Cd57 pe

The following mouse monoclonal antibodies were used: CD56-APC (clone N901, Beckman Coulter, Miami, FL, USA), CD56-Brilliant Violet 421 (clone HCD56, Sony Biotechnology, San Jose, CA, USA), CD56-PE (clone N901 (HLDA6), Beckman Coulter, Miami, FL, USA), CD57-PE (clone TB01, eBioscience, San Diego, CA, USA), CD57-FITC (clone TB03, Miltenyi Biotec, Bergisch Gladbach, Germany), CD57-APC (clone TB03, Miltenyi Biotec, Bergisch Gladbach, Germany), CD16-PE (Sorbent, RF), CD2-PE-Cy7 (clone TS1/8, Sony Biotechnology, San Jose, CA, USA), anti-NKG2A-PE (clone 131411, R&D Systems, Minneapolis, MN, USA), anti-KIR2DL2/DL3-PE (clone DX27, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-HLA-DR-FITC (clone B8.12.2, Beckman Coulter, Miami, FL, CA, USA), NKG2A-PE (clone 131411), NKG2C-AlexaFluor488 (clone 108724), NKG2C-PE (clone 134591; R&D Systems, Minneapolis, MN, USA), NKp46-FITC (clone 9E2; Sony Biotechnology, San Jose, CA, USA), anti-NKG2D-PE (clone REA1175, Miltenyi Biotec, Bergisch Gladbach, Germany). Staining was performed according to the manufacturer’s instructions.

The following anti-human monoclonal antibodies (mAbs) were used for flow cytometry: CD3- PE-Cy7 (Beckman Coulter, USA, clone UCHT1), CD56-APC (Beckman Coulter, USA, clone N901), CD56-Brilliant Violet 421 (Sony, USA, clone HCD56), CD56-PE (Beckman Coulter, USA, clone N901 (HLDA6)), CD57-PE (eBioscience, USA, clone TB01), CD57-FITC (Miltenyi Biotech, Germany, clone TB03), CD57-APC (Miltenyi Biotech, Germany, TB03), CD16-PE (Sony, USA) anti-NKG2A-PE (R&D Systems, USA, clone 131411), anti-KIR2DL2/DL3-PE (Miltenyi Biotech, Germany, clone DX27), anti-HLA-DR-FITC (Beckman Coulter, USA, clone B8.12.2). Cells were incubated in PBS containing 0.5% BSA (bovine serum albumin) and 0.01% sodium azide (labeling buffer) with mAbs for 30 min on ice, then washed twice with labeling buffer and centrifugation. Samples were subsequently analyzed using FACSCalibur flow cytometer (BD Biosciences, San Jose CA, USA) equipped with 488 and 640 nm lasers. At least 30000 events were recorded in lymphocyte gate for total NK cell population and 5000 events for NK cell clones. Acquired data was analyzed using Flowing Software version 2.5.1 (PerttuTerho, Turku Centre for Biotechnology, Finland) and FlowJo software version 7.6 (FlowJo LLC, Ashland, OR, USA). For fluorescence-activated cell sorting, cells were labeled with mAbs in PBS containing 0.5% BSA and 2 mM EDTA.