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.