4.3. Sulforhodamine B (SRB) Cell Proliferation Assay

Cell proliferation was determined by SRB assay. SRB assay was developed by Skehan and colleagues to investigate cytotoxicity and cell proliferation for anticancer drug screening [71]. OSCC cells were seeded at a density of 5 × 10³ cells per well into 96-well plates for 24 h. The cells were then treated with various concentrations of statins (0.1–100 µM) for 48 h, where dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA) was used as the vehicle control group. On the day of the assay, 50 µL trichloroacetic acid (TCA, Sigma, USA) 50% (wt./vol) was gently added to each well directly to the medium supernatant and the plates were incubated at 4 °C for 1 h. The plates were washed four times by submerging the plate in a tub with slow-running tap water and removing excess water by gently tapping the plate into a paper towel. After the last wash, the plates were allowed to air dry at room temperature. Then, 100 µL of sulforhodamine B (SRB, Sigma, USA) 0.4% (wt./vol) solution was added to each well and incubated at room temperature for 1 h, and then the plates were quickly rinsed four times with 200 µL of 1% (vol/vol) acetic acid (Sigma, USA) to remove unbound dye. The plates were allowed to air dry at room temperature. Then, 100 µL of 10 mM Tris base (Amresco, Solon, OH, USA) solution pH 10.5 were added to each well and the plates were shaken on an orbital shaker for 20 min to solubilize the protein-bound dye. The absorbance was measured at 510 nm using the Epoch Microplate Spectrophotometer (Biotek, Taipei, Taiwan). The results of the SRB assay were expressed as the percentage of cell proliferation using dimethyl sulfoxide (DMSO) treatment as the vehicle control group. The half-maximal inhibitory concentration (IC₅₀) was determined using GraphPad Prism 7 software by plotting nonlinear regression log concentration (Log C) of inhibitor (statins) vs. response (cell proliferation, % of control).