Enhanced mitochondrial membrane potential assay kit with jc 1

To evaluate changes in mitochondrial membrane potential (MMP), we employed the Enhanced Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime, China) and measured the relative ratio of red to green fluorescence (PE/FITC) using flow cytometry. Mitochondrial ROS (mtROS) was determined using the MitoSOX™ red mitochondrial superoxide indicator (Invitrogen, USA). MФ was loaded with MitoSOX diluted in HBSS (Gibco, USA) in darkness for 30 min at 25°C, and rinsed with warm HBSS before flow cytometry analysis. Additionally, we used Live Cell Staining Solution Hoechst 33342 (Beyotime, China) to stain the cells during MitoSOX loading, and visualized mtROS production in individual cells using a laser confocal scanning microscope (LSM710) (Carl Zeiss, Germany). Analysis of resulting fluorescence images were performed with ZEN 3.5.

Oocyte mitochondrial membrane potential was detected with the Enhanced Mitochondrial Membrane Potential Assay Kit with JC-1 (C2003S, Beyotime, Shanghai, China). Briefly, about 30 oocytes were transferred into working solution diluted 1:400 with KSOM medium and cultured at 37 °C for 30 min. Following 3 rapid washes with the KSOM medium, the oocytes were imaged using a Nikon BX63 fluorescence microscope. The ratio of red to green fluorescence intensity was calculated to represent mitochondrial membrane potential. The mitochondrial membrane potential was measured four times (n = 20 oocytes for each group).

Analysis of C2C12 myoblast activity was performed using the Enhanced Mitochondrial Membrane Potential Assay Kit with JC-1 (JC-1, 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide, molecular formula C₂₅H₂₇Cl₄IN₄) (Beyotime, China). After exposure to hypoxia for 24 h, the C2C12 myoblasts were washed once with PBS. Next, 1 mL of growth medium and 1 mL of JC-1 staining solution (5 uL/mL JC-1) were added, and the cells were incubated at 37 °C for 20 min. C2C12 myoblasts were then washed twice with PBS. Images were acquired using a fluorescence microscope (Leica Microsystems, Germany), and the relative proportions of red and green fluorescence were used as a measure of mitochondrial depolarization.