Percp cy 5.5 conjugated anti cd56

For flow cytometry analysis, cells were cultured mono/co-culture spheroids, and then collected and trypsinized to obtain a single-cell suspension. Briefly, cells were washed in FACS buffer (PBS supplemented with 2% FBS) and, stained according to the manufacturer’s instructions in 100 µL FACS buffer supplemented with APC-conjugated anti-EPCAM (1:50, BD Biosciences, San Jose, CA, USA), PerCP-Cy 5.5-conjugated anti-CD56 (1:100, BDBiosciences, USA), PE-conjugated anti-CD107a (1:100, BD, Biosciences, USA) and APC conjugated anti α-SMA (1:2000, R&D Biotechne, Minneapolis, MN, USA). Cells were kept in the dark at RT for 20 min. Live/Dead cell analysis, Annexin-V-FITC (1:100, Cell Signaling Technology, Danvers, MA, USA) and Propidium Iodide-PE (1:10, Cell Signaling Technology, USA) were used. Stained cells were washed twice in DPBS to remove unbound antibodies, resuspended in 200 µL FACS buffer and analyzed in BD FACSCalibur Flow Cytometer (BD Biosciences). An unstained negative control was run to establish the fluorescence gates. First, the cells were gated in an FSC-A (forward scatter) and SSC-A (side scatter) dot plot to eliminate doublets. EPCAM (+) cells were chosen to analyze dead/live ratio in cancer cells. In order to identify CD107a (+) NK-92 cells, the CD56 (+) cell population was gated. All data were then analyzed using FlowJo X software Version 9.

To evaluate, by multicolor flow cytometry, the expression of NKG2C, NKG2A, CD57, NKG2D, DNAM-1, KIRs, and PD-1 on NK cells, thawed PBMCs were stained with the LIVE/DEAD™ Fixable Near-IR Dead Cell stain dye (from Invitrogen, Waltham, MA, USA) for 15 min at room temperature (RT). Before fixing for 20 min at RT with 1% PFA, cells were stained for 20 min at 4 °C with PerCP-Cy5.5-conjugated anti-CD56, BV510-conjugated anti-CD16, Alexa Fluor 700-conjugated anti-CD3, BV605-conjugated anti-CD14, BV650-conjugated anti-CD19, PE-CF594-conjugated anti-NKG2D, BV786-conjugated anti-DNAM-1, BV421-conjugated anti-PD-1 (all from BD Biosciences, San Jose, CA, USA), PE-conjugated anti-NKG2C, FITC-conjugated anti-NKG2A (both from Miltenyi Biotec, Bergisch Gladbach, DE), PE-Cy7-conjugated anti-CD57 (from Life Technologies, Carlsbad, CA, USA), APC-conjugated anti-CD158 (KIR2DL1/S1/S3/S5), and anti-CD158b/j (KIR2DL2/L3) (both from BioLegend, San Diego, CA, USA) mAbs. Stained samples were acquired at CytoFLEX Flow Cytometer from Beckman Coulter Life Sciences and all results were analyzed using FlowJo version X.0.7 software.