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Dec 31st Santa Cruz will discontinue a
large number of polyclonal products as
a result of the USDA settlement that was
made public May 19th.

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To Purchase # 45898S

45898S 1 Kit (100 assays) $129.00.0
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Protocol for CellSimple™ Mitochondrial Membrane Potential (II) Assay #45898 and Mitochondrial Membrane Potential (II) Assay Protocol #13296

A. Instrumentation:

The CellSimple™ Mitochondrial Membrane Potential (II) Assay is specially designed for use with the CellSimple™ Cell Analyzer. However, either kit may be used with a flow cytometer or plate reader capable of providing excitation between 550 nm and 580 µM and detecting fluorescent emission at approximately 580 nm.

B. Kit components:

  • TMRE
  • CCCP
  • Phosphate Buffered Saline (PBS-20X)

C. Additional reagents needed, but not supplied.

  • DMSO
  • Reverse osmosis/deionized (RO/DI) water or equivalent

D. Reagent preparation

  1. 1X PBS: To prepare 500 ml 1X PBS add 25 ml PBS-20X to 475 ml RO/DI water, mix. Note: For flow cytometry application, adding 0.5% BSA to 1X PBS buffer may help to prevent cell loss.
  2. TMRE Stock Solution: Add 55 µl DMSO to each vial of TMRE to make a 1 mM stock solution. Aliquot if desired and store at -20°C. Each vial includes enough TMRE for 100 CellSimple™ tests, 50 flow cytometry assays (10 µl/assay) or five 96-well plates (0.1 µl/well), assays. 2 µM is used in this protocol. A titration between 0.1 to 10 µM is recommended to determine the optimal concentration for different cell lines.
  3. TMRE Labeling Solution: Dilute TMRE Stock Solution 1:500 with full cell culture medium to make 2 µM TMRE Labeling Solution.
  4. CCCP: Allow the 50 mM CCCP solution to equilibrate to room temperature before use

E. Protocol for suspension cells

  1. Suspend cells in warm media or PBS at 1 x 106 cell/ml. Prepare 1 ml aliquots; each 1 ml cell aliquot is one assay point. Make sure there are enough cells for your experiment. For example, if one compound is going to be assayed at three different concentrations, a total of 4 x 1 ml samples will be needed (this includes a positive control).
  2. Add test compound(s) to sample tubes at desired concentration and incubate cells for desired time. For best results, a compound titration and incubation time course can help to determine the best assay conditions. To prepare the positive control (mitochondrial membrane potential loss), add 1 µl of 50 mM CCCP to the control tube for a 50 µM final concentration; incubate cells at 37°C for 15 min.
  3. Add 100 µl of the TMRE Labeling Solution to each sample (200 nM final concentration) and incubate cells in the incubator (37°C and 5% CO2) for 15 to 30 min.
  4. Centrifuge sample at 300 x g for 5 min then remove the supernatant.
  5. Wash cells once with 1 ml warm 1X PBS, repeat step 4.
  6. Resuspend cells into 1000 µl warm 1X PBS.
  7. Analyze sample using an appropriate instrument. For analysis using the CellSimple™ Cell Analyzer use the Open Flow Cytometry Application selecting only the 561 nm LP detection channel. Please see the CellSimple™ user guide for more details about using the Open Flow Application. If samples are to be analyzed on a plate reader, transfer 100 µl/ cell suspension/well to a black 96-well plate with a clear bottom and read using the following settings: excitation at approximately 550 nm and emission at approximately 580 nm.

