Jc 1 solution

The nuclei were initially stained for mitochondrial labeling. In this experiment, cells were treated with Hoechst dye, incubated for 16 min at 37 °C, and washed three times with D-PBS. The mitochondria of hiPSC-CMs were then stained for 25 min at 37° with a pre-warmed 0.2 M Mito Tracker working solution (Beyotime, China) or JC-1 solution (Beyotime, China). A confocal microscope A1R was utilized to analyze the staining and fluorescence intensity (Nikon, Tokyo, Japan). We analyze the mitochondria morphology using Mitochondria Analyzer as previously described [46 (link),47 (link),48 (link)]. The formula “(Perimeter²)/(4PiArea)” was utilized to determine the mean form factor (FF) value, which reflects both the length and degree of branching of mitochondria. Using the formula “(Major Axis)/(Minor Axis),” a mean aspect ratio (AR) value was determined for each cell as a measure of mitochondrial length. To measure mitochondrial membrane potential ((ΔΨm), the JC-1 kit was utilized. Green fluorescent monomers signify low ΔΨm for JC-1 staining, but red fluorescent aggregates indicate high ΔΨm.

JC-1 probe was used to detect the changes in the mitochondrial membrane potential [38 (link)]. HepD cells were seeded in a glass-bottomed culture dish at a density is 2 × 10⁵/mL and were incubated with 0, 20, 50, 75, and 100 μg/mL Im and La mixture for 24 h. After that, the HepD cells were washed with PBS three times and incubated with JC-1 solution (10 μg/mL; Beyotime, Shanghai, China) for 30 min at 37 °C. Then, the stained cells were imaged by the fluorescence microscope (Olympus Corporation, Tokyo, Japan). The fluorescence intensity was analyzed with ImageJ software.

The cells were incubated with JC-1 solution (Beyotime) at 37 °C, 30min. A microreader was used to obtain the fluorescence of MMPs at 540 and 490 nm.

Cells were first treated with JC-1 solution (Beyotime, China). Then, the fluorescence-labeled cells were washed and analyzed using an EnSpire Reader. Experiments were repeated three times.

flow cytometry was performed to detect reactive oxygen species (ROS) formation, apoptosis, and mitochondrial membrane potential (MMP) in H9c2 cells. For the ROS generation assay, 1 × 106 H9c2 cells in each group were resuspended in 1 mL of diluted DCFH-DA and maintained at 37°C for 20 min. Thereafter, the cells were treated with 500 μL phosphate-buffered saline (PBS) and analyzed with flow cytometry (ACEA Biosciences, San Diego, CA, USA). For the apoptosis assay, 1 × 106 H9c2 cells in each group were centrifuged at 400 × g at 4°C for 5 min, resuspended in 200 μL PBS, and stained for 30 min with 10 μL annexin V-fluorescein isothiocyanate (FITC) and 10 μL propidium iodide (PI) in the dark at 4°C. After 300 μL PBS was added, the cells were analyzed with flow cytometry (ACEA Biosciences). For the MMP assay, 1 × 106 H9c2 cells in each group were resuspended in 500 μL DMEM and cultured for 20 min with JC-1 solution (Beyotime) at 37°C. Thereafter, the cells were centrifuged at 400 × g at 4°C for 3 min and resuspended in 1 mL of JC-1 solution. The cells were then centrifuged at 400 × g at 4°C for 3 min, resuspended in 400 μL of JC-1 solution, and analyzed with flow cytometry (ACEA Biosciences).