Celltracker blue 4 chloromethyl 6.8 difluoro 7 hydroxycoumarin

The intra-oocyte GSH contents of IVM oocytes were measured by applying previously described methods [17 (link)23 (link)]. Briefly, CellTracker Blue 4-chloromethyl-6.8-difluoro-7-hydroxycoumarin (Invitrogen) was used to detect intra-oocyte GSH based on blue fluorescence. From each treated group, denuded oocytes having visually extruded first polar body were incubated in the dark for 30 min in TLH-PVA supplemented with 10 μM CellTracker, followed by incubation for an additional 30 min in IVC medium. The oocytes were subsequently washed with Dulbecco’s phosphate-buffered saline (Invitrogen) containing 0.1% (w/v) PVA, and placed in 2-µL micro-droplets. Fluorescence was observed under an epifluorescence microscope (TE300; Nikon) with ultraviolet ray filters at 370 nm. The fluorescence intensity of each oocyte was analyzed using the ImageJ software (version 1.49q; National Institutes of Health), and normalized to that of oocytes matured in standard M199.

The intraoocyte GSH contents of IVM oocytes were measured using previously described methods [23 (link)]. Briefly, CellTracker Blue 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (Invitrogen) was used to detect intracellular GSH with blue fluorescence, respectively. A group of 10 to 15 denuded oocytes at the MII stage from each treatment group was incubated for 30 min in TLH containing 0.05% (w/v) polyvinyl alcohol (PVA) supplemented with 10 µM CellTracker in the dark. Oocytes were then washed three times, after which they were incubated for an additional 30 min in IVC medium in the dark. Following incubation, the oocytes were washed with D-PBS (Invitrogen) containing 0.1% (w/v) PVA, then placed into 2-µL droplets, and the fluorescence was observed under an epifluorescence microscope (TE300; Nikon) with ultraviolet ray filters (370 nm). The fluorescence intensities of oocytes were subsequently analyzed by the ImageJ software (ver. 1.46r; National Institutes of Health, USA) and normalized against untreated MPCOCs oocytes.