*2.6. Cellular Toxicity*

HEp-2 cells (National Bank for Industrial Microorganisms and Cell Cultures, No. NBIMCC-95, Sofia, Bulgaria) were grown in medium containing 10% heated calf serum in DMEM (Gibco BRL, Red Bank, NJ, USA) supplemented with 10 mmol/L HEPES buffer (Gibco BRL, Red Bank, NJ, USA) and antibiotics (penicillin, 100 U/mL; streptomycin, 100 μg/mL).

Monolayer cells in 96-well plates (Costar®, Corning Inc., Kennebunk, ME, USA) were inoculated with 0.1 mL/well-containing concentrations (in logarithmic intervals) of the compounds diluted in a maintenance medium. Cells were incubated in a humidified atmosphere at 37 ◦C and 5% CO2 for 48 h. After microscopic evaluation, the maintenance medium containing the test compound was removed, cells were washed, and 0.1 mL maintenance medium supplemented with 0.005% neutral red dye was added to each well and cells were incubated at 37 ◦C for 3 h. After incubation, the neutral red day was removed, and cells were washed once with PBS, and 0.15 mL/well desorb solution (1% glacial acetic acid and 49% ethanol in distilled water) was added. The optical density (OD) of each well was read at 540 nm in a microplate reader (Organon Teknika Reader model 530, Organon, West Chester, PA, USA). The 50% cytotoxic concentration (CC50) was defined as the material concentration that reduced the cell viability by 50% when compared to untreated control.
