Cell Viability Assay with Doxorubicin

Cell viability was determined by quantitation of ATP, an indicator of live cells, using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI) kit, as described previously [32 (link), 58 (link)]. Briefly, cells (4000 cells/well) were grown in 96-well plates with 10% FBS supplemented RPMI-1640 medium. Cells were treated with test agents in 5% FBS medium for 72 hours. Cell viability was determined by the measurement of ATP in a Synergy HT microplate reader (BioTek, Winnooski, VT, USA), following incubation with CellTiter-Glo reagent. For combination treatment, cells (3×10⁶ /100-mm dish; 4000 cells/well in 96-well plates) were grown in 10% FBS RPMI-1640 medium overnight and then cultured in 5% FBS medium containing d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol HCl (PDMP; 5 μM) or fumonisin B1 (FB1, 25 μM) and doxorubicin (Dox; 5∼2.0 μM) for 48 hr. PDMP was purchased from Matreya (State College, PA) and fumonisin B1 (FB1) from Sigma-Aldrich (St. Louis, MO).