Measurement of Cell Proliferation for Adherent Cells (MTT)

Cell cytotoxicity was measured using the3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolio (MTT) assay. L929 cells (5 × 10⁴ cells/well) and Caco2 cells (10⁵ cells/well) were plated in 96-well tissue culture plate in complete medium (100 μL/well). The multiwell plates were incubated at 37°C, 5% CO₂ for 24 h. After 24 h, the culture medium was removed and equal volumes (100 μL) of the treatments were added to each well. In control wells, 100 μL DMEM were added. Control wells consisted of untreated cell cultures. Twenty-four hours later, proliferative cells were detected by MTT assay, according to the ISO 10993-5 International Standard procedure (43). The main purpose of the ISO 10993-5 procedure is to define a scheme for testing in vitro cytotoxicity of different extracts according to a multi-step approach. Briefly, cells were incubated with MTT solution (1 mg/mL, Life Technologies) at 37°C for 2 h. Then, MTT solution was removed and cells were solubilized with 100 μl of isopropanol. The formazan dye formation was evaluated by scanning multiwell spectrophotometer at 540 nm. The results were expressed as percentage of viable cells compared to controls.