H9c2 cardiomyocytes were donated by Dr. He from Hubei university of medicine [17 (link)]. Cells were cultured in high glucose dulbecco’s modified eagle medium (DMEM) supplemented with 10% FBS (Gibco, C11995500), 100 IU/mL of penicillin and 100 μg/mL of streptomycin, and incubated in 95% air, 5% CO2. The detection of reactive oxygen species (ROS) and ∆Ψm in H9c2 cells was carried out according to the specifications of ROS detection kit (Beyotime, S0033) and mitochondrial membrane potential detection kit (Solarbio, CA1310). For the determination of ROS, 1 × 105/well H9c2 cells were first cultured under normal conditions (control group), O2 1% or O2 1% + QLQX, and then incubated with reactive oxygen species sensitive dye 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) solution at 37 °C for 20 min. In order to measure ∆Ψm, cells were treated as described above and then incubated with 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) staining solution (5 mg/ml) at 37 °C for 20 min. After staining, cells were washed twice with JC-1 staining buffer and detected by fluorescence microscope (Olympus FV3000RS). Fluorescence is measured at excitation/emission 485/580 nm (red) and then at excitation/emission 485/530 nm (green). The results were analyzed with Image-Pro Plus software.
Mitochondrial membrane potential detection kit
Manufactured by Solarbio
Sourced in China
The Mitochondrial membrane potential detection kit is a lab equipment designed to measure the electrochemical gradient across the inner mitochondrial membrane, which is an important indicator of mitochondrial function and health. The kit provides the necessary reagents and protocols to quantify the mitochondrial membrane potential in various cell types and samples.
Automatically generated - may contain errors
4 protocols using mitochondrial membrane potential detection kit
1
Assessing Oxidative Stress in H9c2 Cardiomyocytes
2
Mitochondrial Membrane Potential Assay with JC-1
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The mitochondrial membrane potential detection kit (Solarbio, Beijing, China) uses JC-1 as a fluorescent probe to quickly and sensitively detect the changes of MMP. The cells were planked in 96 well plates overnight, paclitaxel solution was added, and incubated at 37 °C. 100 µL JC-1 working solution was added after cleaning. This was followed by incubation for 20 min at 37 °C. The plate was washed twice with JC-1 dye buffer, and 100 µL culture medium was added. The fluorescence spectrophotometer was used to detect fluorescence intensity with 490 nm excitation wavelength and 530 nm emission wavelength.
3
Mitochondrial Membrane Potential Analysis in COPD
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For recruited patients and healthy people, including 17 patients in the normal control group, 17 patients in the COPD group and 17 patients taking YQGB pills, peripheral blood lymphocytes were extracted using a lymph extraction kit (TBD, Tianjin, China). MitoScreen (JC-1) (BD) was used to detect the level of the mitochondrial membrane potential of peripheral blood lymphocytes in different groups. The extracted lymphocytes were stained using JC-1 and incubated for 15 min in the dark. Flow cytometry was performed after resuspension of the assay buffer.
The mitochondrial membrane potential of HBE cells was detected using a mitochondrial membrane potential detection kit (Solarbio, Beijing, China). The cell concentration was adjusted to 1×106 cells/mL and then added to a 2 µm JC-1 probe, and the cells were incubated for 30 min at 37 ℃ in the dark. After being washed three times with phosphate buffered saline (PBS), the cells were stained with 4',6-diamidino-2-phenylindole (DAPI) to determine their nuclei, and then they were photographed and observed under a fluorescence microscope.
The mitochondrial membrane potential of HBE cells was detected using a mitochondrial membrane potential detection kit (Solarbio, Beijing, China). The cell concentration was adjusted to 1×106 cells/mL and then added to a 2 µm JC-1 probe, and the cells were incubated for 30 min at 37 ℃ in the dark. After being washed three times with phosphate buffered saline (PBS), the cells were stained with 4',6-diamidino-2-phenylindole (DAPI) to determine their nuclei, and then they were photographed and observed under a fluorescence microscope.
4
Mitochondrial Membrane Potential in PCOS Granulosa Cells
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A lipophilic cation, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol carbocyanine iodide (JC-1), was used to assess the mitochondrial status in GCs. JC-1 changes its fluorescence reversibly from green (monomeric status) to red (multimeric status) with the change in MMP. Furthermore, JC-1 is rapidly taken up by normal polarised mitochondria to form JC-1 aggregates, and presents a red spectral shift that results in increased levels of red fluorescence emission. In GCs of PCOS, the MMP collapses, and the JC-1 that accumulates within the mitochondria declines. In these cells, JC-1 remains in the cytoplasm in its monomeric form, and subsequently reduces the red fluorescence. Briefly, after culturing for 24 hours, the attached GCs from non-PCOS and PCOS were used to detect MMP using a Mitochondrial Membrane Potential Detection Kit (Solarbio, Beijing, China). GCs in 96-well plates were washed with PBS and incubated with JC-1 working solution at 37°C in a CO 2 incubator for 20 minutes. After rinsing twice with JC-1 staining solution, the MMP was monitored by a fluorescence spectrophotometer, and the relative MMP was calculated. After the GCs were attached, these were treated by the inhibitors of CCCP and BA at 20 minutes to detect MMP using the JC-1 Assay Kit mentioned above and a fluorescent microscope (Olympus, Tokyo, Japan).
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