Staining of human leukemic cell lines for the HLA-E receptor and the acid-washing protocol for loading various peptides. (A) Staining of wild-type (WT) 721.221 and 721.221 HLA-G with mAb 3D12 (green panel) and 4D7 (blue panel). 0.2 × 10⁶ cells per well were incubated with the mAb PE-conjugated mouse anti-human HLA-E (3D12) and APC-conjugated mouse anti-human HLA-E (4D7) or a matched isotype control. (B) Schematic representation of the acid wash protocol, followed by flow cytometry determination of surface expression levels of HLA-E loaded with different nonapeptides. (C) HLA-E staining of acid-washed and peptide-loaded 721.221 HLA-G cells. 721.221 HLA-G cells were treated briefly with an acid buffer (citric acid in Na₂HPO₄ buffer, pH 5). Thirty-two different exogenous peptides, 20 μg/mL, with single amino acid mutations and the WT peptide were loaded on peptide-stripped 721.221 HLA-G cells. The treated cells were then stained with mAb 3D12 and mAb 4D7 or matched isotype control. The results were normalized to staining with commercial antibody to HLA-E. (D) Staining of U266, U937, and RPMI8226 cell lines with mAb 3D12 (green panel) and mAb 4D7 (blue panel). Murine IgG1_k was used as an isotype control. For staining, all the antibodies were used at a concentration of 5 μg/mL. Samples were acquired by a CytoFLEX flow cytometer, and histograms were plotted using Kaluza software. PE, fluorescent dye R-phycoerythrin.