Figure 2. 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 is a commonly used tool used for studying mitochondrial membrane potential, providing valuable insights into mitochondrial health and activity. JC-1 staining 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. JC-1 is a membrane-permeant dye that selectively accumulates in the mitochondria and exhibits a shift in emission color from green to red as the mitochondria membrane potential becomes more polarized. Thermo Fisher Scientific offers JC-1 dye as a bulk chemical or in the MitoProbe JC-1 Assay, optimized for flow cytometry.
See also Mitochondria function assays
Mitochondrial membrane potential changes in apoptosis
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 associated with the opening of the mitochondrial permeability transition pore (MPTP), allowing passage of ions and small molecules (Figure 1). The resulting equilibration of ions leads in turn to the decoupling of the respiratory chain and the release of cytochrome c into the cytosol.
Mitochondrion-selective reagents
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 measured by a variety of fluorescence techniques such as flow cytometry and fluorescence imaging. Mitochondrion-selective reagents enable researchers to probe mitochondrial health, localization, and abundance, as well as to screen and monitor the effects of some pharmacological agents.
Figure 1. Mitochondrial membrane potential
JC-1 dye as a mitochondrial membrane potential indicator
Studying mitochondrial health
The membrane-permeant JC-1 dye is widely used in apoptosis studies to monitor mitochondrial health (1). JC-1 dye exhibits potential-dependent accumulation in mitochondria, at lower internal mitochondrial concentrations or low membrane potential JC-1 dye is present as monomers, indicated by a green fluorescence emission of 529 nm. At higher concentrations (aqueous solutions above 0.1 µM) or higher potentials, JC-1 dye forms red fluorescent "J-aggregates" where it has accumulated within the mitochondria (Figure 2). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.
The ratio of green to red fluorescence depends only on the mitochondrial 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.
Click image to enlarge
JC-1 dye is available in different formats as either a stand-alone reagent or assay kit with a mitochondrial membrane disrupter and buffers.
| JC-1 | MitoProbe JC-1 Assay Kit |
|---|---|---|
| Readout | Active mitochondria exhibit brighter red fluorescence signal compared to mitochondria with lower membrane potential which fluoresce green. Changes in the red/green fluorescence signal ratio can be used to determine healthy versus depolarized mitochondria. | |
| Platform | Imaging Flow cytometry | Flow cytometry |
| Ex/Em (nm) | 514/529 (monomer, green) | |
| 514/590 (J-aggregate, red) | ||
| Common filters and platform | FITC and TRITC (imaging) FITC and PE (flow cytometry) | FITC and PE |
| Sample type | Live cells | Live cells |
| Compatibility with fixation | No | No |
| Format | 5 mg | Kit contents: JC-1, DMSO CCCP (a mitochondria membrane potential disrupter in DMSO) 10x PBS |
| Protocol | JC-1 protocol for imaging includes a description of JC-1 staining conditions in a selection of cell types, including both adherent and suspension cells | JC-1 protocol for flow cytometry includes a description of combining JC-1 staining with annexin V conjugate labeling |
| Cat. No. | T3168 | M34152 |
Experimental data using JC-1 dye
Various types of ratio measurements are possible by combining signals from the green-fluorescent JC-1 monomer (absorption/emission maxima ~514/529 nm in water) and the red-fluorescent J-aggregates (emission maximum 590 nm), which can be effectively excited anywhere between 485 nm and its absorption maximum at 585 nm. Optical filters designed for fluorescein (FITC) and tetramethylrhodamine (TRITC) can be used to separately visualize the monomer and J-aggregate forms, respectively (Figure 3). Alternatively, both forms can be observed simultaneously using a standard fluorescein long-pass optical filter set (Figure 4).
Figure 3. 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.
Figure 4. 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.
In flow cytometry, JC-1 staining to conduct analysis of mitochondrial membrane potential has been detailed using K+/valinomycin-induced depolarization (Figure 5) and/or apoptosis-inducing treatments (Figure 5, 6).
A
B
C
D
Figure 5. Bivariate JC-1 Dye (Mitochondrial Membrane Potential Probe) analysis of mitochondrial membrane potential in HL60 cells by flow cytometry. The sensitivity of this technique is demonstrated by the response to K+/valinomycin (Fluorescent Na+ and K+ Indicators—Section 21.1)–induced depolarization (panels B and D) as compared to the control (panel A). Distinct populations of cells with different extents of mitochondrial depolarization are detectable following apoptosis-inducing treatment with 5 µM staurosporine for two hours (panel C).
Figure 6. 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.
Although JC-1 is widely used, there are alternative reagents to fit different filters in flow cytometry, options for studying detection of dynamic changes using single-emission, non-ratiometric reagents, and also options for using end point assays to assess mitochondrial membrane potential.
Learn more about other functional assays for studying mitochondrial membrane potential
There are additional methods for assessing cells for apoptosis beyond detecting changes in mitochondrial membrane potential. Because no single parameter fully defines apoptosis in all systems and the appearance of these changes can vary with apoptotic pathway or cell types, it is often advantageous to use several different approaches when studying apoptosis.
