Anti cd107a bv421

Clone cells were washed and left in fresh RPMI 1640 CM (5% AB serum) without IL-2 to rest for 5 hours or overnight before being stimulated with RL9 peptide pulsed K562-E (50μM, 20-24 hours at 27°C), K562-E-RL9 or K562-D-RL9 cells at a clone: K562 cell ratio of 1:3 for 1 hour, followed by addition of 5μg/ml Brefeldin A (Biolegend) and 5μg/ml GolgiStop (BD Biosciences) for an additional 8 hours at 37°C. For CD107 staining, anti-CD107a-BV421 and anti-CD107b-BV421 (Biolegend) antibodies were added at the beginning of the co-culture. After 9 hours incubation, cells were washed with PBS and stained with Live/Dead Fixable Aqua, anti-CD8-PerCP-Cy5.5 and anti-CD3-APC-Cy7 for 30 min at RT first, then fixed/permeabilized with Cytofix/Cytoperm 1x Solution (BD Biosciences) for 10 min at 4°C, and stained in Permwash 1x Solution (BD Biosciences) with anti-TNFα-PE, anti-IFN-γ–FITC and anti-CD137-BV650 (All BioLegend) for 30 min at RT. After being washed with PBS and fixed with 2% paraformaldehyde, samples were acquired using an LSR Fortessa (BD Biosciences) and analyzed using FlowJo software v10.3 (Tree Star). In the selected assays to determine HLA-E restriction, K562-E cells were pre-incubated with the VL9 canonical signal peptide (VMAPRTLVL, 50μM, 3 hours at 27°C) prior to addition of RL9 peptide.