JC-10 Mitochondrial Membrane Potential Assay Kit (Microplate) (ab112134)

Overview

  • Product name

    JC-10 Mitochondrial Membrane Potential Assay Kit (Microplate)
    See all Mitochondrial Membrane Potential kits
  • Detection method

    Fluorescent
  • Sample type

    Adherent cells, Suspension cells
  • Assay time

    1h 00m
  • Product overview

    JC-10 Mitochondrial Membrane Potential Assay Kit (Microplate) ab112134 enables researchers to analyze a JC-10 assay with a microplate reader. 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.

  • Notes

    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).

    Related assays

    Review the cell health assay guide to learn about kits to perform a cell viability assaycytotoxicity assay and cell proliferation assay

    Review the metabolism assay guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.

  • Platform

    Microplate reader

Properties

Images

  • 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.

Protocols

References

This product has been referenced in:

  • Wang D & Liu P Ingenol-3-Angelate Suppresses Growth of Melanoma Cells and Skin Tumor Development by Downregulation of NF-?B-Cox2 Signaling. Med Sci Monit 24:486-502 (2018). Human . Read more (PubMed: 29368698) »
  • Li S  et al. Mitochondrial Dysfunctions Contribute to Hypertrophic Cardiomyopathy in Patient iPSC-Derived Cardiomyocytes with MT-RNR2 Mutation. Stem Cell Reports 10:808-821 (2018). Human . Read more (PubMed: 29456182) »
See all 15 Publications for this product

Customer reviews and Q&As

1-10 of 18 Abreviews or Q&A

Answer

Thank you for your enquiry.

JC-1, a lipophiliccationic dye, is widely used to detect mitochondrial de-polarization. However it has poor water solubility which means it precipitates in aqueous buffer even at 1 μM concentration. Compared to JC-1, JC-10 has much better water solubility. As in the case of JC-1, JC-10 is capable of entering selectively into mitochondria, and changes reversibly its color from green to orange as membrane potentials increase.

From assays performed on a fluorescence plate reader and fluorescence microscope, JC-10 outperforms JC-1 in several different cell types (e.g., primary rat hepatocytes, CHO-K1, HeLa, Jurkatand HepG2 cell lines). In most of the cell lines JC-10 has superior signal to background ratios. JC 10 also has smaller assay deviations due to its enhanced water solubility and higher sensitivity.

I am sorry we have no data from primary mouse neurons specifically in this case.

If you have some concerns regarding the results, please do not hesitate to contact me again with further details. The kit is covered by our 12 month guarantee and I will be pleased to provide further support. I would appreciate if it is possible to send:

1. Details of sample preparation.

2. The protocol used, and instrument used for reading.

3. A copy of the data.

I hope this will be helpful. I look forward to hearing from you with the requested information.

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Answer

You will not remove the treatment media prior to step C3.
The final volume in the well is 200 uL (100 uL cells + 50 uL JC-10 + 50 uL assay buffer B).

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Question
Answer

Assay Buffer B is used to quench the reaction and reduce the overall background. It is not necessary to perform the assay successfully however, it will definitely improve the results.

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Question
Answer

Phenol red should be OK; in our experience there has not been any interference.

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Answer

The concentration of 100X JC-10 in ab112134 is 3 mM. The solubility is ˜ 5mg/mL in DMSO.

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Answer



I can confirm that the numbers seems to be alright to me.

The product is used for mitochondria membrane potential measurement.

The dye needs to get into the live cells to exhibit either green (in cytosol) or red (in mitochondria) fluorescence.

Therefore just the solution alone might have some green (monomer) or red (aggregates) fluorescence. This fluorescence has no informational value.

When the mitochondria membrane potential changes, the ratio of the fluorescence in green and red changes. This is the read out the kit provides.

Please read the signal form the bottom of the cell and do not subtract the background signal for the above reasons.

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Answer

Thank you for contacting us about ab112134. Here is some further information: - The blank contains no cells and is growth media only. This gives the background and should be subtracted from all readings. - The control sample should have cells with no treatment. - ex/em = 490/520 (for green) and ex/em = 540/590 (for red) - The data is presented as a ratio of apoptotic (green) / normal cells (red). Therefore divide the 520 emission reading by the 590 emission reading. - In the graph, the ratio for control cells is set as 100%, and other ratio readings are shown relative to that, to show the proportion increase/decrease in apoptosis. I hope this information is helpful. Please do not hesitate to contact me should you have further questions.

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Question
Answer

Yes, you can do it, although the background might increase a bit. 

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Answer

Thank you for your message.

I understand the confusion and apologize for the inconvenience.

We have checked this and the documents have been amended with the correct information: We recommend to incubate at either room temperature for ˜60 minutes or more, or 37oC for 30-60 minutes.

I hope this will be helpful to you. If you have any questions, please do not hesitate to contact me.

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Answer

Q1. Are clear bottomed wells required for plates used in this assay?
A1. Yes

Q2. Serum (1%) will be present in medium when JC10 is added. Will JC10 be effected by serum?
A2. No
Q3. Is there an optimal height setting at which to set the instrument for reading fluorescence?
A3: We have used Flexstation (from Molecular Devices) which don't need to set the hight. you will have to ask the instrument manufacture for the support.  Simply ask them that you are going to read the fluorescent signal from the bottom of the cell layer, which kind of the settings is the best for that purpose.

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1-10 of 18 Abreviews or Q&A

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