air was circulated continuously. All of the procedures used in the experiments were approved by the Institutional Animal Ethics Committee, Medical School of Xi’an Jiaotong University and conformed to accepted ethical standards of the Animals in Research and the Association for Research in Vision and Ophthalmology statement for the use 15557325 of animals in vision and ophthalmic research. H2O2 was purchased from Xi’an Pure Chemical Industries. Fetal Bovine Serum and phenol red free 1:1 DMEM/F-12 were obtained from Hyclone. Poly-lysine, E2, Hoechst 333342 dye and nifedipine, an L-VGCC blocker, were purchased from Sigma. We used 95% ethanol as the solvent to make the E2 stock solution at a concentration of 1×10-2 M. Fluo-3 AM, an indicator of intracellular Ca2+ levels, was purchased from Biotium. We used Dimethylsulfoxide as the solvent for making 5 mM Fluo-3 AM stock solution and 20% Pluronic F-127 in DMSO to facilitate AM ester solubilization. Trypsin, DMSO, 3–2, 5diphenyltetrazolium bromide and ethylene glycol tetraacetic acid, an extracellular Ca2+ chelator, were purchased from Amresco. LY294002, a PI3K inhibitor, was purchased from Cayman. The Annexin V-FITC Apoptosis Assay Kit and bicinchoninic acid Protein Assay Kit were purchased from Zhuhai Joincare Bioscience Ltd, and radio immunoprecipitation assay buffer was purchased from Biotech. Anti-pAkt and anti-Akt antibodies were purchased from Cell Signaling, and Anti–actin antibody was purchased from Santa Cruz Biotechnology. 2.2: Primary Retinal Cells Cultures We cultured primary retinal cells referencing other’s study and making some revision. Neonatal SD rats were sacrificed and then the eyeballs were enucleated and immediately placed into a beaker containing D-Hanks solution. 2 Ca2+ Influx’s Involvement in Retinal Protection The retinas were removed from the pigment epithelium layer with the aid of a dissecting microscope under sterile conditions and were 12876198 placed into a glass tube containing 1:1 Ham’s F-12DMEM medium. The beaker containing the eyeballs and the tube containing the retinas were placed onto ice. The retina fragments were treated with 0.25% trypsin at 37C for 8 mins and the digestion was terminated by adding three times the volume of 1:1 Ham’s F-12-DMEM containing 10% FBS. The suspension was filtered with a 200-mesh screen and centrifuged at 1000 rpm for 10 mins. After the supernatant was discarded, the cells were suspended, diluted with buy 1702259-66-2 medium containing 10% FBS to 1×106 cells/ml and plated onto 24-well or 6-well plates with 1 ml or 3 ml of cell suspension per well. Before culturing, all the plates were coated with poly-lysine and maintained in a humid incubator overnight. Next, we washed the plates three times with sterile double distilled water, once with D-Hanks balanced salt solution, and then with 200 l of medium, which provided a pre-environment for cell growth. The cells were cultured at 37C in a 5% CO2 atmosphere until they were used at 4-6 days in vitro, during which the medium was replaced according to the cell growth and metabolism conditions. Multiskan Spectrum, Thermo, Finland) with Skanlt RE for Mass 2.2 software after the plates were agitated at 37C for 10 mins. All absorbance values were subtracted by the blank value, and the untreated cultures were considered as the control group. The mean cell viability for each condition was determined by averaging at least quadruplicate values, the fold change relative to the control was calculated, and the control value