Anti nkg2d pe cf594

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.

The following antibodies for flow cytometry were used: anti-CD107a-APC (H4A3), anti-CD3-AF700 (UCHT1), anti-CD3-PE-CF594 (UCHT1), anti-CD56-PE-Cy7 (B159), anti-CD56-PerCP Cy5.5 (B159), anti-CD57-PE (NK-1), anti-CD45-PE-Cy5 (HI30), anti-NKG2D-BV605 (1D11), anti-NKG2D-PE-CF594 (1D11), anti-CD16-BV510 (3G8), anti-DNAM-1-BV786 (DX11), anti-PD-1-BV421 (MIH4), anti-IFNγ-BV650 (4S.B3), anti-CD14-BV605 (M5E2), anti-CD19-BV650 (SJ25C1) purchased from BD Biosciences; anti-DNAM-1-APC (11A8), anti-NKp46-PE-Cy7 (9E2), anti-TNFα-AF700 (Mab11), anti-CD96-APC (NK92.39) purchased from Biolegend; anti-NKp30-PE (Z25), anti-KIR2DL1/2DS1-PE Cy5.5 (EB6B), anti-KIR2DL2/L3/S2-PE (GL-183) purchased from Beckman Coulter; anti-NKG2A-AF700 (131411), anti-KIR3DL1-APC (DX9) purchased from R&D Systems; anti-NKG2A-FITC (REA110), anti-NKG2C-PE (REA205) purchased from Miltenyi; anti-TIGIT-APC (MBSA43) purchased from eBioscience. All these antibodies were used according to the manufacturers’ protocol. Prior to surface staining, NK cells were pre-stained with Live/Dead™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen). Flow cytometry was performed by using FACSCantoTM II (BD Biosciences) or Cytoflex (Beckman Coulter) and analyzed by FlowJo Software.