Imagexpress confocal microscope

cGCs were cultured with 10 µm/l Mito-Tracker Red CMXRos staining solution for 30 min at 37°C in 5% CO₂. After washing thrice with PBS buffer. Then, cGCs were stained using 10 µg/ml Hoechst 3342, incubated for 10 min at 37°C in 5% CO₂, and then washed thrice with PBS buffer (Seville) before placing in fresh medium. The fluorescence intensity of mitochondrial Mito-Tracker Red CMXRos was detected immediately with an ImageXpress confocal microscope (Molecular Devices, LLC). The captured images were assessed, using ImageJ Imaging System software (version 1.51; National Institutes of Health). Mito-Tracker Red levels were assessed by calculating the mean red fluorescence intensity. This experiment involved 3 patients with CON and 4 patients with PCOS.

The MMP of cGCs was measured using the fluorescent dye 5,6-dichloro-2-[3[(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-benzimidazole-2-ylidene)-1-propen-1-yl]-1,3-diethyl-1H benzim-idazolium, monoiodide (JC-1; Cayman Chemical Company). cGCs were collected and labeled with 10 µmol/l JC-1 fluorescent probe in 5% CO₂ at 37°C for 30 min. Then were washed thrice with PBS buffer. The aforementioned images were captured using ImageXpress confocal microscope (Molecular Devices, LLC) to demonstrate green fluorescence and red fluorescence. Then, captured images were further analyzed using ImageJ Imaging System software (version 1.51). The fluorescence was expressed as a ratio of red to green. This experiment involved 4 patients with CON and 3 patients with PCOS.

Images were acquired using a 60× objective on a Nikon Eclipse TE2000-U epifluorescence microscope and exported into CellProfiler (Carpenter et al., 2006 (link)) to analyze the number of pixels positive for each antibody normalized by neurite length. To avoid potential biases in results related to distance from the soma or dendritic order, we quantified the signal along neurite segments at various distances from the cell body and averaged. Manual analysis of microscopy data was performed in MetaMorph. N-SIM images were captured on a Nikon N-SIM Structured Illumination superresolution microscope. Confocal images were captured on a Nikon A1R+ confocal laser microscope system. Wide-field fluorescent images were captured using a Molecular Devices ImageXpress confocal microscope at 40×. Western blots were quantified using ImageJ (National Institutes of Health). Numerical data from each experimental repetition were exported and pooled for descriptive and statistical analysis in Prism 5 (GraphPad). All experiments were carried out a minimum of three times. In each experiment, each experimental condition contained at least triplicate samples. qPCR data are reported as geometric means ± 95% confidence intervals. All other data are reported as means ± SEM.

The 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe (Cayman Chemical Company) was employed to detect the intracellular ROS levels. Briefly, cGCs were resuspended using 10 µmol/l DCFH-DA, incubated for 30 min at 37°C in the dark, and then washed thrice with PBS buffer (cat. no. 02-024-1ACS; Biological Industries; Sartorius AG). cGCs were resuspended using 10 µg/ml Hoechst 3342 (Cayman Chemical Company) and incubated for 10 min in the dark at 37°C, and then washed thrice with PBS buffer. The cell fluorescence intensity was detected with an ImageXpress confocal microscope (Molecular Devices, LLC), and the ROS levels were expressed as the mean green fluorescence intensity. This experiment involved 7 patients with CON and 5 patients with PCOS.