Recombinant human igg1 fc protein

Expression of NKp44, NKp30, and NKp46 ligands was detected using recombinant human NKp44 Fc chimera protein, recombinant human NKp30 Fc chimera protein, or recombinant human NKp46 Fc chimera protein (R&D Systems, MN, USA) and stained with PE-conjugated F(ab’)2 fragment goat-anti-human IgG secondary antibodies (Jackson ImmunoResearch, PA, USA). Recombinant human IgG1 Fc protein (R&D Systems) was used as a control. Prior to surface staining, the adherent cells were detached from the bottom of the culture flasks using phosphate-buffered saline containing 0.5 mM ethylenediaminetetraacetic acid. Expression of NKG2D ligands was detected using PE-conjugated MICA/B monoclonal antibody (Biolegend, CA, USA), human ULBP-1 PE-conjugated antibody (R&D systems), human ULBP-2 PE-conjugated antibody (R&D Systems), and human ULBP3 antibody (R&D Systems), followed by staining with PE-conjugated F(ab’)2 fragment goat anti-mouse IgG secondary antibodies (Jackson ImmunoResearch). Purified mouse IgG2a, κ isotype antibody (BioLegend) was used as the control. Antibody staining was detected using a BD FACSCalibur flow cytometer (Becton, Dickinson and Company, NJ, USA).