3.5.2. Cytotoxicity Assay

Cell viability was assessed by SRB assay. Aliquots of 100 μL cell suspension (5 × 103 cells) were in 96-well plates and incubated in complete media for 24 h. Cells were treated with another aliquot of 100 μL media containing EOs and a mixture of them (1:1, *w*/*w*), separately, at various concentrations ranging from (0.01, 0.1, 1, 10, and 100 ug/mL). After 72 h of drug exposure, cells were fixed by replacing media with 150 μL of 10% TCA and incubated at 4 ◦C for 1 h. The TCA solution was removed, and the cells were washed 5 times with distilled water. Aliquots of 70 μL SRB solution (0.4% *w/v*) were added and incubated in a dark place at room temperature for 10 min. Plates were washed 3 times with 1% acetic acid and allowed to air-dry overnight. Then, 150 μL of TRIS (10 mM) was added to dissolve protein-bound SRB stain; the absorbance was measured at 540 nm using a BMG LABTECH®- FLUOstar Omega microplate reader (Ortenberg, Germany) [64,65].
