製品の概要

  • 製品名
    TMRE-Mitochondrial Membrane Potential Assay Kit
    Mitochondrial Membrane Potential キット 製品一覧
  • 検出方法
    Fluorescent
  • サンプルの種類
    Adherent cells, Suspension cells
  • アッセイタイプ
    Cell-based (qualitative)
  • 全工程の試験時間
    0h 35m
  • 製品の概要

    TMRE-Mitochondrial Membrane Potential Assay Kit ab113852 is used for quantifying changes in mitochondrial membrane potential in live cells by flow cytometry, microplate spectrophotometry and fluorescent microscopy. 


    ab113852 uses TMRE (tetramethylrhodamine, ethyl ester) to label active mitochondria. TMRE is a cell permeant, positively-charged, red-orange dye that readily accumulates in active mitochondria due to their relative negative charge. Depolarized or inactive mitochondria have decreased membrane potential and fail to sequester TMRE.


    The TMRE protocol also uses FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone), which is a ionophore uncoupler of oxidative phosphorylation. Treating cells with FCCP eliminates mitochondrial membrane potential and TMRE staining. TMRE is suitable for the labeling of mitochondria in live cells and is not compatible with fixation.


    TMRE protocol summary:
    - add FCCP to appropriate control cell samples and incubate for 10 min
    - incubate with TMRE for 15-30 min, pellet (suspension cells) / remove media (adherent cells) and wash with PBS / 0.2% BSA
    - analyze with micro-plate reader at Ex/Em 549/575 nm, flow cytometer using 488nm laser for excitation and at emission 575 nm, or fluorescent microscope.

  • 特記事項

    TMRE is only suitable for use with live (not fixed) cells.

    Related assays

    Review the cell health assay guide to learn about kits to perform a cell viability assay, cytotoxicity 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.

  • 試験プラットフォーム
    Microplate reader, Fluor. microscope, Flow cyt.

製品の特性

画像

  • P19 neurons (750 cells/mm2) were exposed to MDMA on days 7–9 in serum-free medium for 10 min up to 48 hours. The positive control FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), an uncoupler of mitochondrial oxidative phosphorylation, was applied at the concentration of 5 μM for 10 min. The cells were incubated with 500 μM TMRE for 30–45 min at 37°C, 5% CO2, followed by washing once with 100 μl of HBSS containing 0.2% bovine serum albumin. A volume of 200 μL of HBSS containing 0.2% bovine serum albumin was added to each well, and the fluorescence was measured with excitation/emission: 544/590 nm.

  • A: HeLa cells (adherent) were cultured on coverslips and stained with ab113852 (200nM TMRE) for 20 minutes in media, washed briefly with PBS and immediately imaged. B: Jurkat cells (suspension) were stained and washed as above and then transferred to a slide and immobilized under a coverslip for imaging.
  • Chart showing mean fluorescent intensity +/- standard deviation from quadruplicate measurements of 400 nM TMRE stained Jurkat cells in a 96-well microplate +/- treatment with FCCP.

  • Flow cytometry histogram of Jurkat cells stained with ab113852 (100nM TMRE) with (blue) or without (red) treatment with 100µM FCCP.

プロトコール

参考文献

This product has been referenced in:
  • Ferreira I  et al. Nature and kinetics of redox imbalance triggered by respiratory and skin chemical sensitizers on the human monocytic cell line THP-1. Redox Biol 16:75-86 (2018). Read more (PubMed: 29477863) »
  • Erlich-Hadad T  et al. TAT-MTS-MCM fusion proteins reduce MMA levels and improve mitochondrial activity and liver function in MCM-deficient cells. J Cell Mol Med 22:1601-1613 (2018). Read more (PubMed: 29265583) »
See all 45 Publications for this product

レビューと Q&A

1-10 of 17 Abreviews or Q&A

Question
Answer

Thank you for contacting us. Our lab uses:

BD PureCoat™ Microplates
black/clear, polystyrene
96-well, amine
Cat#354717

The recommendation is for clear bottom, black wall microplate that (a) cells will grow on and (b) is compatible with plate reader.

Please let me know if you have any other questions.

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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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Abreviews
To measure mitochondrial activity in human fibroblast cells, we applied TMRE mitochondrial membrane potential assay kit. It showed great correlation with Seahorse analysis which measured OCR (oxygen consumption rate).

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Verified customer

投稿 May 02 2018

Answer

The samples should be read immediately after the 15-30 minute incubation with TMRE. Since the cells are live, as the health of the cell declined the signal from the dye would also diminish.



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Answer





For another positive control, CCCP is suitable (5-50 μM CCCP for 30 to 60 minutes at 37ºC).

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Answer

It all depends on the sample (embryos vs. explant vs. whole work). We have only worked with this dye using tissue culture cells (monolayer or suspension). If the samples are dissociated cells then TMRE labeling should work. Otherwise, we don’t know how well or deeply the dye can penetrate across cell layers (e.g. into tissues or embryos).


The dye for measuring the potential in the kit is species independent, so it is more a matter of the sample type.

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Answer


We have no experience in using TMRE to stain isolated mitochondria from any source – it is a tricky proposition as you would need intact/non-damaged mitochondria which retain membrane potential therefore we wouldn't recommend this.

As for imaging tissue stained with TMRE, this is also tricky. Flow cytometry is almost certainly out due to the need for single cell suspension – which will be very difficult for most tissue types. For microscopy I suppose it is possible with the proper microscope set up and the ability to adapt the live tissue to microscope stage constraints (e.g. thin slices.)

However, I would like to stress that this kit is only really for mitochondria labelling in intact cultured cells.

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Answer

Thank you for contacting us. My colleague from our MitoSciences lab has provided the following recommendations.

The lack of signal may be due to working outside the peak excitation (549nm) and peak emission (575nm). Getting as close as possible to these parameters is critical for maximal signal.

Many fluorescent spectrophotometers read only in the center of the well. It may help to ensure that your plate reader is capable of reading at the bottom of the plate and ensure the instrument is set up as such. We would also recommend to increase the PMT gain (photomultiplier tube) on the instrument.

Another option is to increase the concentration of TMRE to 1000nM and increase the density of the cells. With mouse embryonic fibroblasts, we would recommend to test the following densities on a 96 well plate to find optimal seeding = 50,000 / 25,000 / 10,000 / 5,000. Using a titration of cells, we recommend testing different TMRE concentrations (1000nM, 200nM, 50nM).

I hope this helps, if not, please let me know and I will be happy to help you further.

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Answer

Thank you for contacting us.

I have received the following information from the lab:

TMRE can only be used with live cells—an active membrane potential is required for staining. This will be true of all membrane potential sensitive dyes (including JC-1).

As for assessing mitochondria in the existing samples:
One possibility is to look at Cytochrome C release from the mitochondria by ICC, e.g. the mouse monoclonal ab110325. (see also Figure 11 in the MitoSciences Apoptosis Playbook here: http://mitosciences.com/PDF/Apoptosis_Playbook.pdf



Another option might be the ApoTrack™ Cytochrome c Apoptosis ICC Antibody Kit (ab110417)
https://www.abcam.com/ApoTrack™-Cytochrome-c-Apoptosis-ICC-Antibody-Kit-ab110417.html

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.

Culture media is known to cause background using kit ab113852. For suspension cells, we recommend pelleting the cells, removing the culture media, resuspending in the same volume of 0.2% BSA in PBS, pelleting again, and resuspending a second time in 0.2% BSA in PBS before transferring to a microplate.

I hope this information helps. Please contact us with any other questions.

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1-10 of 17 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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