Learn more about other Apoptosis Assays
Mitochondrial membrane potential changes in apoptosis
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 associated with the opening of the mitochondrial permeability transition pore (MPTP), allowing passage of ions and small molecules (Figure 1). The resulting equilibration of ions leads in turn to the decoupling of the respiratory chain and the release of cytochrome c into the cytosol.
Mitochondrion-selective reagents
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 measured by a variety of fluorescence techniques such as flow cytometry and fluorescence imaging. Mitochondrion-selective reagents enable researchers to probe mitochondrial health, localization, and abundance, as well as to screen and monitor the effects of some pharmacological agents.
Figure 1. Mitochondrial membrane potential
JC-1 dye as a mitochondrial membrane potential indicator
Studying mitochondrial health
The membrane-permeant JC-1 dye is widely used in apoptosis studies to monitor mitochondrial health (1). JC-1 dye exhibits potential-dependent accumulation in mitochondria, at lower internal mitochondrial concentrations or low membrane potential JC-1 dye is present as monomers, indicated by a green fluorescence emission of 529 nm. At higher concentrations (aqueous solutions above 0.1 µM) or higher potentials, JC-1 dye forms red fluorescent "J-aggregates" where it has accumulated within the mitochondria (Figure 2). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.
The ratio of green to red fluorescence depends only on the mitochondrial 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.
Click image to enlarge
Figure 2. 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 is available in different formats as either a stand-alone reagent or assay kit with a mitochondrial membrane disrupter and buffers.
| JC-1 | MitoProbe JC-1 Assay Kit |
|---|---|---|
| Readout | Active mitochondria exhibit brighter red fluorescence signal compared to mitochondria with lower membrane potential which fluoresce green. Changes in the red/green fluorescence signal ratio can be used to determine healthy versus depolarized mitochondria. | |
| Platform | Imaging Flow cytometry | Flow cytometry |
| Ex/Em (nm) | 514/529 (monomer, green) | |
| 514/590 (J-aggregate, red) | ||
| Common filters and platform | FITC and TRITC (imaging) FITC and PE (flow cytometry) | FITC and PE |
| Sample type | Live cells | Live cells |
| Compatibility with fixation | No | No |
| Format | 5 mg | Kit contents: JC-1, DMSO CCCP (a mitochondria membrane potential disrupter in DMSO) 10x PBS |
| Protocol | JC-1 protocol for imaging includes a description of JC-1 staining conditions in a selection of cell types, including both adherent and suspension cells | JC-1 protocol for flow cytometry includes a description of combining JC-1 staining with annexin V conjugate labeling |
| Cat. No. | T3168 | M34152 |
Experimental data using JC-1 dye
Various types of ratio measurements are possible by combining signals from the green-fluorescent JC-1 monomer (absorption/emission maxima ~514/529 nm in water) and the red-fluorescent J-aggregates (emission maximum 590 nm), which can be effectively excited anywhere between 485 nm and its absorption maximum at 585 nm. Optical filters designed for fluorescein (FITC) and tetramethylrhodamine (TRITC) can be used to separately visualize the monomer and J-aggregate forms, respectively (Figure 3). Alternatively, both forms can be observed simultaneously using a standard fluorescein long-pass optical filter set (Figure 4).
Figure 3. 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.
Figure 4. 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.
In flow cytometry, JC-1 staining to conduct analysis of mitochondrial membrane potential has been detailed using K+/valinomycin-induced depolarization (Figure 5) and/or apoptosis-inducing treatments (Figure 5, 6).
A
B
C
D
Figure 5. Bivariate JC-1 Dye (Mitochondrial Membrane Potential Probe) analysis of mitochondrial membrane potential in HL60 cells by flow cytometry. The sensitivity of this technique is demonstrated by the response to K+/valinomycin (Fluorescent Na+ and K+ Indicators—Section 21.1)–induced depolarization (panels B and D) as compared to the control (panel A). Distinct populations of cells with different extents of mitochondrial depolarization are detectable following apoptosis-inducing treatment with 5 µM staurosporine for two hours (panel C).
Figure 6. 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.
Although JC-1 is widely used, there are alternative reagents to fit different filters in flow cytometry, options for studying detection of dynamic changes using single-emission, non-ratiometric reagents, and also options for using end point assays to assess mitochondrial membrane potential.
Learn more about other functional assays for studying mitochondrial membrane potential
There are additional methods for assessing cells for apoptosis beyond detecting changes in mitochondrial membrane potential. Because no single parameter fully defines apoptosis in all systems and the appearance of these changes can vary with apoptotic pathway or cell types, it is often advantageous to use several different approaches when studying apoptosis.
Learn more about other Apoptosis Assays
JC-1 ordering information
References
- Reers M, Smith TW, Chen LB. (1991) J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry. 30(18): 4480-6.
- Sara De Biasi S, Gibellini L, Cossarizza A (2015) Uncompensated Polychromatic Analysis of Mitochondrial Membrane Potential Using JC-1 and Multilaser Excitation. Curr Protoc Cytom 72(1): 7.32.1-7.32.11.
- Smiley ST, Reers M, Mottola-hartshorn C, et al. (1991) Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci USA. 88(9): 3671-5.
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