Hla abc clone w6 32

To phenotype cervical cancer cell lines, cell suspensions in PBS supplemented with 0.1 % BSA and 0.02 % NaN₃ (FACS buffer) were stained for 30 min at 4 °C using antibodies to HLA-ABC (clone w6/32, Immunotools) (labeled with FITC), HLA-E (clone 3D12HLA-E, eBioscience), HLA-G (clone 87G, Biolegend), EGFR (clone EGFR.1, BD Biosciences), PVR (clone SK11.4, Biolegend), MICA/B (clone 6D4, Biolegend), ULBP2/5/6 (clone #165903, R&D systems), ULBP1 (clone #170818, R&D systems) and ULBP3 (clone #166510, R&D systems) (all labeled with PE). IgG₁, IgG_2a and IgG_2b isotype antibodies were used as negative controls. After incubation, the cells were washed with FACS buffer and analyzed using a flow cytometer LSR Fortessa (BD Biosciences). Phenotypic analyses were obtained from at least two independent experiments performed on each cell line. Data were analyzed using Kaluza software (Beckman coulter) and calculated as specific (geometric) mean fluorescence intensity (MFI) (MFI; geometric mean fluorescence of marker − geometric mean fluorescence of isotype).

Fluorescent-conjugated mAbs used: CD14 (clone OFC14D), CD80 (clone MEM-233), CD86 (clone BU63), and HLA-ABC (clone W6/32) from Immunotools (Friesoythe, Germany); CD163 (clone GHI/61), VISTA (clone B7H5DS8) and HLA-A2 (clone BB7.2) from eBioscience (San Diego, CA, USA); CD206 (clone 15 − 2), CD11b (clone M1/70), SIRPα (clone 15–414), PDL-1 (clone MIH3), TIM-3 (clone F38-2E2), and SIGLEC-10 (clone 5G6) from BioLegend. Unconjugated mAbs used were VISTA (clone ABM5C53) from MyBioSource (San Diego, CA, USA) and HLA-ABC (clone W6/32) from BioLegend, and Rituximab (RTX, human IgG1 chimeric anti-CD20, Roche). The reagents used were: Incucyte® Cytolight Rapid Red Dye for Live-Cell Cytoplasmic Labeling (Cytolight Red) (Sartorius, Göttingen, Germany), Phorbol-12-myristate-13-acetate (PMA) (Thermo Scientific, London, UK), Lipopolysaccharide (LPS) (Merck, MO, USA), TGF-β (Peprotech, Thermo Scientific, London, UK), as well as Interleukin 4 (IL-4), Interleukin 10 (IL-10), Interleukin 13 (IL-13), interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage-colony-stimulating factor (M-CSF) from Immunotools. Secretion of cytokines by macrophages was measured using appropriate ELISA kits (Immunotools).