Jc 1 assay kit

KML001 was a kind gift from Komipharm International (Seoul, Korea). Anti-P53, Horseradish Peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-GSK3β, anti-PTEN, anti-caspase-3, anti-PARP, anti-β-actin were from cell signaling technology (Beverly, MA, USA). FITC annexin V apoptosis detection kit was obtained from BD Pharmingen (San Jose, CA, USA). JC-1 assay kit from Molecular Probes (Eugene, OR, USA). Immobilion-P polyvinylidene difluoride (PVDF) membranes (0.45 µm) were from Millipore (Bedford, MA, USA). Micro-BCA protein assay reagents and Chemiluminescent reagents were from Pierce (Rockford, IL, USA). MG132, Pan-caspase inhibitor, Puromycin, Akt inhibitor II were obtained from Calbiochem (La Jolla, CA, USA). Chloroquine was purchased from Sigma-Aldrich. LY294002 was purchased from LC Laboratories (Woburn, MA, USA).

ALA (463-40-1) (0.914gm/ml) was purchased from TCI Chemicals (India) Pvt. Ltd. RPMI 1640 (Gibco, USA); fetal bovine serum (FBS) (Gibco-10270); trypsin (Gibco, USA); eagle balanced salt solution (EBSS)(Gibco, 2018-11); hank’s balanced salt solution (HBSS) (Himedia, TL1190); EtBr (Himedia, MB071); AO(Himedia, MB116); JC-1 assay kit (Thermo Scientific, M34152); PI (SC-3541); tamoxifen citrate (TMX) (Tammodex 20, Biochem Pharmaceuticals India); penicillin- streptomycin (Thermo Scientific, 15410-163); gentamycin (Thermo Scientific, 15710-049);3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) (Himedia, TC191); RNase (SRL, 58895); dimethyl sulfoxide (DMSO) (Merck, 1.16743.0521); DMBA (Sigma Aldrich, 57-97-6); ponceau S (Himedia, ML045); sodium cacodylate (Sigma Aldrich, C0250); collagenase type 4 (Himedia, TC-214); hyaluronidase (Himedia, TC331); hematoxylin (Himedia, S058); eosin (Himedia, S007); RIPA lysis buffer (Amresco, N653); protein assay kit (Amresco, M173); bovine serum albumin (BSA) (Genetix, PG-2330); transfer buffer (Genetix, GX-9411AR), trizol reagent (Sigma-T9424), cDNA synthesis kit (Genetix-K1612). Caspase 3 (SC-4263) and caspase 8 (SC-4267) assay kits were procured from Santacruz Biotechnology Inc., California, Delaware. All others chemicals were of molecular biology grade and purchased from Genetix Biotech Asia Pvt. Ltd, New Delhi.

Mitochondrial membrane potential was determined using the JC1 assay kit (Thermo Fisher Scientific, San Jose, CA, USA) according to the manufacturer’s protocol. In brief, blastocysts were transferred to PBS+PVA containing JC1 at 38.5°C in a humidified atmosphere of 5% CO2 for 10 minutes. Thereafter, the blastocysts were washed several times with PBS+PVA. The ratio of the intensity of red fluorescence (aggregate molecules) to green fluorescence (monomer molecules) was evaluated using ImageJ (National Institutes of Health, Bethesda, MD, USA).

MMP was measured via staining with the JC-1 assay kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. Briefly, the cells were pretreated with SA (100, 200, and 400 μM) for 24 h followed by treatment with 50 μM of 6-OHDA for another 12 h, after which they were incubated with 10 μg/mL of JC-1 dye for 20 min at 37 °C. JC-1-stained cells were observed using a fluorescence microscope equipped with iXon EMCCD cameras (Oxford Instruments, Oxfordshire, UK). Green fluorescence intensity was measured using a fluorescence spectrophotometer (Spectra Max M5) at 550 nm excitation and 600 nm emission wavelengths, respectively, whereas red fluorescence was measured at 485 nm excitation and 535 nm emission wavelengths, respectively.

The ROS levels, GSH levels, and MMP levels in the oocytes were measured using a ROS detection kit (Beyotime, Shanghai, China), GSH level detection kit (Thermo Fisher Scientific, Waltham, MA, USA), and JC-1 assay kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively. To determine the ROS levels in the oocytes, cumulus cell-free oocytes were cultured in PBS-PVA medium containing DCFH-DA (10 μM) for 30 min. To determine the GSH levels in the oocytes, cumulus cell-free oocytes were cultured in PBS-PVA medium containing CMF2HC (10 μM) for 30 min. To determine the MMP levels in the oocytes, cumulus cell-free oocytes were cultured in PBS-PVA medium containing JC-1 (2 μM) for 30 min. After incubation, the dye was washed off with PBS-PVA medium, and the fluorescence intensity of the oocytes was detected and captured using a fluorescence microscope and then saved as a Tiff file. The fluorescence signal intensity of each group of oocytes was analyzed using ImageJ software.