Thioltracker violet glutathione detection reagent

The intracellular ROS and GSH levels in the control and PGE₂-treated oocytes were measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCHFDA, #D6883; Merck, Sigma-Aldrich, Darmstadt, Germany) and ThiolTracker™ Violet Glutathione Detection Reagent (#T10095; Invitrogen/Molecular Probes, Thermo Fisher Scientific, Waltham, Massachusetts, USA), respectively. After 24 h of IVM, denuded oocytes were incubated with 100 μM DCHFDA or 20 μM ThiolTracker™ Violet dye solution for 30 min at 37 °C in the dark. The oocytes were then washed three times in 0.1% PVA in D-PBS and placed at 4% paraformaldehyde in D-PBS for 30 min at room temperature (RT). After fixation, the oocytes were washed twice in D-PBS and observed under an Axio Observer Microscope System (Carl Zeiss, Poznan, Poland) using appropriate fluorescence filters with excitation/emission at 495 nm/529 nm and 404 nm/526 nm for DCHFDA and ThiolTracker™ Violet, respectively. The fluorescent images obtained were analyzed using ZEN blue 2.5 Pro Software (Carl Zeiss, Poznan, Poland) to determine the intensity of fluorescence in each oocyte. The data were calculated as an average fluorescence intensity ratio. This experiment was repeated three times with 25 COCs per treatment group.