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Protocol for CellSimple™ Mitochondrial Membrane Potential (I) Assay #42239 and Mitochondrial Membrane Potential (I) Assay Protocol #12664

A. Instrumentation:

The CellSimple™ Mitochondrial Membrane Potential (I) Assay was 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 at approximately 480 nm and detecting fluorescent emission at approximately 520 nm and 590 nm.

B. Kit components:

  • JC-1
  • 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. JC-1 Stock Solution: Add 110 µl DMSO to each vial of JC-1 to make a 200 µM stock solution.
  3. 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 10 µl of 200 µM JC-1 stock solution to each sample (2.0 µM final concentration). Incubate cells in an incubator (37°C and 5% CO2) for 15 to 30 minutes.
  4. Centrifuge sample at 300 x g for 5 min then remove the supernatant.
  5. Wash cells once with 1.0 mL warm 1X PBS wash buffer. Repeat step 7.
  6. Re-suspend cells into 1.0 ml warm 1X PBS.
  7. Analyze sample using an appropriate instrument.
    1. For analysis using the CellSimple™ Cell Analyzer use the Open Flow Cytometry Application selecting only the 561 nm LP detection channel. Results may be obtained by choosing the histogram feature (X/Y function on the display screen) or by selecting the control and treated samples in the save data files and using the overlay file feature. Please see the CellSimple™ user guide for more details about using the Open Flow Application.
    2. 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.
    3. Analyze sample on flow cytometer with excitation of ex550/em580
  8. To calculate the red to green ratios using the controls:

    (Red (MFI) control ÷ Green (MFI) control) : (Red (MFI) sample ÷ Green (MFI) sample)

F. Protocol for adherent cells

  1. Plate cells in a 96 well plate in warm culture medium and 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 plate at 100 µl/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 M.
  3. Add 1 µl of JC-1 stock (200 M) to each well to get a final concentration of 2 µM and place the plate in an incubator (37°C and 5% CO2) for 15 to 30 minutes. Note: JC-1 can be diluted 1:10 in media to make a 20 µM solution, add 10 µl of 20 µM JC-1 to each well containing 100 µl media for a final concentration of 2 M.
  4. Aspirate the solution from the plate.
  5. Wash plate 3 times with warm 1X PBS and then add 1X PBS at 100 l/well to the plate.
  6. Analyze sample using an appropriate instrument.
    1. For analysis using the CellSimple™ Cell Analyzer use the Open Flow Cytometry Application selecting only the 561 nm LP detection channel. Results may be obtained by choosing the histogram feature (X/Y function on the display screen) or by selecting the control and treated samples in the save data files and using the overlay file feature. Please see the CellSimple™ user guide for more details about using the Open Flow Application.
    2. 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.
    3. Analyze sample on flow cytometer with excitation of ex550/em580
  7. To calculate the red to green ratios using the controls:

    (Red (MFI) control ÷ Green (MFI) control) : (Red (MFI) sample ÷ Green (MFI) sample)

posted June 2016

revised November 2016

Product Includes Quantity (with Count)
JC-1 3 x 15 µg
CCCP 1 x 100 µl
Phosphate Buffered Saline (PBS-20X) 9808 1 x 25 ml

Product Description

CellSimple™ Mitochondrial Membrane Potential Assay Kit (I) 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 JC-1 and a mitochondrial membrane potential disruptor CCCP (carbonyl cyanide 3-chlorophenylhydrazone). JC-1 is a cell membrane permeable, fluorescent dye with green emission (~520 nm). When JC-1 accumulates in intact mitochondria, the dye forms aggregates that lead to orange-red fluorescence (~590 nm). The mean fluorescence intensity (MFI) of the orange-red emission 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 function as the main cellular powerhouse and play important roles in other processes, such as steroid metabolism, calcium homeostasis, apoptosis, and cellular proliferation. Mitochondrial membrane potential is a key indicator of mitochondrial function and cell health (1,2). The dissipation of mitochondrial membrane potential is considered an early indicator of apoptosis (3).

JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide) is a cell membrane permeable, cationic dye. In normal cells, JC-1 concentrates in mitochondria to form aggregates in response to high membrane potential. Decreased mitochondrial membrane potential results in dispersal of mostly monomeric JC-1 throughout the cell. When excited at 490 nm, JC-1 monomers emit a green fluorescence with a maximum at ~520 nm. Aggregates of JC-1 emit an orange-red fluorescence with a maximum at ~590 nm. Therefore, the fluorescence intensity of the orange-red emission and the ratio of orange-red fluorescence to green fluorescence can be used to measure mitochondrial membrane potential and serve as an indicator of overall 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.  Perelman, A. et al. (2012) Cell Death Dis 3, e430.



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.

42239
CellSimple™ Mitochondrial Membrane Potential Assay Kit (I)