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Cell Biology Apoptosis Mitochondrial
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JC-1 - Mitochondrial Membrane Potential Assay Kit (ab113850)

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  • Protocol Booklet
Reviews (4)Q&A (9)References (36)

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JC-1 assay result in HL60 cells treated with FCCP
  • JC-1 assay result in HepG2 cells treated with CCCP.
  • Using JC1 dye to examine mitochondrial membrane potential depolarization in SH-SY5Y cells
  • JC-1 assay result in HL60 cells treated with Troglitazone
  • JC1 Mitochondrial Membrane Potential Assay Kit using green fluorescence imaging
  • JC1 used to assay in vitro metabolic effect on hepatocyte and hepatoma cells

Key features and details

  • Detection method: Fluorescent
  • Platform: Microplate reader
  • Assay time: 1 hr
  • Sample type: Adherent cells, Suspension cells

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Overview

  • Product name

    JC-1 - Mitochondrial Membrane Potential Assay Kit
    See all Mitochondrial Membrane Potential kits
  • Detection method

    Fluorescent
  • Sample type

    Adherent cells, Suspension cells
  • Assay time

    1h 00m
  • Product overview

    JC1- Mitochondrial Membrane Potential Assay Kit ab113850 contains tetraethylbenzimidazolylcarbocyanine iodide (JC-1), a cationic dye that accumulates in energized mitochondria.


    At low concentrations (due to low mitochondrial membrane potential), JC-1 is predominantly a monomer that yields green fluorescence with emission of 530±15 nm.


    At high concentrations (due to high mitochondrial membrane potential), the dye aggregates yielding a red to orange colored emission (590±17.5 nm).


    Therefore a decrease in the aggregate fluorescent count is indicative of depolarization whereas an increase is indicative of hyperpolarization.


    The JC-1 staining protocol is very simple:
    - wash cells in dilution buffer or PBS
    - add JC solution
    - incubate for 30 min at 37ºC for suspension cells, or 10 min for adherent cells
    - wash cells with dilution buffer
    - treat cells as desired for experimental plan
    - analyze on a fluorescent microplate reader


    The aggregate dye can be excited at 535 nm, the monomer and aggregate together at 475 nm. 

  • Notes

    Review our cell health assays guide to learn more about our other cell viability, cytotoxicity and cell proliferation assay kits.

    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

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 100 tests
    50mM FCCP (in DMSO) 1 x 10µl
    Dilution Buffer (10X, sterile) 1 x 10ml
    DMSO (cell culture tested) 1 x 1ml
    JC-1 (lyophilized) 1 x 500µg
  • Research areas

    • Cell Biology
    • Apoptosis
    • Mitochondrial
    • Kits/ Lysates/ Other
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    • Kits
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    • Cell viability, plasma membrane damage
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Apoptosis
    • Kits/ Lysates/ Other
    • Kits
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    • Cell Damage
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    • Kits
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    • Transmembrane potential
    • Cancer
    • Cell Death
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    • Mitochondrial
    • Cancer
    • Cell Death
    • Apoptosis
    • Metabolism
    • Metabolism
    • Pathways and Processes
    • Mitochondrial Metabolism
    • Membrane potential
  • Relevance

    Mitochondrial Membrane Potential is an important parameter of mitochondrial function used as an indicator of cell death. The collapse of the mitochondrial Membrane potential coincides with the opening of the mitochondrial permeability transition pores, leading to the release of cytochrome c into the cytosol, which in turn triggers other downstream events in the apoptotic cascade.
  • Alternative names

    • mitochondrial membrane potential

Associated products

  • Assay kits

    • JC-10 Mitochondrial Membrane Potential Assay Kit (Flow Cytometry) (ab112133)
    • JC-10 Mitochondrial Membrane Potential Assay Kit (Microplate) (ab112134)
    • TMRE-Mitochondrial Membrane Potential Assay Kit (ab113852)
  • Related Products

    • JC-1, Mitochondrial membrane potential dye (ab141387)

Images

  • JC-1 assay result in HL60 cells treated with FCCP
    JC-1 assay result in HL60 cells treated with FCCP

    JC1 - Mitochondrial Membrane Potential Assay Kit (ab113850). HL60 cells were seeded and labeled according to section 11.1 of the protocol. Cells were then treated for 4 hours with 100 µM FCCP or vehicle/diluent control (DMSO).  Mean and standard deviation is plotted for 3 replicates from each condition.

  • JC-1 assay result in HepG2 cells treated with CCCP.
    JC-1 assay result in HepG2 cells treated with CCCP.

