The mitochondrial membrane potential (MMP, ΔΨm) of MPC5 podocytes was assessed using the JC-1 staining kit (Beyotime), following the standard kit instructions. Podocytes were washed twice with PBS and plated in a 6-well plate. JC-1 dye was added to each well, and the podocytes were subsequently incubated in the dark at 37 °C for 20 minutes. After washing with cold JC-1 staining buffer, cultured podocytes were quantified by flow cytometry to detect the fluorescent cells.
Jc 1 staining kit
Manufactured by Beyotime
Sourced in China
The JC-1 staining kit is a laboratory tool used to assess mitochondrial membrane potential in cells. It contains the JC-1 dye, which exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green to red. This kit provides the necessary components for performing this type of mitochondrial function analysis in a research setting.
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Lab products found in correlation
Fluorescence microscope, by Olympus (4 mentions)
Fluorescence microscopy, by Olympus (2 mentions)
β actin 13e5 rabbit mab, by Cell Signaling Technology (2 mentions)
Triton x 100 solution, by Beyotime (2 mentions)
Sulforhodamine b, by Solarbio (2 mentions)
Ca2 specific fluorescent probe fluo 4 am, by Beyotime (2 mentions)
Western blot antibody diluent, by Ipsen (2 mentions)
Reactive oxygen species assay kit, by Beyotime (2 mentions)
One step tunel apoptosis assay kit, by Beyotime (2 mentions)
Rnase a, by Solarbio (2 mentions)
Propidium iodide, by Solarbio (2 mentions)
Fluorescence microscope, by Nikon (2 mentions)
Laser confocal microscopy, by Leica (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Fv3000, by Olympus (1 mentions)
Mito tracker red cmxros, by Beyotime (1 mentions)
Tissue mitochondrial isolation kit, by Beyotime (1 mentions)
Fv1000 ix81, by Olympus (1 mentions)
Evos m7000 imaging system, by Thermo Fisher Scientific (1 mentions)
Greennuc caspase 3 assay kit, by Beyotime (1 mentions)
54 protocols using jc 1 staining kit
1
Mitochondrial Membrane Potential in Podocytes
2
Mitochondrial Membrane Potential in Chondrocytes
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The mitochondrial membrane potential of chondrocytes was assessed using a JC-1 staining kit (Beyotime). Briefly, the cells were collected and washed three times with PBS. Thereafter, the cells were stained with JC-1 (5 μg/ml) for 20 min at 37°C. The cells were then washed again three times with PBS to remove the residual JC-1. Cells were observed under confocal microscopy (Olympus, FV3000) (magnification, ×200). In addition, the mitochondrial membrane potential of chondrocytes was evaluated using the BD Accuri C6 Plus flow cytometer, and the data were analyzed using the FlowJo software (FlowJo LLC, version 10.6.0).
3
Mitochondrial Membrane Potential Assay
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JC-1 and Mito-Tracker Red CMXRos staining of mitochondria is dependent on the mitochondrial membrane potential. The cells transfected PER2-sh2 and the control lentiviral vector exhibited fluorescence upon excitation with a 488 nm laser. However, this laser’s spectral range overlapped with the fluorescence emission spectra of the JC-1 monomer, rendering it unsuitable for detecting MMP in the PER2-sh2 and the control cells. As a result, Mito-Tracker Red CMXRos dye was sought for MMP detection in these cells. Mito-Tracker Red CMXRos is suitable for fluorescent double-labeling experiments, and similar to JC-1, the staining of mitochondria with Mito-Tracker Red CMXRos is dependent on MMP. Cells were cultured in OM medium for 3 days and then stained with a JC-1 staining kit (monomer: 490/530; aggregate: 525/590; C2006, Beyotime, China) or Mitotracker Red CMXRos (100 nM, 15 min, 579/599; C1035, Beyotime, China) according to the manufacturer’s protocol. After that, living cells were viewed and imaged with a confocal laser scanning microscope.
4
Mitochondrial Membrane Potential Assessment
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The MMP levels in cells and kidney tissues were assessed using the JC-1 staining kit (Beyotime, Shanghai, China). For cellular MMP detection, cells were evenly seeded in six-well plates. JC-1 staining working solution was added following the instructions provided with the kit. After incubation, cells were washed with JC-1 staining buffer. The samples were observed under a fluorescence microscope to distinguish JC-1 monomers (green fluorescence) and JC-1 polymers (red fluorescence). MMP in kidney tissues was assessed using the following method. Firstly, kidney tissues were processed using a tissue mitochondrial isolation kit (Beyotime, Shanghai, China) to extract mitochondrial organelles. The total protein concentration in the mitochondrial samples was determined using the BCA protein quantification method. Subsequently, purified mitochondria were suspended in diluted JC-1 working solution. Finally, the fluorescence intensity values of JC-1 polymers and monomers were measured using a fluorescence microplate reader. Calculate the ratio of JC-1 polymers and monomers.
