D 100 M FeSO4 for 8 hours. Then, after removal of H2O
D 100 M FeSO4 for 8 hours. Then, after removal of H2O2 and FeSO4, cells were cultured in DMEM/F12 medium containing 10 Heshouwuyin-containing serum for 72 hours. The protective effect of Heshouwuyin on the aging of Leydig cells in the testes was observed. The -galactosidase staining kit was used, which purchased from Genmed Scientifics Inc., USA(GMS10010.1). According to the experimental requirements, the Leydig cells were cultured primarily using Petri dishes. After the culture medium was aspirated, the cells were washed twice with 0.1 M PBS, and fixed using 1 mL -galactosidase fixative for 15 minutes. Then, the fixative was aspirated, and the cells were washed with 0.1 M PBS three times for 3 minutes each. -galactosidase dye working solution was added, 1 mL per well, along with 10 L A solution, 10 L B solution, 930 L C solution, and 50 L X-Gal solution. Cells were incubated at 37 overnight. The six-well plates were covered with plastic wrap to prevent evaporation. Then, the working fluid was removed. Under the optical microscope (Leica DM 6000 M, Germany), the cytoplasm of -galactosidase positive cells was blue. The number of blue cells out of 200 cells was observed and recorded. The SP600125 site experiments were repeated five times for robust statistical analysis.Fluorescence immunocytochemistry for observation of StAR and P450scc expression -galactosidase enzyme assayH2O2 and FeSO4 solutions, at a final concentration of 50 M and 100 M, respectively, were added into the six-well culture plates containing Leydig cells cultured for 48 hours. After culture 8 hours, H2O2 and FeSO4 were removed. Then, the cells were placed in DMEM/ F12 containing 10 fetal bovine serum for 72 hours.Sterilized 20 mm coverslips were placed in 90 mm Petri dishes. The cells were seeded in the Petri dishes at density of 2 ?104/mL. The corresponding treatment was given in each group. Samples were fixed in 4 paraformaldehyde for 30 minutes, incubated in 0.5 Triton X-100 for 20 minutes, and cultured in 3 H2O2 for 15 minutes toNiu et al. BMC Complementary and Alternative Medicine 2014, 14:250 http://www.biomedcentral.com/1472-6882/14/Page 5 ofeliminate endogenous peroxidase activity. The samples were rinsed with 0.1 M PBS three times for 2 minutes each before each experimental procedure. After rinsing with 0.1 M PBS three times for 2 minutes each, normal goat serum was added, and the samples were inoculated at 37 for 30 minutes. Filter paper was used to absorb the serum. Diluted rabbit anti-StAR, P450scc monoclonal antibody (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was added and incubated with samples at 4 overnight. Then, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25636517 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28404814 samples were rinsed with PBS three times, for 5 minutes each. Fluorescent dye-labeled secondary antibody working solution (goat anti-rabbit IgG, Santa Cruz) was added and incubated with samples at 37 in darkness for 30 minutes. Green granules in the cytoplasm were observed under the fluorescence microscope, and the number of positive cells out of 200 cells was recorded. The experiments were repeated five times for robust statistical analysis.Western blot assay for determination of StAR and P450scc protein expression in Leydig cellsCultured cells in each group were digested with 0.25 trypsin and centrifuged to remove the supernatant. The total protein of each sample was quantified by a BCA protein assay kit. In each group, 50 g Leydig cell proteins were mixed with loading buffer, placed in a boiling water bath for 5?0 minutes and co.

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