Vero Kidney Cell Cytotoxicity Assay

Vero kidney epithelial cells (ATCC CCL-81) kindly provided by Dr. Alexander Rouvinski, The Hebrew University of Jerusalem, Israel, were seeded at 4 × 10⁵ cells per well in a 96-well flat-bottomed tissue culture plate (Corning) in 200 µL DMEM (Sigma) supplemented with 8% heat-inactivated fetal calf serum (Sigma), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin and 0.1 mg/mL streptomycin (Biological Industries, Beth HaEmek, Israel) and incubated at 37 °C in the presence of 5% CO₂. On the following day, when a cell monolayer was formed, the medium was exchanged with 200 µL fresh medium containing the indicated concentrations of triclosan and/or CBD followed by a 24 h incubation. At the end of incubation, the cell morphology was inspected under a light microscope, and the cells were either stained with 0.1% CV solution for 20 min at room temperature or MTT was added to the medium at a final concentration of 0.5 mg/mL followed by a 30 min incubation at 37 °C. The cells were washed in PBS and the absorbance at 595 and 570 nm was measured for the CV-stained and MTT-exposed cells, respectively, using the M200 infinite Tecan plate reader.