Annexin v 7 aminoactinomycin d 7 aad

rDPCs were cultured in αMEM or rDFSC-CM with or without 0.5 mg/L LPS for 24 h, harvested by trypsinization, and fixed with 70% cold ethanol for 1 h on ice. After washing, the cells were incubated with Annexin V/7-aminoactinomycin D (7-AAD, BD Biosciences, USA) for 30 min at 4 °C in the dark. Cell apoptosis and the cell cycle distribution were analyzed using a FACSCalibur flow cytometer (BD Biosciences, USA) and Cell Quest version 3.1 software (BD Biosciences, USA) according to the manufacturer’s instructions.

For proliferation analysis, primary AML and normal CD34+ cells were stained with CellTrace Violet (Thermo Fisher Scientific) as per manufacturer’s protocol. For establishing a maximum point of fluorescence staining, cells were cultured with Colcemid (100 ng/mL, Sigma-Aldrich) to determine non-dividing cells. All primary cells were treated with fadraciclib, VEN, AraC, AZA or fadraciclib in combination with VEN, AraC or AZA in physiological growth factor conditions. Annexin V/7-aminoactinomycin D (7-AAD) or DAPI (4’,6-diamidino-2-phenylindole) (BD Biosciences) staining to assess apoptosis by flow cytometry using 1 × 10⁵ cells per condition. Propidium iodide (PI) staining buffer to assess cell cycle progression as per manufacturer’s protocol. Fixation/Permeabilization Solution Kit (BD Biosciences) was used prior to MCL-1 or active caspase-3 staining. For staining reagents used, see Supplementary Table S3.