Cytotoxicity Assay for Leukemia Drugs

Fifty percent inhibitory concentration (IC50) values of prednisolone (Pred), dexamethasone (Dex), vincristine (VCR), daunorubicin (DNR), L-asparaginase (L-Asp), cytarabine (AraC), methotrexate (MTX), mercaptopurine (6MP), and mafosfamide [Maf; active metabolite of cyclophosphamide (CY)] were determined using the alamarBlue cell viability assay (Bio-Rad Laboratories, Hercules, CA) as previously reported [17 (link)]. Cells (1–4 × 10⁵) were placed onto 96-well flat bottom plates in the presence or absence of seven separate concentrations of each drug in triplicate. The cells were cultured for 44 h to determine the DNR, VCR and CY (Maf) sensitivities and for 68 h to determine Pred, Dex, L‐Asp, MTX, and 6MP; 20 µL of alamarBlue was then added. After incubation for an additional 6 h in the presence of alamarBlue, the optimal density was read on a spectrophotometer at 570 nm using 600 nm as a reference wavelength. Cell viability was calculated by the ratio of the optical density of the treated wells to that of the untreated wells as a percentage. The concentration of each agent required to reduce the viability of the treated cells to 50% of the untreated cells (IC50 value) was calculated and the median IC50 value of three independent assays was determined.