Anti cd56 pe cy7 clone b159

Changes in TIGIT expression levels on CD226⁺ NK cells were detected by flow cytometry. For staining, PBMCs were seeded in 96-well round-bottom plates (50,000–60,000 per well) and stimulated with IL-10 (10 ng/mL; R&D Systems), IL-12 + IL-15 (10 ng/mL and 50 ng/mL, respectively), or transforming growth factor-beta (TGF-β) (50 ng/mL; R&D Systems) respectively. Unstimulated cells were used as negative controls. Cells were incubated in RPMI media containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin/streptomycin (PS) for 24 h at 37°C with 5% CO₂ in a total volume of 200 μL, then harvested, washed, and stained with anti-CD3-PerCP (clone SK7; BD Biosciences), anti-CD56-PE-Cy7 (clone B159; BD Biosciences), anti-CD16-APC-Cy7 (clone 3G8; BD Biosciences), and anti-TIGIT-APC (clone A15153G; Biolegend) in staining buffer on ice in the dark for 20 min. APC mouse IgG1, κ isotype control (clone MOPC-21, Biolegend) was used to define gates. Cells were then washed twice with PBS containing 2% FBS, re-suspended in staining buffer, and analyzed using a BD FACS Canto II cytometer (BD Biosciences).

PBMCs were stained with anti-CD3 Pacific Blue (clone UCHT1, BD Biosciences), anti-CD56 PE-Cy7 (clone B159, BD Biosciences) for 30 min at room temperature. CD56+ T cells, CD56+ NK cells and CD56− T cells were sorted by BD FACS AriaIII (BD Biosciences, San Jose, CA) and only cells with purity >95% were used in subsequent experiments.

Lymphocytes present in PBMC (BC group) and ascites (ASC and ASC-CA groups) were phenotyped for the identification of their subsets. A flow cytometric-based assay was used according to standard procedures [37 (link)]. Briefly, the cells were mixed with 50 µL of staining solution containing a mix of fluorochrome-conjugated monoclonal antibodies at a 1:50 dilution; anti-CD3 APC-Cy7 (clone SK7), anti-CD4 PerCP-Cy5.5 (clone RPA-T4), anti-CD25 PE (clone M-A251), anti-CD56 PE-Cy7 (clone B159), anti-CD127 Alexa Fluor647 (clone HIL-7R-M21) (BD Pharmingen™), and anti-CD8 FITC (clone OKT8) (Miltenyi Biotec). Cells were incubated for 30 min on ice and protected from light. After the incubation, cells were washed twice with PBS and the final pellets suspended for acquisition in a FACSVerse cytometer using the FACSuite software (Becton Dickinson, San Jose, CA, USA). FlowJo software was used for the data analysis. The lymphocyte population was identified by the FSC and SSC parameters, and then FSC-Area vs. FSC-Height was used to eliminate doublets. Within the lymphocyte populations, the CD3⁺ lymphocyte population was identified by anti-CD3 APC-Cy7. Within the CD3⁺ lymphocytes, CD4⁺ and CD8⁺ populations were distinguished. Within the CD4⁺ population, the T-reg population was quantified by the parameters anti-CD25 PE vs. anti-CD127 Alexa Fluor647.