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

Images

  • 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.

  • 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

  • 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

  • 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.

Protocols

References

This product has been referenced in:
  • Dey T  et al. Anti-Proliferative Activities of Vasicinone on Lung Carcinoma Cells Mediated via Activation of Both Mitochondria-Dependent and Independent Pathways. Biomol Ther (Seoul) 26:409-416 (2018). Read more (PubMed: 29310422) »
  • Goswami P  et al. Betulinic acid induces DNA damage and apoptosis in SiHa cells. Mutat Res 828:1-9 (2018). Read more (PubMed: 29555058) »
See all 18 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Abreviews
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
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

Abreviews
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
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.

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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.

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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.

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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.

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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).

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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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Answer

Thank you for your inquiry. I heard back from the lab regardingthese two distinct dyes that both measure mitochondrial membrane potential. TMRE stains mitochondria only when there is a membrane potential. JC-1 is slightly different in that it has distinct emission spectra depending on whether mitochondria have high or low potential. TMRE can be measured in a spectophotometer, fluorescent microscope or flow cytometry. JC-1 can be measured in a spectrophotometer. Details are in the respective protocol booklets. https://www.abcam.com/ps/products/113/ab113850/documents/ab113850%20Protocol%20(Website)%20v2.pdf https://www.abcam.com/ps/products/113/ab113852/documents/ab113852%20protocol%20final%20v2%20(website(.pdf For you, wewould recommend TMRE with analysis on a flow cytometer. Flow cytometry is both sensitive and amenable to very few cells. I hope this information helps. Please contact us with any other questions.

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1-10 of 12 Abreviews or Q&A

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