5
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential (MMP) was monitored by using JC-1staining kit (Beyotime, Shanghai, China). Cells were stained by JC-1 (10 μg/mL) at 37 °C for 20 min. MMP was visualized by fluorescence microscopy (Olympus, Tokyo, Japan). JC-1 monomers appear green fluorescence, indicating low MMP. JC-1 aggregates present red fluorescence, suggesting high MMP. Data are represented as the ratio of red to green fluorescence intensity.
6
Mitochondrial Membrane Potential Assay in LO2 Cells
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The JC-1 staining kit (C2006, Beyotime, Shanghai, China) was used to measure the mitochondrial membrane potential of LO2 cells. Cells (2.5 × 105) were resuspended in 0.5 mL of cell culture medium containing serum and phenol red. They were then incubated with 0.5 mL of JC-1 staining working solution (5×) for 20 min at 37 °C. After incubation, the cell suspension was centrifuged at 600g for 4 min at 4 °C to precipitate the cells. Afterward, the cell supernatant was aspirated and washed with ice-cold 1 × JC-1 staining buffer. Next, cells were resuspended by adding 1 mL of JC-1 staining buffer (1 ×), centrifuged at 600×g for 4 min at 4 °C, repeated twice. The precipitated cells were resuspended in 1 × JC-1 staining buffer again and analyzed with a flow cytometer (BD Biosciences, San Jose, CA, USA).
7
Measuring Mitochondrial Membrane Potential
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JC‐1 spontaneously creates J‐aggregates that produces red fluorescence in the mitochondria of healthy cells with elevated mitochondrial membrane potential (MMP). Nevertheless, the MMP decreases, and JC‐1 is discharged from the mitochondria of unhealthy cells and resides as a green fluorescent monomer in the cytoplasm. A JC‐1 staining kit (Beyotime Biotechnology) was utilized to monitor the MMP according to the instructions stipulated by the manufacturer. Adult mouse cardiomyocytes were stained with JC‐1 (10 μg/ml) for 20 min at 37°C. A fluorescence microscope (Olympus) was utilized to visualize MMP. JC‐1 monomers exhibit green fluorescence, indicative of reduced MMP, while the red fluorescence of JC‐1 aggregates indicates elevated MMP. The data are displayed as the red‐to‐green fluorescence intensity ratio.
8
Mitochondrial Membrane Potential Assay
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The MMP was determined using a JC-1 staining kit (Beyotime, Nanjing, China). Mitochondria were extracted from liver tissue and purified by differential centrifugation. Cells (1 × 105) were washed twice in phosphate-buffered saline (2000 rpm, 5 min each) and suspended in 500 μL of JC-1 working solution. The cells were incubated in 5% CO2 at 37 °C for 20 min. After washing them twice with the incubation buffer, the cells were resuspended in 1 mL of incubation buffer and subjected to flow cytometry. The red-to-green fluorescence intensity ratio was analyzed using Cell Quest and ImageJ software (NIH). For detecting JC-1 monomers and polymers, the excitation wavelength was 490 and 525 nm, and the emission wavelength was 530 and 590 nm, respectively. The MMP changes were calculated by the relative ratio of red-to-green fluorescence.
9
Assessing Mitochondrial Membrane Potential in HIF-1α Knockdown Cells
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Changes in ΔΨm were detected using a dual‐emission potential‐sensitive probe, 5, 5′, 6, 6′‐tetra‐chloro‐1,1′,3,3′‐tetraethyl‐imidacarbocyanine iodide (JC‐1) staining kit (Beyotime Biotechnology, Shanghai, China). CMECs were seeded in 6‐well plate and transfected with HIF‐1α siRNA for 48 hours. After being treated with QL and subjected to hypoxia for 12 hours, cells were incubated with dyeing working fluid for 20 minutes at 37°C. For images of JC‐1 monomers, the wavelengths were green (Ex = 514 nm, Em = 529 nm) and for J‐aggregates were red (Ex = 585 nm, Em = 590 nm), and images from 5 randomly chosen fields were photographed under a microscope (Olympus). Then, the fluorescence intensity of aggregates and monomers was analysed using ImageJ software using the same method as DHE staining, and the ratio of aggregates/monomers fluorescence intensity was calculated as an indicator of mitochondrial transmembrane potential.
10
Assessment of Mitochondrial Membrane Potential
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To assess mitochondrial membrane potential (MMP), a JC-1 staining kit (C2005, Beyotime) was used. After treatment, cells were collected and rinsed with PBS. The cells were then stained with 5 μM JC-1, as provided in the kit, for 30 minutes at 37 °C in the dark. As a positive control, 10 μM CCCP was applied 20 minutes prior to the assay. For flow cytometry, 1 × 104 cells were prepared and measured using a flow cytometer (BD Biosciences). For fluorescence microscopy, nuclei were stained with DAPI (D212, Dojindo), and images for every sample (400× magnification) were captured with a fluorescence microscope (IX81-FV1000, Olympus).
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