Evaluating Cell Growth Inhibition

The influence on cell growth of selected samples was evaluated following the protocol described by Monks et al. [12 (link)] with adaptations [13 (link)]. In 96-well plates, CHO-K1 cells (4 × 10⁴ cell mL⁻¹, 100 µL well⁻¹) were exposed to each sample (0.25, 2.5, 25 and 250 µg mL⁻¹, final concentrations) for 4 and 48 h. In the 4 h exposure experiment, after the first 4 h, the medium was removed, and CHO-K1 cells were kept in fresh complete medium for 20 h. A T0 plate representing the cell density at the time of sample treatment was made (T0_{untreated cells}). At each experimental plate (4 + 20 h and 48 h), untreated cells were kept in the complete medium (T1_{untreated cells}). The cell viability was evaluated by total protein quantification at 540 nm with the sulforhodamine assay. For each sample, the influence of each concentration on cell growth was expressed in percentage, considering (T1_{untreated cells}–T0_{untreated cells}) as 100% cell growth. The sample concentration required by eliciting 50% cell growth inhibition (GI₅₀) was calculated by sigmoidal regression.