Mitochondrial membrane potential detection kit

R2C cells at a density of 3 × 10⁵ cells per well were seeded in 6-well plates and cultured in a 37 °C, 5% CO₂ incubator for 24 h. The medium was replaced with 160 μM of AAPH and 50 μM of different anthocyanins. After 24 h, the cells were digested and resuspended with 0.5 mL of medium. According to the instructions of the mitochondrial membrane potential detection kit (Beyotime Biotechnology, Shanghai, China), 500 μL of staining working solution was added to each tube, the tube was then incubated at 37 °C for 20 min, centrifuged at 4 °C, 600× g for 5 min, and the supernatant was discarded. Then, 1 mL of staining buffer was added to each group and centrifuged under the same conditions, and the supernatant was discarded and repeated again. Cells were resuspended in a 500 μL staining buffer, and the changes in MMP were detected by flow cytometry (Beckman Coulter, Brea, CA, USA). The red fluorescence excitation wavelength was 585 nm, emission wavelength was 590 nm; and the green fluorescence excitation wavelength was 514 nm, emission wavelength was 529 nm. Data were analyzed with Flowjo software (B&D Biosciences, Franklin Lakes, NJ, USA).

The changes of sperm mitochondrial membrane potential (ΔΨm) were evaluated using a JC-1 (lipophilic cation 5,5^’, 6,6’- tetrachloro-1,1’,3,3’ -tetraethylbenzimidazolcarbocyanine iodide) Mitochondrial Membrane Potential Detection Kit (Beyotime Institute of Biotechnology, China), following the manufacturer’s instruction [27 (link)–28 (link)]. Briefly, the sperm samples (2×10⁶/mL) were stained with 28 μL of JC-1 (stock solution) in PBS (final volume, 100 μL). After incubation at 37°C for 30 min in the dark‚the samples were centrifuged at 600 x g for 5 min, and re-suspended in JC-1 buffer and placed on ice. Sperm samples were immediately analyzed using a flow cytometer (FAC SCalibur, BD Biosciences) with excitation at 525 nm and emission at 590 nm. A total of 20,000 sperm-specific events were evaluated and calculated as percentages. Data were processed using the CellQuest program (BD Biosciences).

Flow cytometry was used to analyze mitochondrial membrane potential (MMP). Briefly, GC2 cells were harvested after transfection of miR-574 mimics or inhibitors for 24 h, and then stained with JC-1 according to the manufacturer’s instructions. The mitochondrial membrane potential detection kit (Beyotime) was used for cell staining. The stained cells were detected by a FACS Caliber (Becton Dickinson, Mountain View, NJ, USA) and analyzed using Modfit LT software (Becton Dickinson). All experiments were performed in three replicates.

The changes of sperm mitochondrial membrane potential (ΔΨm) were evaluated using a JC-1 (lipophilic cation 5,5^’,6,6’-tetrachloro-1,1’,3,3’ -tetraethylbenzimidazolcarbocyanine iodide) Mitochondrial Membrane Potential Detection Kit (Beyotime Institute of Biotechnology), by following the manufacturer’s instruction [23 ]. Briefly, the sperm samples (2×10⁶/mL) were stained with 28 μL of JC-1 (stock solution) in PBS (final volume, 100 μL). After being incubated at 37°C for 30 min in the dark, the samples were centrifuged at 600 × g for 5 min, and re-suspended in JC-1 buffer and placed on ice. Sperm samples were immediately analyzed in a flow cytometer (FAC-SCalibur, BD Biosciences) with excitation at 525 nm and emission at 590 nm. A total of 20 000 sperm-specific events were analyzed and the percentage of spermatozoa with red fluorescence in total sperm number was calculated. Data were processed by using the CellQuest program (BD Biosciences).

Mitochondrial membrane potential (Δψm) was measured using a Mitochondrial Membrane Potential Detection Kit (Beyotime, No. C2006, Haimen, China). Briefly, membrane potential was monitored by determining the relative amount of dual emissions from mitochondrial JC-1 monomers or aggregates using an Olympus fluorescent microscope (Olympus, Tokyo, Japan) under Argon-ion 520 nm laser excitation. Mitochondrial depolarization was expressed as the increased ratio between green and red fluorescence intensities.

The lipophilic cationic fluorescent dye 5, 5′, 6, 6′-terachloro-1, 1′, 3, 3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1) was used to detect changes in the MMP. Cells were cultured in 6-well plates. Measurements were performed using the Mitochondrial Membrane Potential Detection Kit (Beyotime Institute of Biotechnology), according to the manufacturer’s instructions. In brief, cells were collected and incubated with 10 μg/ml of the MMP-sensitive fluorescent dye JC-1 at 37°C for 20 min, washed twice in PBS, and subjected to flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).