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To monitor mitochondrial health, JC-1 dye (invitrogen) was used to assess mitochondrial membrane potential. UCMSCs or F-Mito were incubated with JC1 (5 μM or 1 μM) for 20 min at 37°C. JC1 dye exhibits potential-dependent accumulation in mitochondria, indicated by fluorescence emission shift from green (Ex 485 nm/Em 516 nm).to red (Ex 579 nm/Em 599 nm).
Mitochondria membrane potential (MMP) in UCMSCs was determined by the flow cytometry and fluorescence microscopy of SLIDEVIEW (VS200, OLYMPUS) and MMP in F-Mito was detected by fluorescence microscopy of SLIDEVIEW (VS200, OLYMPUS).

The Leydig cell coverslips and testicular sections (4 μm) were fixed with 4% (v/v) paraformaldehyde for 30 min at 37 °C The slides were blocked using Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) before being incubated for 16–18 h at 4 °C with the primary antibodies anti-MT1/-MT2 (1:500 dilution) and anti-3β-HSD (1:500 dilution). The slides were incubated with secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 594 for 60 min at room temperature after being washed with PBS (1:400 dilution; ZSGB-BIO, Beijing, China). Finally, the nuclei were stained with DAPI (Thermo, 1:5000 dilution) for 5 min at room temperature. Then, the slides washed twice with PBS. The fluorescence images were captured using digital slide-scanning equipment (model: VS200, SLIDEVIEW, Olympus, Tokyo, Japan).

Cells with the marker of interest were identified using SLIDEVIEW (VS200, OLYMPUS). Mean fluorescence intensity of Mitotracker Green for F-Mito or MitoSOX red for intracellular ROS levels was quantified in 300 random cell samples of MitoSOX red positive. Background was first corrected by segmenting images so that the entireties of cells were broadly outlined and averaging fluorescence signal from the background and then subtracted it from the images. This procedure was repeated for all images at each channel. Next, cells that divided, balled up, or displayed some abnormality (e.g., double-nucleated, abnormally large) were excluded. A mask in the WGA blue fluorescence image was used to identify the whole cell and to calculate single cell area. Mean fluorescence intensity (mean gray value) was calculated by dividing the Mitotracker or MitoSOX integrated fluorescence density by the single cell area. We performed all segmentation and fluorescence quantitation steps using the ImageJ of 6 version by the method described previously (Anguissola et al., 2011 (link)).