NIH 3T3 fibroblasts stained with JC-1 showing the progressive loss of red J-aggregate fluorescence and cytoplasmic diffusion of green monomer fluorescence following exposure to hydrogen peroxide. Images show the same field of cells viewed before H2O2 treatment and 5, 10, and 20 minutes after treatment.
JC-1 Dye for Mitochondrial Membrane Potential
The membrane-permeant JC-1 dye is widely used in apoptosis studies to monitor mitochondrial health. JC-1 dye can be used as an indicator of mitochondrial membrane potential in a variety of cell types, including myocytes and neurons, as well as in intact tissues and isolated mitochondria. We offer Invitrogen JC-1 dye as a bulk chemical and in the Invitrogen MitoProbe JC-1 Assay Kit, optimized for flow cytometry.
Early stages of apoptosis
A distinctive feature of the early stages of programmed cell death is the disruption of active mitochondria. This mitochondrial disruption includes changes in the membrane potential and alterations to the oxidation–reduction potential of the mitochondria. Changes in the membrane potential are presumed to be due to the opening of the mitochondrial permeability transition pore (MPTP), allowing passage of ions and small molecules. The resulting equilibration of ions leads in turn to the decoupling of the respiratory chain and the release of cytochrome c into the cytosol.
Probes that detect mitochondrial membrane potential are positively charged, causing them to accumulate in the electronegative interior of the mitochondrion. Changes in the mitochondrial membrane potential can be measure by a variety of fluorescent techniques such as flow cytometry and fluorescent imaging. Mitochondrion-selective reagents enable researchers to probe mitochondrial activity, localization and abundance, as well as to monitor the effects of some pharmacological agents, such as anesthetics that alter mitochondrial function.
Studying mitochondrial health
The membrane-permeant JC-1 dye is widely used in apoptosis studies to monitor mitochondrial health. JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~529 nm) to red (~590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. The potential-sensitive color shift is due to concentration-dependent formation of red fluorescent J-aggregates.
The versatility of JC-1 dye
JC-1 dye can be used as an indicator of mitochondrial potential in a variety of cell types, including myocytes and neurons, as well as in intact tissues and isolated mitochondria. JC-1 dye is more specific for mitochondrial versus plasma membrane potential and more consistent in its response to depolarization than some other cationic dyes such as DiOC6(3) and rhodamine 123. The ratio of green to red fluorescence depends only on the membrane potential and not on other factors such as mitochondrial size, shape, and density, which may influence single-component fluorescence signals. Use of fluorescence ratio detection therefore allows researchers to make comparative measurements of membrane potential and determine the percentage of mitochondria within a population that respond to an applied stimulus.
Subtle heterogeneity in cellular responses can be discerned in this way. For example, four distinct patterns of mitochondrial membrane potential change in response to glutamate receptor activation in neurons have been identified using confocal ratio imaging of JC-1 dye fluorescence. The most widely implemented application of JC-1 dye is for detecting mitochondrial depolarization occurring in apoptosis.
Reagent selection guide
Cultured human pre-adipocytes loaded with the ratiometric mitochondrial potential indicator JC-1 at 5 µM for 30 minutes at 37°C. In live cells, JC-1 exists either as a green-fluorescent monomer at depolarized membrane potentials or as an orange-fluorescent J-aggregate at hyperpolarized membrane potentials. Cells were then treated with 50 nM FCCP, a protonophore, to depolarize the mitochondrial membrane. Approximately 10 minutes after the addition of the uncoupler, the cells were illuminated at 488 nm and the emission was collected between 515/545 nm and 575/625 nm. (Image contributed by Bob Terry, BioImage A/S, Denmark.)
Potential-dependent staining of mitochondria in CCL64 fibroblasts by JC-1. The mitochondria were visualized by epifluorescence microscopy using a 520 nm longpass optical filter. Regions of high mitochondrial polarization are indicated by red fluorescence due to J-aggregate formation by the concentrated dye. Depolarized regions are indicated by the green fluorescence of the JC-1 monomers. (Image contributed by Lan Bo Chen, Dana Farber Cancer Institute, Harvard Medical School.)
Flow cytometric analysis of Jurkat cells using the MitoProbe JC-1 Assay Kit.Jurkat cells were stained with 2 μM JC-1 for 15 min at 37°C, 5% CO2, and then washed with phosphate-buffered saline (PBS) and analyzed on a flow cytometer using 488 nm excitation with 530 nm and 585 nm bandpass emission filters. (A)Untreated cultured cells. (B) Cells induced to apoptosis with 10 μM camptothecin for 4 hr at 37°C.
The following protocol describes introducing JC-1 reagent into cultured cells and analyzing the stained cells by flow cytometry. Suggested initial conditions may require modifications because of differences in cell types and culture conditions. The concentration of probe for optimal staining will vary depending upon the application. A concentration range should be tested starting around 2 µM JC-1. CCCP controls should be used to confirm that the JC-1 response is sensitive to changes in membrane potential.
Allow the JC-1 powder and DMSO solutions to come to room temperature before use. Prepare a 200 µM JC-1 stock solution immediately prior to use by dissolving the contents of one vial in 230 µL of the DMSO provided.
Labeling of cells
Before beginning the experiment, ensure that the vial of CCCP has equilibrated to room temperature.
- 1.1 For each sample, suspend cells in 1 mL warm medium, phosphate-buffered saline, or other buffer at approximately 1×106 cells/mL.
- 1.2 For the control tube, add 1 µL of 50 mM CCCP (supplied with the kit, 50 µM final concentration) and incubate the cells at 37°C for 5 minutes. Note: CCCP can be added simultaneously with JC-1. Titration of the CCCP may be required for optimal results with an individual cell system.
- 1.3 Add 10 µL of 200 µM JC-1 (2 µM final concentration) and incubate the cells at 37°C, 5% CO2, for 15 to 30 minutes. If performing additional labeling, for example with an annexin V conjugate, follow the protocol below, beginning with step 2.1. If no additional staining is to be performed, proceed with step 1.4.
- 1.4 OPTIONAL: Wash cells once by adding 2 mL of warm phosphate-buffered saline (PBS) or other buffer to each tube of cells.
- 1.5 Pellet the cells by centrifugation.
- 1.6 Resuspend by gently flicking the tubes. Add 500 µL PBS (or other suitable buffer) to each tube.
- 1.7 Analyze on a flow cytometer with 488 nm excitation using emission filters appropriate for Invitrogen Alexa Fluor 488 dye and R-phycoerythrin. Gate on the cells, excluding debris. Using the CCCP-treated sample, perform standard compensation.
Additional labeling with an annexin V conjugate
It is possible to label the JC-1–stained cells with other markers for apoptosis or viability, as long as the fluorescence emission of the additional label is spectrally resolved from JC-1. The example below is a protocol for labeling with annexin V-allophycocyanin.
- 2.1 After step 1.3 (above), wash cells once by adding 2 mL of warm phosphate-buffered saline or other buffer to each tube of cells.
- 2.2 Pellet the JC-1–stained cells and resuspend in 100 µL of 1X annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl2, pH 7.4).
- 2.3 Add 5 µL annexin V conjugate (e.g., Invitrogen annexin V-allophycocyanin). Note: 5 µL is appropriate for Invitrogen annexin V conjugates. Conjugates purchased from other suppliers may require a different volume to be effective.
For Research Use Only. Not for use in diagnostic procedures.