To measure cell proliferation, cells were plated in 96- or 384-well plates (3000 or 1250 cells/well, 3–6 wells/sample) with drugs and incubated for 48 hours at 37° C in 5% CO2. After incubation, relative numbers of viable cells were measured using a tetrazolium-based colorimetric assay (CellTiter Aqueous One Solution Cell Proliferation Assay, Promega). Inhibitor combination screening was carried out in pre-treated 384 well plates with inhibitors prepared in a 7-point concentration series ranging from 10 μM to 0.014 μM for each drug (23 (link)).
To measure cell apoptosis, cells (in duplicates) were resuspended in 100 μL of Annexin V binding buffer containing 1 μL of Annexin V-PE, 1 μL of 7-aminoactinomycin D (7-AAD) (Southern Biotech) and 1 μL of CD19-mAbs (BD Bioscience) followed by flow cytometry on FACSAria (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star).
AZD5991 and navitoclax were purchased from Active Biochem; venetoclax and Q-VD-OPh were from MedChemExpress; AZD4320, IACS-010759 were from Selleck Chemicals. CCCP (carbonyl cyanide m-chlorophenyl hydrazine), NAC (N-Acetyl-L-cysteine) were from Sigma.
To measure cell apoptosis, cells (in duplicates) were resuspended in 100 μL of Annexin V binding buffer containing 1 μL of Annexin V-PE, 1 μL of 7-aminoactinomycin D (7-AAD) (Southern Biotech) and 1 μL of CD19-mAbs (BD Bioscience) followed by flow cytometry on FACSAria (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star).
AZD5991 and navitoclax were purchased from Active Biochem; venetoclax and Q-VD-OPh were from MedChemExpress; AZD4320, IACS-010759 were from Selleck Chemicals. CCCP (carbonyl cyanide m-chlorophenyl hydrazine), NAC (N-Acetyl-L-cysteine) were from Sigma.
