Celltiter glo 2.0 luminescent cell viability assay kit

Drug cytotoxicity was evaluated by measuring ATP as a marker of metabolically active cells. RD cells (5x10³) were cultured with diluted drugs (total 25 μl/well, triplicate) in a 384-well plate (Corning) at 37°C for 16 h. ATP levels were measured using a CellTiter-Glo 2.0 luminescent cell viability assay kit (Promega) according to the manufacturer’s instructions.

HL60 and HL-60R cells were seeded at 3.0 × 10³ cells/well in 96-well plates and incubated for 24 h. ATRA, MK-4, MKH-DMG, and MKH-SUC were initially dissolved in ethanol. Stock solutions (15 mM) of each drug were diluted to the intended final concentrations with medium. Cells were exposed to a medium containing ATRA, MK-4, MKH-DMG, or MKH-SUC for 72 h, and cell viability was measured using the CellTiter-Glo 2.0 Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. The CellTiter-Glo assay detects intracellular ATP, thereby enabling identification of living cells. The doubling times of the HL60 and HL-60R cells using CellTiter-Glo were 24.9 ± 3.68 and 26.2 ± 2.69 h, respectively. These values were almost consistent with the number of cells counted by staining them with trypan blue after each subculturing. The IC₅₀ values were determined using a log (drug) vs. normalized response-variable slope analysis with GraphPad Prism, version 6.0 (GraphPad Software, San Diego, CA, USA). Resistance index was calculated using the following equation: