Pe conjugated anti nkg2a

Frozen PBMCs were thawed, washed and stained with the following combination of monoclonal antibodies, Pacific Blue-conjugated anti-CD3 (Biolegend), PE-Cy7-conjugated anti-CD56 (BD pharmingen), APC-Cy7-conjugated anti-CD16, PE-conjugated anti-NKG2A (Beckman Coulter), APC-conjugated anti-CD158a/h (Biolegend), APC-conjugated anti-CD158b1/b2/j (Biolegend), APC-conjugated anti-KIR3DL1 (Biolegend), Alexa Fluor 488-conjugated anti-NKG2C (R&D Systems), APC-Cy7-conjugated anti-CD27 (BD pharmingen), and PerCP-Cy5.5 conjugated anti-CD57 (Biolegend) monoclonal antibody. LIVE/DEAD Fixable Aqua was used for dead cell exclusion in every phenotypic analysis. The stained cells were then washed, fixed and permeabilized by BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) according to the manufacturer's protocol. Subsequently, cells were washed and stained intracellularly with FITC-conjugated anti-Ki67 (BD Pharmingen) for 30 min at 4°C. Cells were washed and re-suspended in PBS prior to flow cytometric analysis. Cells were acquired on FACS LSR-II and LSR Fortessa (BD Biosciences) and FACS data was analyzed by FlowJo v10 (BD). Samples with <20% live lymphocytes were excluded from the analysis.

After incubation for 6 h, cells were stained with the following combination of monoclonal antibodies, FITC-conjugated anti-CD3 (Biolegend), PE-Cy7-conjugated anti-CD56 (BD pharmingen), PE-conjugated anti-NKG2A (Beckman Coulter), APC-conjugated anti-CD158a/h (Biolegend), APC-conjugated anti-CD158b1/b2/j (Biolegend), and APC-conjugated anti-KIR3DL1 (Biolegend) monoclonal antibody for surface marker staining. LIVE/DEAD Fixable Aqua (Invitrogen) was stained for dead cell exclusion in each sample. The stained cells were subsequently washed, fixed, and permeabilized by BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) according to the manufacturer's protocol. Cells were washed and re-suspended in PBS prior to flow cytometric analysis. Cells were acquired on FACS LSR-II flow cytometer (BD Biosciences) and FACS data were analyzed by FlowJo v10 (BD). Samples with <20% live lymphocytes were excluded from the analysis. The spontaneous degranulation signals from PBMCs only were subtracted in each analysis.