Anti cd56 percp cy5

The cetuximab (Bristol-Myers Squibb, New York, NY), trastuzumab (Genentech, San Francisco, CA), pertuzumab (Genentech) and isotype (rituximab, Genentech) antibodies were purchased from the National Institutes of Health Pharmacy. Flow cytometry of haNK cells was performed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed in FlowJo 9.9 (TreeStar Inc., Ashland, OR). Flow cytometry of tumor cells was performed on a BD FACSVerse flow cytometer (BD Biosciences) and analyzed in BD FACSuite (BD Biosciences). Staining of haNK cells was performed with four panels, and the antibodies used were: Anti-CD56-PErCP-Cy5.5, anti-CD16-APC-Cy7, anti-NKG2D-APC, anti-NKG2D-FITC, anti-CD107a-PE-Cy7, anti-GranzymeB-FITC, anti-perforin-APC, anti-NKp46-PE-Cy7, anti-NKp30-APC, anti-NKp44-PE, anti-2B4-FITC, anti-CD158a-PE, anti-CD158d-PE, anti-CD158j-APC, anti-PD-1-PE-Cy7, anti-PD-L1-APC, and anti-MICA-PE; all were obtained from BioLegend (San Diego, CA). Anti-CD56-PE, anti-CD16-PE-Cy7, CD11a-PE, Ki67-FITC, anti-HLA-ABC-FITC and anti-4-1BB-BV421 were obtained from BD Biosciences. In controlled experiments to detect CD16 on haNK cells, the anti-CD16 MAb was CD16-FITC (BD Biosciences) and the isotype control antibody was MIgG1k-FITC (BD Biosciences).

Cells were stained for surface markers for 20 min at room temperature in PBS (Sigma). Extracellular antibodies used were as follows: LIVE/DEAD Fixable Near-IR (Thermo Fischer Scientific; L10119), Anti-CD3-BUV737 (BD Biosciences; 612750), Anti-CD14-BUV737 (BD Biosciences; 612763); Anti-CD14-BUV395 (BD Biosciences; 563561); Anti-CD19-BUV737 (BD Biosciences; 564303), Anti-CD19-BUV395 (BD Biosciences; 563549); Anti-CD56-PerCP-Cy5.5 (Biolegend; 304626), Anti-CD16-Pe-Cy7 (Biolegend; 302016), Anti-CD107a-BV510 (Biolegend; 328632), Anti-CD69-BV650 (Biolegend; 310934). Samples were then washed and fixed using 4% formaldehyde or BD Cytofix/Cytoperm Kit (BD biosciences) according to manufacturer’s directions. Flow cytometry analysis was performed on a LSRII instrument (BD Biosciences). A total of 50,000 to 250,000 events were acquired and analyzed using FlowJo software. The analysis was performed on gated cells that fell within the lymphocyte population, stained as live cells using Live/Dead stain. Cells were then gated for negative CD3/CD14/CD19 expression to exclude other cell populations, and NK cells were identified using CD56/CD16 gating strategy. Gating strategies are shown in Supplementary Figure S1. Within the NK cell population, we analyzed expression of CD107a and CD69 for each sample compared to their corresponding unstimulated control.

The isolated mononuclear lymphocytes from both the decidua and peripheral blood were directly stained for sorting. For FACS sorting, they were incubated with conjugated mouse anti⁻human antibodies, including anti-CD3 FITC (BioLegend, UK), anti-CD56 PerCp/Cy5.5 (BioLegend, UK), anti-CD4 BV421 (BD Biosciences, U.S.A.), and anti-CD8a PE/Cy7 (BioLegend, UK), for 30 min at 4 °C. After incubation, they were treated with LIVE/DEAD^® Fixable Aqua Dead Cell Stain (Invitrogen Life Technologies, U.S.A.) for 10 min. CD3⁺CD56⁻CD4⁻CD8⁺ cells were sorted on a BD FACS Aria-II machine to obtain a purity > 95%. For coculturing with either DSCs or trophoblast cells, CD8⁺T cells from the decidua and PBMC via magnetic affinity cell were sorted using CD8 MicroBeads (MiltenyiBiotec, Germany), according to the manufacturer’s instructions.