Anti pd 1 percp cy5.5 eh12 2h7

A total of 1 x 10⁶ PBMCs were stained with LIVE/Dead fixable violet stain (Invitrogen) for 15 minutes at room temperature (RT), followed by a wash step with phosphate-buffered saline (PBS). Cells were then re-suspended in 50 μL of human serum and incubated with PE-conjugated HLA class II tetramer for 1 h at 37°C and 5% CO₂. After three washes with staining buffer (1xPBS supplemented with 0.5% BSA and 2 mM EDTA), the cells were co-stained for 15 minutes at 4°C with saturating amounts of different combinations of the following antibodies: anti-CD3 AmCyan (SK7, BD Biosciences), anti-CD4 PerCPCy5.5 (RPA-T4, eBioscience), anti-CD4 PE-CF594 (RPA-T4, BD Biosciences), anti-CD4 APC-Cy7 (RPA-T4, Biolegend), anti-CD14 Pacific Blue (HCD14, Biolegend), anti-CD19 eFluor450 (H1B19, eBioscience), anti-CD25 APC-Cy7 (BC96, Biolegend), anti-CD27 APC-eFluor780 (O323, eBioscience), anti-CD28 PE-Cy7 (28.2, Biolegend), anti-CD45RA AF700 (H1 100, Biolegend), anti-CD57 APC (HCD56, Biolegend), anti-CD69 PE-Cy7 (FN50, Biolegend), anti-CD127 PerCP-Cy5.5 (HIL-7R-M21, BD), anti-CCR7 FITC (150503, R&D), anti-CX3CR1 PerCP-Cy5.5 (2A9-1, Biolegend), anti-FasL AF488 (14C2, AbD Serotec), anti-NKG2D PE-CF594 (1D11, BD Biosciences), anti-PD-1 PerCP-Cy5.5 (EH12.2H7, Biolegend) and anti-Tim3 APC (F38-2E2, eBioscience). Following a final wash cells were re-suspended in 200 μL of staining buffer for acquisition.