The Sulphorhodamine B assay (SRB assay) for assessing cytotoxicity
The cytotoxicity of THA was assessed using MRC-5 cell lines (ATCC, CCL-171, Virginia, USA) as previously described 20. Briefly, MRC-5 cells were seeded at 5000 cells/well and co-cultured with varying concentrations of THA (25 to 400 µg/mL) diluted in DMSO. The cytotoxicity of THA was compared with that of Paclitaxel (doses ranging from 1.25 to 20 µg/mL), which was again diluted in DMSO. As DMSO itself it cytotoxic, the concentrations were kept below 0.05% in all experiments. Untreated MRC-5 cells were considered as the negative controls. All experiments were carried out in triplicate. The cytotoxicity was assessed by the Sulforhodamine B assay at 24, 48 and 72 hours according to the manufacturer’s instructions. The absorbance was measured at 540 nm using a microplate reader (Synergy TMHT multimode microplate reader, Bio Tek, USA). The percentage inhibition of cell growth for each concentration of the test substance was calculated. Half-maximal inhibitory concentrations (IC50) values were determined by analyzing sigmoid dose-response inhibition curves using GraphPad Prism software version 9.