Immunostaining and flow cytometry analysis

AML cell lines and primary cells were treated with CPI-637, NEO1132, or NEO2734 for 96 hours before analysis. Cells were harvested and washed in PBS with 0.1% human serum albumin (PBS/HSA). Samples were incubated for 15-30 minutes at room temperature with monoclonal antibody combinations consisting of antihuman 7AAD-PerCP (1:10), CD45-HV500c (2D1, 1:20), CD33-PE (1:20) or CD33-PC7 (1:20), CD34-APC (8G12, 1:50) or CD34-BV421 (581, 1:20), CD38-APC (HB7, 1:50), CD15-FITC (HI98, 1:100), CD7-APC (M-T701, 1:50) or CD7-FITC (M-T701, 1:20), CD56-PE (MY31, 1:20), HLA-DR-FITC (L243, 1:100), CD3-FITC (SK7, 1:50) or CD3-PE (1:50), CD19-APC-H7 (SJ25C1, 1:10) or CD19-FITC (89B-B4, 1:20), or antimouse CD45-PerCP (30-F11, 1:50), from BD Biosciences, and washed once with PBS/HSA. For detection of apoptosis, cells were stained with 7AAD-PerCP (1:10) for 15-30 minutes, washed with PBS/HSA, and subsequently stained with AnnexinV-FITC 1:1000 (Tau Technologies) in AnnexinV-binding buffer (Invitrogen) for 15 minutes on ice. Flow-count fluorosphere beads (Beckman Coulter, Brea) were added according to manufacturer instruction directly before analysis using a FACS-Fortessa flow cytometer (BD Biosciences). Data analysis was performed with FACS Diva software (BD Biosciences).