Anti cd16 apc h7

Staining and flow cytometric analysis was performed as described before [10 (link)]. The Following monoclonal antibodies were used in this study: Via probe PerCP, anti-CD19 PerCP, anti-CD14 PerCP and anti-CD3 PerCP (BD Biosciences, CA, USA) to exclude dead cells, B cells, monocytes and T cells, respectively, and anti-CD56 PC7 (Beckman Coulter, CA, USA) and anti-CD16 APC-H7 (BD Biosciences, CA, USA) to identify NK cells. Additional antibodies that were used include: anti-CXCR3 APC (BD Biosciences, CA, USA), anti-CD69 PE (Invitrogen, CA, USA), anti-CD127 PacBlue (BD Biosciences, CA, USA), anti-CD95 APC (Biolegend, CA, USA), anti-NKp30 APC (Beckman Coulter, CA, USA), anti-NKp46 PE (Beckman Coulter, CA, USA), anti-NKG2D APC (BD Biosciences, CA, USA). At least 1 million events were acquired for each sample, using the BD Canto II (BD Biosciences, CA, USA). Data were analysed with FlowJo 8.8.4 (TreeStar, Or, USA). Lymphocytes were defined by forward and side scatter. CD3⁺, CD14⁺, CD19⁺, dead cells and cell aggregates were removed from analysis based on PerCP and Viaprobe cell viability staining and pulse width analysis. Fluorescence minus one (FMO) staining was used to determine threshold values for the expression of each surface marker.

Flow cytometry of NK cells was performed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed in FlowJo 9.9 (TreeStar Inc., Ashland, OR). Staining of NK cells was performed with four panels, and the antibodies used were: anti-CD56-BV605, anti-NKG2D-APC, anti-CD107a-PE-Cy7, anti-GranzymeB-FITC, anti-perforin-APC, anti-NKp46-PE-Cy7, anti-NKp30-APC, anti-NKp44-PE, anti-2B4-FITC, anti-4-1BB-PerCP-Cy5.5, anti-CD95-BV421, anti-Tim3-BV421, anti-TRAIL-PE, anti-CD122-PerCP-Cy5.5, anti-CD122-BV510, anti-CD95L-PE, anti-Ki67-BV421, anti-CD40L-BV510, anti-PD-1-PE-Cy7; all were obtained from BioLegend (San Diego, CA). Anti-CD16-APC-H7, PD-L1-PE-Cy7, and CD11a-FITC, were obtained from BD Biosciences (San Jose, CA). Anti-NKG2A-PE was from R&D Systems (Minneapolis, MN), and anti-CD158a-PerCP-Cy5.5 from eBioscience.