Anti hla abc

Live cells (10⁵–10⁶) were suspended in 100 μL PBS, mixed with 3 μL fluorescein isothiocyanate (FITC)-conjugated antibody, and subsequently incubated in the dark at 25°C for 30 min. The antibodies used were anti-CD133/1-VioBright/FITC (Miltenyi Biotec), anti-HLA-ABC (BD Biosciences, Franklin Lakes, NJ, USA), anti-HLA-DR (BD Biosciences), anti-CD34 (BD Biosciences), anti-CD45 (BD Biosciences), anti-CD73 (BD Biosciences), and anti-CD105 (BD Biosciences). After 30 min, the cells were washed with 3 mL PBS and analyzed using the MACSQuant VYB (Miltenyi Biotec).

Cells were washed with fluorescence-associated cell sorting (FACS) buffer (BD Bioscience, 554656) and blocked with Fc blocker (BD Bioscience, 564220) for 30 min at room temperature. Cells were stained with fluorescence-conjugated primary antibodies for 30 min on ice, washed twice with FACS buffer, and fixed with 2% PFA for 15 min. For non-conjugated primary antibodies, the cells were incubated with primary antibodies overnight at 4°C and incubated with secondary antibodies for 60 min at room temperature. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter). Primary antibodies used were as follows: anti-CD107a (BD Biosciences, 560664), anti-IFNγ (BioLegend, 554701, San Diego, CA, USA), anti-NKG2A (BioLegend, 375103), anti-CD96 (BioLegend, 562379), anti-NKG2D (BD Biosciences, 557940), anti-CD16 (Invitrogen, MHCD1604), anti-ULBP-1 (R&D Systems, MAB1380), anti-ULBP-2/5/6 (R&D Systems, MAB1289), anti-ULBP-3 (R&D Systems, MAB1517), anti-MIC-A/B (Invitrogen, 12-5788-42), anti-HLA-ABC (BD Biosciences, 557349), anti-HAL-E (Invitrogen, 12-9953-41), anti-HLA-G (Invitrogen, 12-9957-41), and CXCR6 (BioLegend, 151103). For CD107a and IFNγ staining, cells were treated with brefeldin A (BioLegend, 420601) for 5 h before incubation with the primary antibody.