Therefore, in subsequent analyses we deliberately used gene targets obtained from different cell lines

spase inhibitor Z-VADFMK at a final concentration of 1 ml/ml to inhibit caspase activation. All the test and control groups were incubated with 1 ml of the fluorescent maker in a CO2 incubator for 1 h, and subsequently washed twice with the rinse buffer on ice. Analyze samples by flow cytometry. The above experiment was repeated five times. Estimation of ROS in sperm cells The intracellular ROS level in sperm cells was measured using a ROS assay kit. Briefly, the enriched sperm cells were divided into six groups: four groups were exposed to 0, 25, 50, 100 mg/ml HBs for 3 h, respectively, and two groups were pretreated with HBs MAb and NAC for 30 min followed by exposure to 25 mg/ml HBs for 3 h, respectively. After removing the supernatant, the sperm cells were washed with phosphate buffered saline and incubated with DCFH-DA at a final concentration of 10 mM at 37uC in the dark for 20 min. Then sperm cells were centrifuged and washed three times with PBS. The labeled sperm cells were analyzed by flow cytometry. The above experiment was repeated five times. Determination of DNA fragmentation in sperm cells The FragELTM DNA Fragmentation Detection assay kit was used to investigate the impact of HBs exposure on nuclear apoptosis in sperm cells according to the manufacturer’s protocol with some slight modifications. ” Briefly, the washed sperm cells in the test and control groups were fixed with 4% formaldehyde-PBS at room 14521400” temperature for 30 min. Then the cells were washed once with 1 ml of PBS followed by permeabilization with 100 ml of 20 mg/ml proteinase K at room temperature for 5 min. After washing with equilibration buffer, the labeling reaction was performed by incubating cells with 60 ml of terminal deoxynucleotidyl transferase labeling reaction mixture at 37uC for 1.5 h in the dark. TdT enzyme was not added to the negative control. The positive control was obtained by incubating one sample with 10 mg/ml DNAse at room temperature for 10 min. Estimation of lipid peroxidation in sperm cells Aldetect Lipid Peroxidation assay was used to measure LP in sperm cells. Sperm cells in the test and control groups were lysed with Western and immunol precipitation lysis buffer, respectively. The lysates were homogenized, and the homogenates were centrifuged at 1,6006g at 4uC for 10 min. The supernatants were collected and determined with Lipid Peroxidantion MDA Assay Kit. A 200 ml of thiobarbituric acid reagent was added to 100 ml of the sperm suspension. The mixture was treated in a boiling water bath for 15 min. After cooling, the suspension was centrifuged and the supernatant Effects of HBs on Sperm Functions After labeling, the samples were washed twice with Tris-buffered saline and analyzed with a flow cytometer equipped with a 488 nm argon-ion laser source. The above experiment was repeated five times. FITC-IETD-FMK and FITC-LEHD-FMK, and using the FL2 channel at 488/620 nm excitation/emission for PI. purchase Indirubin-3′-oxime Statistical analysis Data were presented as mean values 6 SEM. SPSS 17.0 programs were used in the statistical analysis. A paired-samples T test was used to determine whether there is a significant difference between the average values of the test group and the control group. P-value of less than 0.05 was considered to be significant. Analysis of Flow cytometry All flow cytometric analyses were performed using a FACScan FlowCytometer. Cells were isolated from fragments by gating on the forward and side scatter signals, and then cells were detect