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JC-10 Mitochondrial Membrane Potential Assay Kit (Microplate) (ab112134) enables researchers to run JC-10 assay in the format of microplate reader, and it provides the most robust assay method for monitoring mitochondria membrane potential changes. This assay 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.
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A microplate reader with bottom-reading mode is essential to perform this assay.
If you would like to use JC-10 on a flow cytometer, we recommend JC-10 Mitochondrial Membrane Potential Assay Kit (Flow Cytometry) (ab112133).
|Components||5 x 96 tests|
|100X JC-10 in DMSO||1 x 250µl|
|Assay Buffer A||1 x 25ml|
|Assay Buffer B||1 x 25ml|
CGN were cultured on 96-well white-walled clear-bottom plates in phenol-red free Neurobasal until 7 DIV. Thirty minutes before the end of the treatment, 50 μl of JC-10 dye-loading solution was added to each well and incubated for 30 minutes before measuring fluorescence intensities (Ex/Em = 485/515 nm and Ex/Em = 540/590 nm). The change of mitochondrial membrane potential was measured as the ratio between aggregate (Em = 515 nm) and monomeric forms (Em = 590 nm) of JC-10. Increasing ratios indicate mitochondrial membrane depolarization.
JC-10 Mitochondrial Membrane Potential Assay Kit (Microplate) (ab112134). Camptothecin-induced mitochondria membrane potential changes with JC-10 and JC-1 in Jurkat cells. JC-1 (blue bar) and JC-10 (red bar) dye loading solutions were added to Jurket cells untreated (0 µM) or treated with camptothecin (10 µM for 4 hours) and incubated for 30 minutes. The fluorescent intensities for both J-aggregates and monomeric forms of JC-1 and JC-10 were measured at Ex/Em = 490/525 nm and 490/590 nm with a microplate reader.
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