Anti cd107a texasred antibody

To test the effect of specific peptides, T2 cells were incubated in serum-free RPMI media overnight at 26°C in the presence of 100μM synthetic peptide (Genscript). Cells were washed and stained with the HLA-C specific DT9 antibody (EMD Millipore). Peptide stabilization was assessed by flow cytometry on an Accuri C6 benchtop flow cytometer (BD Biosciences).
Natural killer cells were isolated from five healthy KIR A haplotype and HLA-C1 homozygous donors and stimulated overnight with 500 units/ml recombinant human IL-2 (R&D Systems) in RPMI 10% complete media. T2 cells (3 × 10⁴) were incubated with 100μM peptide (Genscript) overnight in serum-free media at 26°C, washed, and re-suspended in serum-free media with anti-CD107a-TexasRed antibody (eBioscience). T2 cells were incubated with NK cells at a target: effector ratio of 10:1 for 3.5 hours in serum-free media with 500 units/ml recombinant human IL-2. Surface staining was performed using anti-KIR2DL2/3-APC GL183 (Beckman Coulter) and anti-KIR3DL1-FITC DX9 (Biolegend) to identify KIR2DL3+/KIR3DL1- NK cells. Following surface-staining, cells were treated with live/dead orange (Life Sciences) and fixed (Cytofix, BD Biosciences). Cells were analyzed on an LSR II cytometer (BD Biosciences) at the Stanford Flow Cytometry Core Facility. A representative gating strategy is shown in Figure S5B.