4.3. CellTiter-Glo Viability Assay (CTG)

Cytotoxicity evaluation was performed using the CellTiter-Glo Luminescent Cell Viability Assay kit (G7570, Promega, Madison, WI, USA), according to the manufacturer’s instructions. Briefly, the cell concentrations used were experimentally determined to ensure logarithmic growth during the 72-h duration of the experiment, and avoid adverse effects on cell growth by DMSO exposure. 1 × 10³–4.8 × 10³ or 4 × 10²–1.2 × 10³ cells/well were seeded in 96 or 384-well white flat-bottomed plates (3610 or 8804BC, Corning, Corning, NY, USA) in 100 µL or 30 µL/well, respectively. The plates were incubated at 37 °C in 5% CO₂ for 24 h before drugging. Test compounds (10 mM in DMSO) in nine 3-fold serial dilutions were dispensed via pintool (Biomek liquid handler, Beckman, Indianapolis, USA) to assay plates. The final concentration of DMSO was 0.3% (v/v) in each well. The positive controls included staurosporine (10 μM), and gambogic acid (10 μM). The plates were incubated for 72 h at 37 °C in 5% CO₂, then quenched with CellTiter-Glo^® (Promega, Madison, WI, USA, 50 µL/96 or 30 µL/384), centrifuged at 1000 rpm for 1 min and incubated at RT for 20 min. Luminescence was recorded with a plate reader (Envision, Perkin Elmer, Waltham, MA, USA). The mean luminescence of each experimental treatment group was normalized as a percentage of the mean intensity of untreated controls. EC₅₀ values were calculated by Pipeline Pilot software (Accelrys, Enterprise Platform, San Diego, CA, USA).