105 cells was collected and incubated in the buffer containing 200 mL Annexin V solutions and 300 mL PI in the dark at room temperature for 15 minutes. Untreated cells were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19683258 used as the control. Then the samples were analyzed using a FAC Sort flow cytometer within 45 min after staining. Morphological Observation Cell morphology was evaluated by light microscopy following crystal violet staining. Briefly, HL-7702 cells were grown on 12-well glass slides and cultured. After that time, cells were washed with PBS and fixed with cold methanol. Then, cells were stained with crystal violet, and incubated for 20 min at room temperature. Cells were examined with light microscope. Apoptotic cells were identified by characteristic features of apoptosis. Determination of ROS Generation Generation of ROS was determined by two methods: fluorometric analysis and microscopic fluorescence imaging, using 2,7-dichlorofluorescin diacetate 4 / 22 Protective Efficacy of Vitamins C and E on p,p9-DDT as described by our previous method. DCFH-DA was a cellpermeable probe, and was widely used to detect the ROS generation. For fluorometric analysis, cells were seeded in 60 mm dish. After treatment for 24 h, cells were washed twice with PBS and incubated 
in 500 mL 10 mmol/L DCFH-DA at 37 C for 30 min. Then cells were collected, and 200 mL of cells in suspension were placed in a 96-well plate to assess cell fluorescence intensity, using a fluorescence microplate reader with excitation at 488 nm and emission at 525 nm. The values were expressed as a percent of fluorescence intensity relative to control wells. A parallel set of cells, which were placed in 12-well glass slides, were analyzed for intracellular fluorescence using by a fluorescence microscope by grabbing the images at 206magnification. Western Blotting To analyse total protein expression, lysates of HL-7702 cells were prepared with the protein concentrations determined using BCA protein assay. Equal amounts of each total protein lysates were determined, mixed with 56SDS sample buffer, boiled for 5 min and resolved by 10% SDSPAGE then transferred onto nitrocellulose membranes. The blots were blocked for 1 h in PBS containing 5% non-fat dry milk and incubated at 4 C overnight, then probed with antibody for 1 h at room temperature or overnight at 4 C. After washing, membranes were incubated at 37 C for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody. Protein loading was controlled by probing the membranes for a-tubulin protein. Immune-reactive proteins were detected using ECL western blotting detection PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19681941 system. ONO4059 chemical information mitochondrial Membrane Potential Assay Dym is an important parameter of mitochondrial function used as an indicator of cell health. Measurement mitochondrial membrane potential was performed in HL-7702 cells, according to the manufacturer’s protocol for a commercial kit. For fluorometric analysis, HL-7702 cells were seeded in 60 mm dish. After treatment for 24 h, cells were washed twice with PBS and then incubated with 1.0 mg/mL JC-1 for 15 min at 37 C. Cells were collected, and 200 mL of cells in suspension were placed in a 96-well plate to assess cell fluorescence intensity. The fluorescence was determined at an excitation wavelength of 490 nm and an emission wavelength of 590 nm. The values of relative monomer fluorescence intensity were used for data presentation, which were expressed as a percent of fluorescence intensity relative to control wells. A parallel set of c
