*2.11. Cytotoxicity*

The assay detects the reduction of MTT (3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide) by mitochondrial dehydrogenase within the fungi to form blue formazan, which indicates that the mitochondria are functioning normally, and hence the measurement of cell viability. Briefly, 25 cells/μL HEp-2 in RPMI-1640 were added to each well in a 96-well plate and incubated for 24 h. After incubation, the cells were washed twice with phosphate-buffered saline (PBS), and fresh RPMI-1640 was added. Bacterial cellulose 4 mm disks, which had been loaded with 50 mg/L solutions of thymoquinone, ocimene, or miramistin, were then added and incubated for a further 24 h in triplicate (*n* = 4). Following incubation, MTT (0.5 mg/mL PBS) was added to each well and incubated at 37 ◦C for 3 h. The solubilisation of formazan crystals was achieved by mixing DMSO (100 μL/well), which was gently agitated for 10 min, and the absorbance was read at 570 nm using a microplate scanning spectrophotometer (SPECTROstar® Nano, BMG Labtech). The following formula calculated toxicity level:

$$\text{Cytotoxicity\%} = 1 - \frac{\text{mean abs of antibugal}}{\text{mean abs of negative}} \times 100\\\text{Vaidbility\%} = 100 - \text{cytotoxicity\%} \tag{1}$$

To reduce test error level, MTT was added to wells without cells, and along with other wells, absorbance level was read and ultimately subtracted from the whole absorbance.
