Determination of cytotoxicity by MTT assay

Exponentially growing cell lines were seeded into 96-well plates at the concentration of ~ 1 × 10⁴ cells per well and allowed to attach for 24 h. Test fractions isolated from antioxidant Lactobacillus strain were prepared in dimethyl sulfoxide (DMSO) (Sigma, USA) and serially diluted with basic media to obtain appropriate concentrations (400, 200, 100, 50, 25 μg/mL) in such way that DMSO concentration was lower than 0.2%.[23]

Cells were treated with different concentrations of fractions and incubated for 24 h. Cells in the control group received only media containing 0.2% DMSO. The test compound containing media was removed and washed with 200 μl of PBS followed by addition of 20 μl of 3-(4, 5-dimethyl-2-thiazolyl)-2 and 5-diphenyl-2H-tetrazolium bromide (MTT) reagent (5 mg/mL MTT in PBS) (Sigma, UK) and incubated for 3h at 37°C. The medium was removed and 100 μl DMSO was added into the wells to solubilize the purple crystal formazan and the absorbance was measured using a microplate reader (Bio-Tek Instruments Inc., USA) at the wavelength of 570 nm.[24] The effect of the samples on the proliferation of cell lines was expressed as the% cell viability, using the following equation:

% cell viability = [(A_t–A₀)/(A_c-A₀)] ×100.

Where A_c = Absorbance of cells treated with 0.2% DMSO medium, A_t = Absorbance of cells treated with extract/fractions, and A₀= Absorbance of background. 0.1% (v/v) DMSO in the medium was used as negative control. Each treatment was performed in triplicate. The 50% growth inhibition concentrations (IC₅₀) of the partial-purified fractions were estimated from the graphical interpolation.[25]