were simultaneously stimulated using PMA (25 ng/mL) and ionomycin (500 ng/mL) for 6 hours at 37°C in 96-well microplates. After 6 hours, plates were washed in phosphate buffered saline (PBS) before cells were stained with 1:500 of either MR1 6-FP (PE) or MR1 5-OP-RU (PE) tetramer (NIH Tetramer Core Facility). Tetramer staining was performed in the dark at room temperature for 40 minutes, followed by surface staining at 4°C for 20 minutes. The surface staining antibody master-mix included: 1:25 of anti-CD4 BV711 (OKT4, BioLegend), 1:100 of anti-CD8 APC-H7 (SK1, BD Biosciences), 1:50 of anti-CD26 FITC (BA5b, BioLegend), 1:50 of anti-CD161 PE-Cy7 (HP-3G10, BioLegend) and 1:800 of a fixable viability dye (Live/Dead Aqua, Invitrogen). Cells were then fixed (Fix & Perm Medium A, Invitrogen) at room temperature in the dark for 15 minutes and permeabilized (Fix & Perm Medium B, Invitrogen) for 20 minutes at 4°C. Intracellular antibodies were added during permeabilization and included 1:50 each of: anti-CD3 AlexaFluor700 (UCHT1, BioLegend), anti-IFN-γ PE-Dazzle594 (4S.B3, BioLegend), anti-granzyme B AlexaFluor647 (GB11, Biolegend), and anti-IL-17 BV421 (BL168, BioLegend). Cells were PBS washed, acquired using the LSRFortessa™ (BD Biosciences) and the data then analyzed using FlowJo (v10.4).