Anti human cd45 hi30

The following antibodies were used in this study: anti-B7-H3 (FM276, Miltenyi Biotech), anti-GD2 (14.G2a, BD Biosciences), human Ig (polyclonal, Thermo Fisher Scientific), anti-mouse IgG (polyclonal, R&D), anti-CD3 (UCHT1, BioLegend), anti-HisTag (J095G45, BioLegend), anti-CD34 (QBEnd10, R&D), anti-ab-TCR (IP26, BioLegend), anti-CD107a (H4A3, BioLegend), anti-cD25 (BC96, BioLegend), anti-CD69 (FN50, BioLegend), anti-Tim3 (F38-2E2, BioLegend), anti-Lag3 (11C3C65, BioLegend), anti-PD-1 (EH12.1, BD Biosciences), anti-mouse CD45 (30-F11, BioLegend), anti-human CD45 (HI30, BioLegend), Ghost Red 780 (Tonbo Biosciences), Zombie Yellow Viability Dye (BioLegend), propidium iodide (Gibco), Cell Trace Violet (Thermo Fisher Scientific), and Precision Count Beads (BioLegend).

Live immune cells from peripheral blood, spleen, and BM were determined by staining with live/dead fixable blue dead cell stain kit (Life Technologies) for 30 min prior to cell-specific marker labeling. Cells were labeled with anti-human CD34 (clone 581; BD Biosciences), anti-human CD3 (UCHT1; Biolegend), anti-human CD56 (MEM-188, Biolegend), anti-human CD14 (63D3; Biolegend), anti-human CD19 (SJ25C1; BD Biosciences), anti-human CD117 (104D2; Biolegend), anti-human CD38 (HB-7; Biolegend), anti-human CD33 (WM53; BD Biosciences), mouse CD45.1 (A20; BD Biosciences), anti-human CD8 (SK1; Biolegend), anti-human CD4 (SK3; BD Biosciences), and anti-human CD45 (HI30; Biolegend) monoclonal antibodies for 30 min at room temperature. After incubation, cells were washed and resuspended in FACs buffer containing phosphate buffered saline (PBS), 0.2% bovine serum albumin (GE Healthcare Life Sciences), and 0.05% sodium azide (Merck) for flow cytometry data acquisition. Data was acquired using a LSR II flow cytometer (BD Biosciences) with FACSDiva software, and analysis was performed using FlowJo software (version 10; Tree Star Inc). Absolute count of cells in peripheral blood was determined using CountBright™ Absolute Counting Beads (Thermo Fisher Scientific).