MTT test (cell viability assay)

The MTT test was used to analyze cell survival and the half-maximal inhibitory concentration index (IC50). The IC50 value shows the concentration of tested substances which is needed to inhibit, in vitro, the biological activity of cells by 50%. The cytotoxicity level was established by estimating the percentage of dead cells and the degree of growth inhibition. The MTT test was performed according to the protocol provided with the reagent (Sigma-Aldrich). The MTT test measures the reduction of MTT tetrazolium salts soluble in water (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to blue-violet insoluble formazan crystals by active mitochondrial dehydrogenase. This test makes it possible to carry out a quantitative assessment of cell proliferation by measuring the linear relationship between cell activity and the absorbance of color reaction [20]. Before the absorbance was read, formazan crystals were extracted from the cells and solubilized with 10% SDS in 0.01M HCl solution. The absorbance was measured with a microplate reader (Multiscan, Labsystems, Thermo Fisher Scientific Inc.) at the wavelenghts of 570/690 nm. Jurkat cells were placed on the culture wells in the amount of 5×10³ cells/well. Then, cells cultured with the addition of Au/ZnO/Ag nanoparticles in the above-described conditions were treated with 10μl of MTT solution (5 mg/ml thiazolyl blue Tetrazolium Bromide). Following 4 hours of incubation, formazan crystals were released from the cells using 100 μl of a solubilizing solution. The results of the assay were presented as Relative Viability of Cells (RVC) value, i.e. the ratio between the absorbance value for cells that were cultured in the presence of the tested nanoparticles, to the absorbance value for control samples [21, 22]. The RVC value was calculated according to the formula specified below proposed by us, in which (a) means the absorbance of the tested sample; (b) means the absorbance of the blank control (pure medium with no cells); and (c) means the absorbance of control cells with no addition of Au/ZnO/Ag nanoparticles.