Specific active caspase 3 antibody

Apoptosis assays were performed as described previously (10 (link)). Briefly, after 24 to 48 hours of treatment, primary cells were stained for the surface antibodies CD34-allophycocyanin (APC), CD38-phycoerythrin with cyanin-7 (PECy7), CD123-phycoerythin (PE), and CD45-allophycocyanin-Hilite.7-BD (APC-H7; Becton Dickinson,) for 15 minutes. Cells were washed in cold PBS and resuspended in 200 μL of Annexin-V buffer (0.01mol/L HEPES/NaOH, 0.14 mol/L NaCl, 2.5 mmol/L CaCl₂) containing Annexin-V-FITC or Annexin-V-PE (Becton Dickinson) and 7-aminoactinomycin (7-AAD, Life Technologies). Cells were incubated at room temperature for 15 minutes then analyzed on a BDLSRII flow cytometer using the high throughput attachment. To analyze human cell engraftment in the NOD/SCID xenotransplant model, bone marrow cells were blocked with the anti-Fc receptor antibody 2.4G2 and 25% human serum and later labeled with anti-human CD45-PE and anti-mouse CD45-FITC antibodies (Becton Dickinson). For reactive oxygen species (ROS) detection, the cells after treatment were incubated with 5 mmol/L CellROX Green Reagent (Life Technologies). The staining for CD117 was performed on MV4–11 cells after treatment with AR-42 or HSP90i using anti-human CD117-PE antibody (Becton Dickinson). Specific active caspase-3 antibody was used according to the manufacturer's protocols (BD Pharmingen).