Cell proliferation assay

Cell proliferation rates were quantified using colorimetric MTT proliferation assay. HeLa cells were placed in 96-well cell culture plates at the density of 8000 cells per well and were grown overnight. Cells were exposed to different concentrations clotrimazole or ethanol and grown for 24, 48, and 72 h. Cells were washed with basal solution containing: 135 mM NaCl, 3.8 mM KCl, 1.2 mM MgSO₄, 1.3 mM CaCl₂, 1.2 mM KH₂PO₄, 10 mM HEPES, and 10 mM glucose (pH=7.4, adjusted with NaOH). Aliquots of basal solution containing 0.5 mg/ml thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich) were added to each well and incubated at 37°C MTT for 4 h. Then, the solution was removed, cells were solubilized in 150 μl dimethyl sulfoxide to dissolve newly formed MTT-formazan particles, and incubated at 37°C for 10 min. Next, culture plates were gently agitated and cell proliferation was measured by examining absorbance at 490 nm using an ELx800 Absorbance microplate reader (BioTek Instruments, Winooski, VT, USA). The rate of cell proliferation inhibition (“R”) was calculated using the equation “R=[(A0−At)/A0]×100%”. “A0” is the average absorbance value of the control group. “At” is the average absorbance value of the experimental group.