Hla g clone 87g

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).

Cells were stained with mAbs to CD3 (clone UCHT1), CD69 (clone TPI-55-3) (Beckman-Coulter, Miami, FL), CD8 (clone HIT8a), CD161 (clone DX12), IL-10 (clone JES3-9D7), interferon (IFN)-γ (clone B27), tumor necrosis factor (TNF)-α (clone MAb11) (BD Pharmingen, San Diego, CA, USA), CD14 (clone TuK4), CD45 (clone H130) (Invitrogen, Carlsbad, CA), and HLA-G (clone 87G), CD85j (ILT2) (clone GHI/75), and TCRα7.2 (clone 3C10)(Biolegend, San Diego, CA). Polyclonal antibody to the Salmonella Common Structural Antigens (CSA) (KPL, Gaithersburg, MD, USA) was used to measure S. Typhi infection. Antibodies conjugated to the following fluorochromes were used in these studies: Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), PE-Cy5, Peridinin chlorophyll protein (PerCP)-Cy5.5, PE-Cy7, Pacific Blue, Brilliant Violet (BV) 570, BV605, BV650, Quantum dot (QD) 800, Alexa 647, allophycocyanin (APC)-Alexa 700, or APC-H7.