    JC1 - Mitochondrial Membrane Potential Assay Kit (ab113850). HepG2 cells were seeded and labeled according to section 11.2 of the protocol. Cells were then treated for 4 hours with a titration series of CCCP (carbonyl cyanide 3-chlorophenylhydrazone) and both monomer and aggregate forms were read on a Perkin Elmer-Wallac 1420 Victor 2 Multilabel plate reader. Mean and standard deviation of aggregate/monomer ratios is plotted for 12 replicates for each concentration. IC50 of CCCP in HepG2 cells was calculated at 8.7 µM.

  • Using JC1 dye to examine mitochondrial membrane potential depolarization in SH-SY5Y cells
    Using JC1 dye to examine mitochondrial membrane potential depolarization in SH-SY5Y cellsImage courtesy of Son M S et al. Sci Rep. 2017; 7: 2075. doi: 10.1038/s41598-017-02129-w. Reproduced under the Creative Commons License http://creativecommons.org/licenses/by/4.0/.

    Son MS et al. (2017) used JC1 Mitochondrial Membrane Potential Assay Kit ab113850 to stain:
    - untreated SH-SY5Y cells (control),
    - SH-SY5Y cells treated with 1mM MPP+ (MPP+) and,
    - SH-SY5Y cells pretreated with BDS-II followed by 1mM MPP+ treatment (MPP+ +BDSII).

    Normal mitochondrial membrane potential is shown in red with JC-1 dimers and depolarized membrane potential is shown in green in JC-1 monomers.

  • JC-1 assay result in HL60 cells treated with Troglitazone
    JC-1 assay result in HL60 cells treated with Troglitazone

    JC1 - Mitochondrial Membrane Potential Assay Kit (ab113850). HL60 cells were seeded and labeled according to section 11.1 of the protocol. Cells were then treated for 4 hours with a titration series of the thiazolidinedione Troglitazone and both monomer and aggregate forms were read on a Perkin Elmer-Wallac 1420 Victor 2 Multilabel plate reader. Mean and standard deviation of aggregate/monomer ratios is plotted for 3 replicates for each concentration. IC50 of Troglitazone in HL60 cells was calculated at 1.2 µM.

  • JC1 Mitochondrial Membrane Potential Assay Kit using green fluorescence imaging
    JC1 Mitochondrial Membrane Potential Assay Kit using green fluorescence imagingThis image is courtesy of an Abreview submitted by Dimitra Kalamida

    The JC1- Mitochondrial membrane potential assay kit has been tested using HepG2 cells, control cells and FCCP-treated cells (100uM for 4h) have been used as a positive control. The company's instructions were followed for JC1 mitochondrial membrane potential assay. Imaging was performed on a customized Andor Revolution Spinning Disk Confocal System built around a stand (IX81 Olympus) with a 60x lens and a digital camera (Andor Ixon+885) (CIBIT Facility, MBG-DUTH). Image acquisition was performed in Andor IQ 2 software. Optical sections were recorded every 0.3 µm. All confocal microscopy images presented in this work are 2D maximum intensity projections of z-stack images (ImageJ 1.47v National Institute of Health,USA).
    Personal feedback: A green laser with the appropriate emission filter (530nm) has been used to detect the monomer of the JC1 dye, following FCCP treatment the mitochondrial membrane potential of the cells was eliminated, as demonstrated by the increase of the monomer emission.

  • JC1 used to assay in vitro metabolic effect on hepatocyte and hepatoma cells
    JC1 used to assay in vitro metabolic effect on hepatocyte and hepatoma cellsImage courtesy of Koukourakis M I et al. Sci Rep. 2016; 6: 30986doi: 10.1038/srep30986. Reproduced under the Creative Commons License http://creativecommons.org/licenses/by/4.0/

    Koukourakis MI et al. (2016) used the MMP assay kit to stain with JC-1 in NCTC hepatocytes exposed to amifostine (100 μg/ml) over a time course of 20 minutes in vitro. Confocal microscopy used to assess mitochondrial membrane potential in the cells.

    Serial images confirmed a rapid drop of both green and red fluorescence, one minute after exposure, an effect that was restored to normal at 20 minutes, after a small period of a rebound increase.

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (36)

Publishing research using ab113850? Please let us know so that we can cite the reference in this datasheet.

ab113850 has been referenced in 36 publications.

  • Rodriguez-Terrones D  et al. A distinct metabolic state arises during the emergence of 2-cell-like cells. EMBO Rep 21:e48354 (2020). PubMed: 31849178
  • Li Y  et al. Imaging of macrophage mitochondria dynamics in vivo reveals cellular activation phenotype for diagnosis. Theranostics 10:2897-2917 (2020). PubMed: 32194843
  • Lee YJ  et al. Down-regulation of the mitochondrial i-AAA protease Yme1L induces muscle atrophy via FoxO3a and myostatin activation. J Cell Mol Med 24:899-909 (2020). PubMed: 31725201
  • Liu HW  et al. Enhanced Hsa-miR-181d/p-STAT3 and Hsa-miR-181d/p-STAT5A Ratios Mediate the Anticancer Effect of Garcinol in STAT3/5A-Addicted Glioblastoma. Cancers (Basel) 11:N/A (2019). PubMed: 31783691
  • Chen H  et al. Apoptotic-like changes in epididymal spermatozoa of soft-shelled turtles, Pelodiscus sinensis, during long-term storage at 4?ºC. Anim Reprod Sci 205:134-143 (2019). PubMed: 31060923
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 13 Abreviews or Q&A

Mitochondrial membrane potential in human immortalized cardiomyocytes

Good Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
JC-1 assay to evaluate mitochondrial membrane potential (MMP) in cardiomyocytes cells under different treatment conditions for 24 hours. Data are mean ± SEM of four different experiments. FCCP 100μM was used as Depolarization Control. The values of fluorescence intensity are indicated as ratio between aggregate form of JC-1 dye on monomer form of JC-1 dye. Briefly, cells were plated (seeding density 2.5 x 104 cells/cm2) in culture medium w/o phenol red onto a 96 black well plate. The cells were exposed to the treatments of interest in culture medium w/o phenol red for 24 hours. For depolarization control FCCP 100μM was used, and the cells were exposed to FCCP for 4 hours. At the end of the treatments, the wells were washed with PBS, and then the cells were incubated with JC-1 dye 10 μM for 20 min at 37° C protected from light.Following incubation the wells were washed with 1X dilution buffer (provided in the kit) and the fluorescence related to MMP was measured immediately by using a fluorimeter. Each single experiment was performed in quadruplicate. To measure the fluorescence of aggregate species the Ex-531 and Em-595 nm wavelengths were set. To measure the fluorescence of monomer species the Ex-485 and Em-535 nm wavelengths were set. The values of fluorescence intensity were indicated as ratio between aggregate form of JC-1 dye on monomer form of JC-1 dye.

Abcam user community

Verified customer

Submitted Feb 12 2020

mitochondrial membrane potential in human fibroblast cells

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
mitochondrial membrane potential in human fibroblast cells was determined by JC-1.
FCCP treated cells showed increased monomeric (green) signal which supported change of mitochondrial membrane potential compared to control cells.

Dr. Min-Joon Han

Verified customer

Submitted Jun 04 2018

Good and easy to use kit

Good Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
We used JC-1 on primary mouse astrocyte culture to rate the mitochondrial stress under different inflammatory stimuli. Following the protocol recommended, we fount this kit an useful and easy to use tool.

Ms. Maria Velasco

Verified customer

Submitted Apr 09 2018

JC1 Mitochondrial Membrane Potential Assay Kit using green fluorescence imaging

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
The JC1- Mitochondrial membrane potential assay kit has been tested using HepG2 cells, control cells and FCCP-treated cells (100uM for 4h) have been used as a positive control. The company's instructions were followed for JC1 mitochondrial membrane potential assay. Imaging was performed on a customized Andor Revolution Spinning Disk Confocal System built around a stand (IX81 Olympus) with a 60x lens and a digital camera (Andor Ixon+885) (CIBIT Facility, MBG-DUTH). Image acquisition was performed in Andor IQ 2 software. Optical sections were recorded every 0.3 µm. All confocal microscopy images presented in this work are 2D maximum intensity projections of z-stack images (ImageJ 1.47v National Institute of Health,USA).
Personal feedback: A green laser with the appropriate emission filter (530nm) has been used to detect the monomer of the JC1 dye, following FCCP treatment the mitochondrial membrane potential of the cells was eliminated, as demonstrated by the increase of the monomer emission.

Dr. Dimitra Kalamida

Verified customer

Submitted Nov 09 2016

Question

any volume to be added before reading th eplate?
would the cells not dry out?

Read More

Abcam community

Verified customer

Asked on Jul 12 2013

Answer


The lab confirmed that the protocol is not very clear in that part you pointed out. We will update the protocol shortly.