F. Protocol for adherent cells

  1. Plate cells in a 96 well plate in warm culture medium place in incubator overnight to allow cells to attach to the plate. A typical cell number is between 1 x 104 and 5 x 104 cells/well. A cell number titration may be necessary for optimal results.
  2. Aspirate media from the plate and add test compounds in growth medium or 1X PBS to the plate at 100 l/well and incubate cells for desired time. Compound titration and incubation time course can help determine the best assay conditions. For a positive control (mitochondrial membrane potential loss), add CCCP to the control wells at 50 µM final concentration and incubate cells at 37°C for 15 min. For example, add 1 µl of 50 mM stock CCCP to 100 µl medium to make 500 µM CCCP; then add 10 µl of this 500 µM CCCP to each well containing 100 µl medium to get final concentration of 50 µM.
  3. Add 10 µl of TMRE Labeling Solution to each well to get a final concentration of 200 nM and place plate in an incubator (37°C and 5% CO2) for 20 min. Note: 200 nM TMRE is recommended in this protocol. For best results, a titration of TMRE is recommended.
  4. Aspirate the solution from the plate.
  5. Wash plate 3 times with warm 1X PBS and then add 100 µl/well 1X PBS to the plate.
  6. Analyze sample with an appropriate instrument. For analysis using the CellSimple™ Cell Analyzer, use the Open Flow Cytometry Application selecting only the 561 nm LP detection channel. Please see the CellSimple™ user guide for more details about using the Open Flow Cytometry Application. If samples are to be analyzed on a plate reader, transfer 100 µl cell suspension/well to a black 96-well plate with a clear bottom and read using the following settings: excitation at approximately 550 nm and emission at approximately 580 nm.

posted June 2016

revised November 2016

Product Includes Quantity (with Count)
TMRE 1 x 29 µg
CCCP 1 x 100 µl
Phosphate Buffered Saline (PBS-20X) 9808 1 x 25 ml

Product Description

The CellSimple™ Mitochondrial Membrane Potential Assay Kit (II) is a fluorescent assay designed for use with the CellSimple™ Cell Analyzer. It detects the mitochondrial membrane potential in living cells. The kit includes the cationic dye TMRE (tetramethylrhodamine ethyl ester perchlorate) and a mitochondrial membrane potential disruptor CCCP (carbonyl cyanide 3-chlorophenylhydrazone). TMRE is a cell membrane permeable, fluorescent dye that accumulates in intact mitochondria. Depolarized or inactive mitochondria exhibit decreased membrane potential, resulting in reduced TMRE accumulation. The mean fluorescence intensity (MFI) of the red emission (561 nm LP) can be used as an indicator for mitochondrial membrane potential.


Product Usage Information

Storage: All components in this kit are stable for at least 12 months when stored at the recommended temperature and left unused. Upon receipt, #9808 should be removed from kit box and stored at room temperature. Remaining components should be stored at -20ºC.

Specificity / Sensitivity


Species Reactivity: Human
Species predicted to react based on 100% sequence homology: All Species Expected

The CellSimple™ Cell Analyzer is a benchtop instrument that utilizes a disposable thin-film cassette and a combination of a 488 nm laser, two photomultiplier tubes (525/45 nm and 561 nm LP filters), Coulter Principle-based cell measurements, and on-board software to provide easy-to-run applications and data analysis. Data acquisition occurs within approximately 10 seconds per test. The instrument relies on disposable cassettes for sample handling, which alleviates the need for flow cell cleaning and fluidics maintenance and the instrument is small enough to be portable between the lab bench and the hood. Applications include quantitative assessments of cell viability, apoptosis, other labeled antibody markers and single and multiplexed bead-based assays for protein and cellular analysis.


Mitochondria are the main power house in cells and play important roles in processes such as steroid metabolism, calcium homeostasis, apoptosis and cellular proliferation. Mitochondrial membrane potential is a key indicator of cell health (1,2). The dissipation of mitochondrial membrane potential is established as an early indicator for apoptosis (3).

TMRE (tetramethylrhodamine, ethyl ester) is a cell membrane permeable cationic dye. In normal cells, TMRE accumulates in the mitochondria in response to their high membrane potential and negative charge. When excited at 550 nm, TMRE emits an orange-red fluorescence with a maximum at 575 nm (orange-red). Cells that have lost membrane potential or mitochondria activity cannot accumulate TMRE. Therefore, the fluorescence intensity of the orange-red emission can be used to measure mitochondrial membrane potential and is an indicator for cell health (4).


1.  Perry, S.W. et al. (2011) Biotechniques 50, 98-115.

2.  Nesti, C. et al. (2007) Biosci Rep 27, 165-71.

3.  Petit, P.X. et al. (1995) J Cell Biol 130, 157-67.

4.  O'Reilly, C.M. et al. (2003) Biophys J 85, 3350-7.



For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
CellSimple is a trademark of Cell Signaling Technology, Inc.

45898
CellSimple™ Mitochondrial Membrane Potential Assay Kit (II)