Cell Meter™ Mitochondrion Membrane Potential Assay Kit Red Fluorescence Optimized for Microplate Reader

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Shipping	Standard overnight for United States, inquire for international

Spectral properties

Excitation (nm)	613
Emission (nm)	631

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Alternative formats

Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Orange Fluorescence Optimized for Microplate Reader*

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

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

Overview SDS Protocol

Excitation (nm)

613

Emission (nm)

631

Our Cell Meter™ assay kits are a set of tools for monitoring cell viability. There are a variety of parameters that can be used. This particular kit is designed to detect cell apoptosis by measuring the loss of the mitochondrial membrane potential (MMP). The collapse of MMP 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™ Mitochondrial Membrane Potential Assay Kit provides all the essential components with an optimized assay method. This fluorimetric assay uses our proprietary cationic MitoTell™ Red for the detection of apoptosis in cells with the loss of MMP. In normal cells, the red fluorescence intensity is increased when MitoTell™ Red is accumulated in the mitochondria. However, in apoptotic cells, the fluorescence intensity of MitoTell™ Red decreases following the collapse of MMP. Cells stained with MitoTell™ Red can be either visualized with a fluorescence microscope Cy5 channel, or with a fluorescence microplate reader. The kit is optimized for screening apoptosis activators and inhibitors with a fluorescence microplate reader. And the assay can be performed in a convenient 96-well and 384-well fluorescence microtiter-plate format without a wash step.

Platform

Fluorescence microplate reader

Excitation	610 nm
Emission	650 nm
Cutoff	630 nm
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Bottom read mode

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare cells
Add test compounds
Add MitoTell™ Red working solution (100 µL/well/ 96-well plate or 25 µL/well/384-well plate)
Incubate the plate in a 5% CO₂, 37°C incubator for 30 minutes
Add Assay Buffer B (50 µL/well/96-well plate or 12.5 µL/well/384-well plate)
Monitor the fluorescence increase (bottom read mode) at Ex/Em = 610/650 nm (Cutoff = 630 nm) or fluorescence microscope with Cy5 filter

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

PREPARATION OF WORKING SOLUTION

Add 50 µL of 200X MitoTell™ Red (Component A) into 10 mL of Assay Buffer A (Component B) and mix well to make MitoTell™ Red working solution. Protect from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

Treat cells with test compounds for a desired period of time to induce apoptosis, and set up parallel control experiments. Note: We treated HeLa cells with 20 µM CCCP for 15 minutes to change the mitochondrial membrane potential. See Figure 1 for details. CCCP or FCCP can be added simultaneously with MitoTell™ Red. To get the best result, titration of the CCCP or FCCP may be required for each individual cell line.
Add 100 µL/well/96-well plate or 25 µL/well/384-well plate of MitoTell™ Red working solution into the cell plate.
Incubate the plate at 37ºC for 15 - 30 minutes, protected from light. Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
Add 50 µL/well/96-well plate or 12.5 µL/well/384-well plate of Assay Buffer B (Component C) into the cell plate. Note: DO NOT wash the cells after loading. For non-adherent cells, it is recommended to centrifuge cell plates at 800 rpm for 2 minutes with brake off after adding Assay Buffer B (Component C).
Monitor the fluorescence intensity with a fluorescence microplate reader (bottom read mode) at Ex/Em = 610/650 nm (Cutoff = 630 nm) 10 to 30 minutes after adding Assay Buffer B (Component C) or observe the fluorescence signal under a fluorescence microscope with Cy5 filter set.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	613
Emission (nm)	631

Images

Figure 1. Hela cells were dye-loaded with MitoTell™ Red alone or in the presence of 20 μM CCCP for 30 minutes. The fluorescence intensity of MitoTell™ Red was measured 5 minutes after adding Assay Buffer B (Component C) using (A) a fluorescence microscope with Cy5 filter set or (B) FlexStation microplate reader at Ex/Em = 610/650 nm (Cutoff = 630 nm, bottom read mode).

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