*4.4. Cell Viability Assay (MTT)*

The cell viability was determined by the MTT assay as described in [65]. This test is based on the ability of living cells to metabolize the yellow tetrazolium salt to a blue formazan via the mitochondrial succinate dehydrogenase which is a member of mitochondrial electron transfer system complex. To determine the neuroprotective effect of HE, PC12 cells (10<sup>5</sup> cells/well in a 24-well plate) were incubated at 37 ◦C after pretreatment with HE (1 μM) for 2 h and then incubated with DEHP (83 μM) for 24 h. A negative control containing only cells was also evaluated. After treatment, cells were incubated with 5 mg/mL MTT for 3 h at 37 ◦C, the medium was removed carefully after the incubation and the formazan crystals were dissolved in 150 μL of DMSO and absorbance of formazan reduction product was measured by spectrophotometer at 570 nm using a microplate reader (Biotek, Elx 800, USA). The results were expressed as the percentage of MTT reduction relative to the absorbance measured from negative control cells. All assays were performed in triplicate. Based on the results obtained from cell viability assay, the effective dose of HE against DEHP toxicity was utilized to study the effect of HE by assessing reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and apoptotic protein marker expression.