You should add 100 uL of the 1X buffer to the plate after washing - to prevent the cells from drying out.

Read More

Sybille Rex

Abcam Scientific Support

Answered on Jul 12 2013

Question

Custome rkindly contacted us to inquire whether they may be able to use this kit to determine membrane potential and then follow up with flow cytometry staining. Will the dye remain for the 3-4 hours needed to perform the flow cytometry? Would the kit interfere with 405 or 633 fluorochromes?

Read More

Abcam community

Verified customer

Asked on Jun 04 2013

Answer

Looking at JC1 staining it appears that flow cytometry would be possible to perform as long as the cytometer is capable of the proper excitation/emission required. Protocol booklet mentions on Pg 5 that Excitation = 540 – 590 nm and Emission = 570 – 610 nm and on Pg 12. Set excitation wavelength at 535 ± 17.5nm (aggregate excitation only) or at 475 ± 20nm (for simultaneous aggregate and monomer excitation) or to Set emission wavelength at 590 ± 17.5nm (aggregate emission only). If reading of the monomer species is also desired, set a second emission reading at 530 ± 15nm. Looking at the protocol booklet pg 12 step 4. FCCP treatment for 4 hours is recommended after JC1 staining to observe a decrease. So the dye appears to be detectable for at least 4 hours. The JC-1 aggregate can be detected with similar settings to those used to detect rhodamine (excitation/emission = 540/570nm) or texas red (590/610nm). This may interfere with 405 or 633 fluorchromes, although you should check via a spectra graph for the specific fluorchromes to see if compensation may solve any interference.

Read More

Abcam Scientific Support

Answered on Jun 04 2013

Question

is fixation with PFA in well possible (after live imaging, for storage)?
or does this interfere with the dye signal

Read More

Abcam community

Verified customer

Asked on May 01 2013

Answer



If you wanted to keep the relation between JC-1 dye and the detected mitochondrial membrane potential after fixation this is not possible. This is because JC-1 is dependent on mitochondrial membrane potential which will be abolished by the fixation.

Read More

Abcam Scientific Support

Answered on May 01 2013

Question

I am using primary cells and am afraid that 10 minutes without media is too much. Can I mix the JCI with media rather than buffer so my cells aren't affected?

Read More

Abcam community

Verified customer

Asked on Apr 24 2013

Answer

Thank you for contacting us. It is fine to go ahead and use media without phenol red instead of buffer. Please let me know if you have any further questions.

Read More

Abcam Scientific Support

Answered on Apr 24 2013

Question

1. My treatment involves incubating cells in serum free medium. Can I dilute JC-1 in a 1X supplemented buffer which does not contain
FBS?
2. Is this kit designed to do a cell treatment before or after JC-1 incubation? Or does it matter?

Read More

Abcam community

Verified customer

Asked on Mar 18 2013

Answer

The most important thing with the protocol is to read membrane potential in the presence of compound (or treatment). The reason is that we have found in our experience that changes in membrane potential can be reversible as soon as the treatment is removed from the cells. The easiest thing to do this is to stain first with JC1 and then treat (ideally short treatment). If they can do this, then they could follow the protocol as shown and then simply do the treatment in the absence of FBS. If they do it the way around and treat first and then stain w/o the FBS they run the risk of not finding the true effects of the treatment due to reversal during the staining and washes following the treatment (unless they stain and wash in the presence of the compound).

Read More

Abcam Scientific Support

Answered on Mar 18 2013

Question

Dear sir/madam,
I just bought a JC1 - Mitochondrial Membrane Potential Assay Kit (ab113850). The protocol said it can be used for 100 times, but I find that if follow the protocol steps the 1*buffer solution will be used up very soon.Can I use PBS or some other buffer to replace the 1*buffer solution for JC-1mix?How to storage the 1*buffer solution,4℃ or -20℃?Waiting for your reply.
Best Regards,

Read More

Abcam community

Verified customer

Asked on Nov 20 2012

Answer

Thank you for contacting us.


The kit contains enough buffer to perform 100 tests. We give 10mL of buffer at 10X which should give a total of 100mL of buffer at 1X (20mL are used to dilute compounds for toxicity assays, 10mL are used for diluting JC1, 15mL are used for washing after JC1 staining and resuspending the cells). Following the protocol, you should not have to use more than 50mL of buffer. You could also use the media without phenol red to resuspend the cells and dilute the compounds.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Abcam Scientific Support

Answered on Nov 20 2012

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