Description
Key facts
Detection method:Fluorescent,
Sample types:Suspension cells, Adherent cells,
Assay time:1h,
Assay Platform:Microplate reader
Product details:
JC-10 Mitochondrial Membrane Potential Assay Kit (Microplate) ab112134 enables researchers to analyze a JC-10 assay with a microplate reader.
How the assay works
The JC-10 assay provides the most robust assay method for monitoring mitochondria membrane potential changes.
This mitochondrial membrane potential assay protocol is based on the detection of the mitochondrial membrane potential changes in cells by the cationic, lipophilic JC-10 dye. In normal cells, JC-10 concentrates in the mitochondrial matrix where it forms red fluorescent aggregates. However, in apoptotic and necrotic cells, JC-10 diffuses out of mitochondria. It changes to monomeric form and stains cells in green fluorescence.
Although JC-1 is widely used in many labs, its poor water solubility causes great inconvenience. Even at 1 μM concentration, JC-1 tends to precipitate in aqueous buffer. JC-10 is developed to be a superior alternative to JC-1 when high dye concentration is desired. Compared to JC-1, JC-10 has much better water solubility. JC-10 is capable of selectively entering mitochondria, and reversibly changes its color from green to orange as membrane potentials increase. This property is due to the reversible formation of JC-10 aggregates upon membrane polarization that causes shifts in emitted light from 520 nm (i.e. emission of JC-10 monomeric form) to 570 nm (i.e. emission of J-aggregate form). When excited at 490 nm, the color of JC-10 changes reversibly from green to greenish orange as the mitochondrial membrane becomes more polarized.
In normal cells, JC-10 concentrates in the mitochondrial matrix where it forms red fluorescent aggregates. However, in apoptotic and necrotic cells, JC-10 exists in monomeric form and stains cells green. The green emission can be analyzed in fluorescence channel 1 (FL1) and greenish orange emission in channel 2 (FL2). Besides its use in fluorescence microplate platform, it can also be used in fluorescence imaging and flow cytometry.
JC-10 Mitochondrial Membrane Potential Assay Kit protocol summary
- Prepare cells
- Add test compounds
- Add JC-10 dye-working solution (50 μL/well/96-well plate or 12.5 μL/well/384-well plate)
- Incubate at 37°C, 5% CO2 incubator for 30 to 60 minutes
- Add Assay Buffer B (50 μL/well/96-well plate or 12.5 μL/well/384-well plate)
- Monitor fluorescence intensities (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm) and 540/590 nm (Cutoff = 570 nm)
How other researchers are using ab112134
JC-10 Mitochondrial Membrane Potential Assay Kit has been used in a variety of sample type including:
- ciPTEC cell lines 1
- HepG2 cells 2
- Ovarian cancer cells 3
References: 1 - Faria j et al. 2023; 2 - Abasi U et al. 2023; 3 - Walker T et al. 2023.
Related and recommended products
If you would like to use JC-10 on a flow cytometer, we recommend
ab112133
JC-10 Mitochondrial Membrane Potential Assay Kit (Flow Cytometry)
See other alternative kits to quantify Mitochondrial Membrane Potential:
- JC-1 - Mitochondrial Membrane Potential Assay Kit
ab113850
- NIR Mitochondrial Membrane Potential Assay Kit (Flow Cytometry)
ab112149
- Orange Mitochondrial Membrane Potential Assay Kit (Flow Cytometry)
ab138898
- TMRM Assay Kit (Mitochondrial Membrane Potential)
ab228569
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C, Storage information--20°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
The mitochondrial membrane potential also known as Δ ψm is the electrical potential difference across the inner mitochondrial membrane. This potential results from the electrochemical gradient produced by the proton pumps during electron transport chain activity. The mechanical function of the mitochondrial membrane potential is important to ATP production through oxidative phosphorylation. Mitochondrial membranes are widely expressed in almost all eukaryotic cells and are an essential component of cellular metabolism. The inner membrane is structured to facilitate its function housing integral proteins that are key to maintaining the potential.
Biological function summary
The mitochondrial membrane potential drives ATP synthesis by powering ATP synthase an enzyme complex embedded in the mitochondrial membrane. This potential also plays a vital role in other processes such as calcium homeostasis and regulation of mitochondrial biogenesis. The mitochondrial membrane itself forms part of the larger mitochondrial respiratory chain complex coordinating with components like complex I (NADH: ubiquinone oxidoreductase) and complex II (succinate dehydrogenase) to maintain cell energy needs and respond to metabolic demands.
Pathways
The mitochondrial membrane potential is integral to cellular energy metabolism pathways such as the Krebs cycle and oxidative phosphorylation. Mitochondrial membrane potential modulation can affect signaling proteins like cytochrome c which is instrumental in apoptosis. Apoptotic signaling pathways involving proteins such as Bax and Bcl-2 influence the mitochondrial membrane potential and regulate cell survival or death in response to cellular stress or damage.
Changes in the mitochondrial membrane potential relate significantly to conditions like neurodegenerative diseases and cancer. In neurodegenerative diseases such as Parkinson's and Alzheimer's dysregulation of mitochondrial membrane potential can lead to impaired energy production and increased oxidative stress. Cancer cells often exhibit altered mitochondrial membrane potential affecting processes like apoptosis and enabling survival in adverse conditions. These alterations in potential impact proteins such as p53 which play critical roles in cancer progression and neurodegenerative disease pathology.
