8F4 mediated CDC of both AML blasts and AML stem cells. AML or ND bone marrow cells were incubated with no rabbit complement (C′), C′ only, isotype control antibody, or 8F4 (2.5 μg/mL) for 1 hour at 37°C. Cells were then washed, labeled with lineage-specific mAbs (Lin cocktail was CD3, CD4, CD7, CD8, CD10, CD14, CD16, CD19, and CD20), CD38, CD34, and Live/Dead Fixable Aqua for 30 minutes on ice, and resuspended in 200 μL of 1% PFA in PBS. Before analysis, 50 μL of Caltag Counting Beads was added to each sample for single-platform determination of absolute cell numbers. (A) Representative flow cytometric plots showing scatter distribution and counting beads (top panels) and phenotypes (bottom panels) of AML2 from CDC assay. Beads (FSC^lo/SSC^hi gate) were counted, and debris was excluded using the FSC^hi/SSC^lo gate. Gates for viable and Lin⁻ cells are not shown. LSCs were identified as viable Lin⁻CD34⁺CD38⁻ cells, as shown in the bottom panels. The cytotoxicity of 8F4 increased the bead-to-cell ratio, and CD34⁺/CD38⁻ LSCs constituted a reduced fraction of the few remaining viable cells in the 8F4-treated samples. (B) 8F4 mediates CDC of HLA-A2⁺ patient-derived AML blasts (●). Normal HLA-A2⁺ PBMCs (▵) and bone marrow cells (◊) were not affected (P = .0007). Leukemia cell line U937–transfected HLA-A2 was used as a positive control. Cells from an HLA-A2⁻ AML patient (□) and bone marrow cells from an HLA-A2⁻ normal donor (○) were used as negative controls. (C) Flow cytometric analysis of the same samples gated on live Lin⁻CD34⁺CD38⁻ cells shows preferential CDC of LSCs. HSCs from 3 of 6 healthy donors are also affected by 8F4-mediated CDC. (B-C) CDC assay for each sample and enumeration of cell populations was performed as shown in panel A. For each sample, isotype-mediated background lysis was subtracted from the measured value. Each point is the calculated mean value from 1-3 independent experiments (based on available cells) from individual patients. Error bars show SEM for each group.