Evaluating Cell Viability with ICA

HC-A were cultured in a humidified incubator at 37 °C and 5% CO₂ in 96-well plates. The cells (1 × 10⁶ cells mL-1) were maintained in Dulbecco Minimal Eagle’s Medium (Gibco, Carlsbad, CA, USA) plus 10% fetal bovine serum (Australia origin, Gibco, Carlsbad, CA, USA). ICA (10⁻¹⁰~10⁻⁷ M, 72 h) or simple saline (control) was added to the HC-A cultures. Cell viability was assessed using a modified MTT assay. Briefly, 10 μl of MTT solution (5 mg/mL in phosphate-buffered saline) was added to the 96-well plates and incubated continually for 4 h at 37 °C. The MTT solution was then removed, and formazan dye was dissolved in dimethyl sulfoxide (Sigma-Aldrich) for 10 min by shaking. The absorbance values were measured at 570 nm using a Bio-Rad Model 450 Microplate Reader (Bio-Rad). The cell viability was indirectly determined by examining the ratio of the absorbance value of ICA-treated cells relative to the control. The final results were determined by analyzing three independent experiments. GAPDH used as control and none of treatment group affect the GAPDH.31 (link)