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Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*

The decrease in fluorescence intensity of MitoTell™ Orange with the addition of FCCP in Jurkat cells. Jurkat cells were loaded with MitoTell™ Orange alone (Blue) or in the presence of 30 µM FCCP (Red) for 15 minutes. The fluorescence intensity of MitoTell™ Orange was measured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer using FL2 channel.
The decrease in fluorescence intensity of MitoTell™ Orange with the addition of FCCP in Jurkat cells. Jurkat cells were loaded with MitoTell™ Orange alone (Blue) or in the presence of 30 µM FCCP (Red) for 15 minutes. The fluorescence intensity of MitoTell™ Orange was measured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer using FL2 channel.
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Catalog Number22804
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


This particular kit is designed to detect cell apoptosis by measuring the loss of the mitochondrial membrane potential(MMP). The collapse of 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. Our Cell Meter™ Orange Mitochondrial Membrane Potential Assay Kit provides all the essential components with an optimized assay method. This fluorimetric assay uses our proprietary cationic MitoLite™ Orange for the detection of apoptosis in cells with the loss of mitochondrial membrane potential. In normal cells, the red fluorescence intensity is increased when MitoLite™ Orange is accumulated in the mitochondria. However, in apoptotic cells, the fluorescence intensity of MitoLite™ Orange is decreased following the collapse of MMP. Cells stained with MitoLite™ Orange can be visualized with a flow cytometer at 488 nm excitation with red emission (FL2 channel). The kit can be used together with other reagents, such as Cell Meter™ Phosphatidylserine Apoptosis Assay Kit (22835) for multi-parametric study of cell vitality and apoptosis. The kit is optimized for screening apoptosis activators and inhibitors with a flow cytometer.

Platform


Flow cytometer

Excitation488 nm or 532 nm laser
Emission575/26 nm filter
Instrument specification(s)PE channel

Components


Component A: 500X MitoTell ™ Orange in DMSO1 vial (200 µL)
Component B: Assay Buffer1 bottle (100 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds at the density of 5 × 105 to 1 × 106 cells/mL
  2. Add 2 µL of 500X MitoTell™ Orange into 1 mL of cell solution
  3. Incubate the cells in a 37 °C, 5% CO2 incubator for 15 - 30 minutes
  4. Pellet the cells, and resuspend the cells in 1 mL of growth medium
  5. Analyze cells using flow cytometer with FL2 channel

Important notes
Thaw all the kit components at room temperature before starting the experiment.

SAMPLE EXPERIMENTAL PROTOCOL

  1. For each sample, prepare cells in 1 mL of warm medium or buffer of your choice at the density of 5×105 to 1×106 cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.

  2. Treat cells with test compounds for a desired period of time to induce apoptosis, and set up parallel control experiments.

    For Negative Control: Treat cells with vehicle only.

    For Positive Control: Treat cells with FCCP or CCCP at 5-50 µM in a 37 oC, 5% CO2 incubator for 15 to 30 minutes. Note: CCCP or FCCP can be added simultaneously with MitoTell™ Orange. To get the best result, titration of the CCCP or FCCP may be required for each individual cell line.

  3. Add 2 µL of 500X MitoTell™ Orange (Component A) into the treated cells.

  4. Incubate the cells in a 37 °C, 5% CO2 incubator for 15 to 30 minutes. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact and wash the cells once with serum-containing media prior to the incubation with MitoTell™ Orange dye-loading solution.

  5. Centrifuge the cells at 1000 rpm for 4 minutes, and then re-suspend cells in 1 mL of Assay Buffer (Component B) or buffer of your choice.

  6.  Monitor the fluorescence intensity using a flow cytometer wih FL2 channel (Ex/Em = 540/590 nm). Gate on the cells of interest, excluding debris.

Citations


View all 2 citations: Citation Explorer
Evaluation of the oncolytic property of recombinant Newcastle disease virus strain R2B in 4T1 and B16-F10 cells in-vitro
Authors: Ramamurthy, Narayan and Pathak, Dinesh C and D'Silva, Ajai Lawrence and Batheja, Rahul and Mariappan, Asok Kumar and Vakharia, Vikram N and Chellappa, Madhan Mohan and Dey, Sohini
Journal: Research in Veterinary Science (2021): 159--165
Mitochondrial Dysfunction Contributes to Aging-Related Atrial Fibrillation
Authors: Liu, Chuanbin and Bai, Jing and Dan, Qing and Yang, Xue and Lin, Kun and Fu, Zihao and Lu, Xu and Xie, Xiaoye and Liu, Jianwei and Fan, Li and others,
Journal: Oxidative Medicine and Cellular Longevity (2021)

References


View all 91 references: Citation Explorer
Safranine O as a fluorescent probe for mitochondrial membrane potential studied on the single particle level and in suspension
Authors: Perevoshchikova IV, Sorochkina AI, Zorov DB, Antonenko YN.
Journal: Biochemistry (Mosc) (2009): 663
Determination of high mitochondrial membrane potential in spermatozoa loaded with the mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) by using fluorescence-activated flow cytometry
Authors: Guthrie HD, Welch GR.
Journal: Methods Mol Biol (2008): 89
The mitochondrial membrane potential and Ca2+ oscillations in smooth muscle
Authors: Chalmers S, McCarron JG.
Journal: J Cell Sci (2008): 75
Computer-assisted live cell analysis of mitochondrial membrane potential, morphology and calcium handling
Authors: Koopman WJ, Distelmaier F, Esseling JJ, Smeitink JA, Willems PH.
Journal: Methods (2008): 304
How DASPMI reveals mitochondrial membrane potential: fluorescence decay kinetics and steady-state anisotropy in living cells
Authors: Ramadass R, Bereiter-Hahn J.
Journal: Biophys J (2008): 4068
Life cell quantification of mitochondrial membrane potential at the single organelle level
Authors: Distelmaier F, Koopman WJ, Testa ER, de Jong AS, Swarts HG, Mayatepek E, Smeitink JA, Willems PH.
Journal: Cytometry A (2008): 129
Effects of eprosartan on mitochondrial membrane potential and H2O2 levels in leucocytes in hypertension
Authors: Labios M, Martinez M, Gabriel F, Guiral V, Ruiz-Aja S, Beltran B, Munoz A.
Journal: J Hum Hypertens (2008): 493
Mitochondrial membrane potential in axons increases with local nerve growth factor or semaphorin signaling
Authors: Verburg J, Hollenbeck PJ.
Journal: J Neurosci (2008): 8306
Evaluation of sperm mitochondrial membrane potential by JC-1 fluorescent staining and flow cytometry
Authors: Xia XY, Wu YM, Hou BS, Yang B, Pan LJ, Shi YC, Jin BF, Shao Y, Cui YX, Huang YF.
Journal: Zhonghua Nan Ke Xue (2008): 135
Cyclosporin A-induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential
Authors: van der Toorn M, Kauffman HF, van der Deen M, Slebos DJ, Koeter GH, Gans RO, Bakker SJ.
Journal: Febs J (2007): 3003