Gsh level detection kit

The intracellular ROS and GSH levels after oocyte maturation (non-activated) were detected by ROS detection kit (Thermo Fisher Scientific, #C400) and GSH level detection kit (Thermo Fisher Scientific, #C12881), respectively. To determine intracellular ROS levels, oocytes were incubated in 0.1% PBS-PVA medium containing 10 μM 2′,7′ dichlorodihydrofluorescein diacetate (H2DCFDA) for 30 min. To determine intracellular GSH levels, oocytes were incubated in 0.1% PBS-PVA medium containing 10 μM 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CMF2HC) for 30 min. After washing thrice with PBS-PVA, fluorescence microscopy (Eclipse Ti2; Nikon, Tokyo, Japan) and ImageJ software (NIH, Bethesda, MD, USA, https://ij.imjoy.io/) were used to analyze the fluorescence intensities.

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.