2.3.6. MTT cytotoxicity assay (TC50)

The prepared AgNPs samples were diluted with Dulbecco's Modified Eagle's Medium (DMEM). Stock solutions of the test compounds were prepared in 10% DMSO in bi distilled H₂O. The cytotoxic effect of the extracts was tested in Vero E6 cells by using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) according to Mossman method [⁴⁴] with a minor modification. Briefly, the cells were seeded in 96 well-plates (100 μl/well at a density of 3 × 10⁵ cells/ml) and incubated for 24 h at 37 °C in 5% CO₂. After 24 h, the cells were treated with various concentrations of the tested AgNPs compounds in triplicates. After further 24 h, the supernatant was discarded and cell monolayers were washed with sterile phosphate buffer saline (PBS) 3 times, then MTT solution (20 μl of 5 mg/ml stock solution) was added to each well and incubated at 37 °C for 4 h followed by medium aspiration. In each well, the formed formazan crystals were dissolved with 200 μl of acidified isopropanol (0.04 M HCL in absolute isopropanol = 0.073 ml HCL in 50 ml isopropanol). Absorbance of formazan solutions was measured at λ_max 540 nm with 620 nm as a reference wavelength using a multi-well plate reader. The percentage of cytotoxicity compared to the untreated cells was determined according to the following equation:

The plot of cytotoxicity % versus sample concentration was used to calculate the concentration which exhibited 50% cytotoxicity (TC₅₀).