Jc 1 mitochondrial membrane potential detection kit

Apoptosis of hDPCs was evaluated using an Annexin V-FITC/propidium iodide (PI) double staining assay (BD, Bergen, NJ, USA). After treatments with melatonin and (or) H₂O₂, hDPCs were collected, rinsed, and incubated with Annexin V-FITC and PI for 15 min at room temperature. Cell samples were then analyzed by flow cytometry (Beckman Coulter, Brea, CA, USA). The proportions of Annexin V-positive cells were recorded as apoptotic rates.
Intracellular ROS levels were measured using CellROX® Green (Invitrogen, Grand Island, NY, USA). Upon H₂O₂ and melatonin treatment, hDPCs were collected and incubated with CellROX® reagent for 1 h at 37 °C, washed with PBS for three times, and analyzed by flow cytometry. Intracellular ROS levels were indicated by the mean intensity of green fluorescence.
A Mitochondrial Membrane Potential Detection Kit (JC-1; Beyotime, Shanghai, China) was employed to analyze mitochondrial membrane potential (ΔΨm) in hDPCs. After various treatments, hDPCs were collected and incubated with JC-1 staining solution for 20 min at 37 °C. After rinsing, the cells were resuspended for flow cytometry. The ratio of red (J-aggregates)/green (JC-1 monomer) fluorescence was calculated, which represents ΔΨm.
A total of 10,000 cells were harvested from each sample for these flow cytometric analyses. Experiments were repeated at least three times.

The Mitochondrial Membrane Potential Detection Kit (JC-1; Beyotime Institute of Biotechnology, Shanghai, China) was used to observe changes in the mitochondrial potential.32 (link) Briefly, 5 mg/mL JC-1 working solution was added to the medium and incubated for 30 minutes at 37°C with CO₂. The cells were subsequently washed with PBS to remove the JC-1 probe, and images were obtained via fluorescence microscopy (BX-61; Olympus Corporation). The ratio of red to green fluorescence was analyzed using Image-Pro Plus version 4.5 (Media Cybernetics, Inc., Rockville, MD, USA).33 The LDH release assay was used to observe cell death according to the manufacturer’s guidelines.34 (link) The relative LDH release was recorded as the ratio to that of the control group. The experiments were performed in triplicate and repeated three times with similar results.