Nanodrop uv vis spectrophotometry

ROS generation in GCs was measured using 2′,7′-diclorodihydrofluorescein di-acetate (H2-DCFDA) method by ROS assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). Briefly, GCs were resuspended in PBS and incubated with 10 μM H2-DCFDA in the dark for 25 min at 37 °C, and then incubated with 10 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) (NeoFroxx, Frankfurt, Germany) for 5 min. After washed three times with PBS, GCs suspensions were added to glass slides, and examined by fluorescence microscopy (Olympus Corporation, Tokyo, Japan). The examination wavelength was 488 nm, and the emission wavelength was 525 nm.
Similar with the above protocol but without DAPI, NanoDrop UV-Vis spectrophotometry (Thermo Scientific, MA, USA) was used to measure the intracellular ROS level of GCs. The relative ROS levels were presented as the fluorescence intensity of the PCOS group relative to that of non-PCOS controls.

With a dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe, ROS levels in GCs were detected by ROS assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). Briefly, GCs were resuspended in PBS and incubated with 10 μmol/L DCFH-DA in the dark for 25 min (37 °C) and then incubated together with 10 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) (NeoFroxx, Frankfurt, Germany) for 5 min. After the cells were washed three times with PBS, GC suspensions were added to glass slides, and fluorescence was examined by fluorescence microscopy (Olympus Corporation, Tokyo, Japan). The examination wavelength was 488 nm, and the emission wavelength was 525 nm.
Similar to the above protocol but without DAPI, another set of GCs was used to measure the intracellular ROS level by NanoDrop UV-Vis spectrophotometry (Thermo Scientific, Massachusetts, USA). The fluorescence intensities are shown as the intensity in the POR group relative to that in the control group (non-POR group).