After this procedure, the cells were dried at room temperature and subsequently fixed in a 1% methanol in 1% acetic acid solution for 2 h. The fixed cells were stained with a 0.5% SRB in 1% acetic acid solution, and then washed with a 1% acetic acid solution to GSK-3 beta pathway remove the excess probe. The SRB attached to the cell membranes was extracted using 1 ml of a 10 mM Tris
solution, pH 10.0. The absorbance of the dye was then measured at a wavelength of 540 nm in a microplate reader (Varian Cary 50MPR, Varian, USA). Cell viability was assessed using a (4,5 dimethylthiazole-2-il)-2,5 diphenyltetrazolium bromide dye, according to Denizot and Lang (1986). HepG2 cells were seeded with a density of 1 × 105 cells and exposed to BDE-99 at final concentrations ranging from 0.5 to 25 μM. At least three replicates were made for each sample and cultured for 24 and 48 h. The cells were subsequently incubated with a 0.5% MTT (5 mg/mL) solution in an atmosphere containing 5% CO2 at 37 °C for 3 h. After this period, the medium in the wells
was discarded and the formazan crystals formed dissolved in a DMSO solution in 0.2 M glycine buffer, pH 10.2. The final absorbance Quizartinib supplier was evaluated at 570 nm wavelength in a microplate reader (Varian Cary 50MPR, Varian, USA). The results were shown as the percentage difference from the control group. Indications of cell damage can be evaluated by mitochondrial depolarization, since the collapse of the membrane potential compromises the cell energy and consequently damages cell integrity. Mitochondrial depolarization can be measured using the fluorescent dye TMRM, a cation compound permeable to cell membranes, which is rapidly sequestered by the mitochondria of intact cells, and produces a stoichiometric relationship between the fluorescence and the mitochondrial membrane potential (Imberti et al., 1993). The HepG2 cells were cultured to a density of 1 × 105 cells and then exposed to BDE-99 at final concentrations
ranging from 0.5 to 25 μM. Each sample was tested with at least three replicates. The cells were then washed with PBS, trypsinised and incubated with a 6.6 μM TMRM solution at 37 °C for 30 min. The samples were subsequently lysed with a 0.1% Triton X-100 solution (v/v) and the TMRM captured Niclosamide and retained by the mitochondria measured at the excitation and emission wavelengths of 485 and 590 nm, respectively, using a F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The results are shown as the percentage of fluorescence in relation to the control group. The accumulation of ROS can be evaluated using CM-H2DCFDA, a reactive oxygen species indicator that becomes fluorescent in the presence of intracellular oxidation (Chernyak et al., 2006). The HepG2 cells were cultured to a density of 1 × 105 cells. After incubation with BDE-99, the cells were further incubated with a 2 mM CM-H2DCF-DA solution at 37 °C for 1 h.