Microscopic view of fibroblasts with green, blue and orange fluorescence

Thermo Fisher Scientific offers an array of reagents and assays for detecting and monitoring changes in mitochondria function on imaging and flow cytometry platforms. Options include probes for real-time measurements in live cells and live-cell stains that are compatible with fixation and immunodetection.

Overview of mitochondria function assays

A distinguishing feature of apoptosis is the disruption of mitochondria function, resulting in an increase of mitochondrial membrane permeability as well as decreases in membrane potential and oxidation-reduction activity. These changes can be detected using a variety of fluorescence-based assays including measurement of calcium flux with fluorescent Ca2+ indicators, superoxide detection probes, mitochondria transition pore opening assays and membrane potential dyes. A summary of these assays can be seen in Table 1 below.

Table 1. Summary of mitochondria function assays.

  Mitochondria function assays
(See corresponding tabs for more information)
  Membrane potential Superoxide production Calcium flux Transition pore opening
What can be identified? Cells with healthy, metabolically active mitochondria Detection of mitochondrial superoxide Calcium influx into mitochondria Changes in the membrane potential are presumed to be due to the opening of the mitochondrial permeability transition pore, allowing release of cytochrome c and other small molecules.
What is the basis of assay? Measure fluorescence of reagents that accumulate in active (healthy) mitochondria with intact membrane potentials Measure the increasing fluorescence of a superoxide detection reagent with increasing superoxide concentration in the mitochondria Measure the increasing fluorescence of a calcium indicator with increasing calcium concentration in the mitochondria In healthy cells the mitochondria take up a probe and fluoresce brightly. When the mitochondrial transition pore opens, fluorescence is lost due to influx of a quenching reagent through the pore.

Healthy mitochondria membranes maintain a difference in electrical potential between the interior and exterior of the organelle, referred to as a membrane potential. Mitochondrion-selective stains that accumulate and are concentrated by active mitochondria with intact membrane potentials can be used to assay mitochondria health including detection of cells going through apoptosis. There are different options available to detect the mitochondria membrane potential state, including ratiometric versus single wavelength dyes, fixable versus nonfixable dyes, and dyes that are formatted for fluorescence imaging, flow cytometry or microplate reader instrumentation.

In this section you will find:

Ratiometric dyes

JC-1 dye

The most popular dye for detecting mitochondria membrane potential is the cell-permeant JC-1 dye which is used to detect apoptotic cells with flow cytometry and microscopy platforms (Figure 1). JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a concentration dependent–fluorescence emission shift from the monomer (green, ~529 nm) that is prominent at lower dye concentrations to aggregates (red, ~590 nm) which are formed as the dye concentration increases. Consequently, mitochondria depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.

Learn more about the JC-1 dye for mitochondria membrane potential

Microscopic view of mitochondria stained with green-red fluorescence at 4 different time course points.
A
Dot plot of JC-1 stained cells showing 2 cell populations-healthy cells colored red and apoptotic cells colored green.
B

Figure 1. Detection of mitochondria membrane potential using JC-1 dye. (A) NIH 3T3 fibroblasts stained with JC-1 dye show 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 min after treatment. The images were contributed by Ildo Nicoletti, Perugia University Medical School. (B) MitoProbe JC-1 Assay Kit was used to stain Jurkat cells (T-cell leukemia, human) which were then analyzed on a flow cytometer using 488 nm excitation with 530 nm and 585 nm bandpass emission filters. Green = apoptotic cells (reduced mitochondria membrane potential), red = normal cells.

MitoProbe DiOC2(3) Assay Kit

Another cationic carbocyanine dye that can be used as a ratiometric probe for mitochondria membrane potential is DiOC2(3). The mechanism is similar to that of JC-1 dye in that the monomer exhibits green fluorescence at lower concentrations where mitochondria membrane potential is low and the aggregate of the dye in more active mitochondria causes the emission to shift toward the red. In the case of DiOC2(3), the shift is farther in the red than JC-1, above 650 nm. The MitoProbe DiOC2(3) Assay Kit has been specifically developed to work in flow cytometry applications and includes a mitochondrial membrane-potential disrupter, CCCP.

Ratiometric dye selection guide

  JC-1 MitoProbe JC-1 Assay Kit MitoProbe DiOC2(3) 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.
Ex/Em (nm) 514/529 (monomer, green) 485/497 (monomer, green)
514/590 (aggregate, red) 485/>650 (aggregate, far–red)
Common filters TRITC FITC and PE FITC and APC
Instrument platform Imaging microscopy Flow cytometry Flow cytometry
Sample type Live cells Live cells Live cells
Compatibility with fixation No No No
Format 5 mg Kit contents:
JC-1, DMSO
CCCP (a mitochondria membrane potential disrupter in DMSO)
10x PBS
Kit contents:
DiOC2(3) in DMSO
CCCP (a mitochondrial membrane potential disrupter in DMSO)
Cat. No. T3168 M34152 M34150

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Fixable mitochondria membrane potential dyes

MitoTracker probes are small (<1kDa), cell-permeant mitochondrion-selective dyes that contain a mildly thiol-reactive chloromethyl moiety that is thought to be responsible for keeping the dye associated with the mitochondria after fixation. To label mitochondria, cells are simply incubated in submicromolar concentrations of the MitoTracker probe, which passively diffuses across the plasma membrane and accumulates in active mitochondria. Once their mitochondria are labeled, the cells can be treated with aldehyde-based fixatives to allow further processing of the sample without losing the pattern of staining achieved in the live cells. (We should note that MitoTracker Red FM dye is not fixable and should only be used in live cells.) These single wavelength emission reagents can be used for flow cytometry, imaging microscopy, and high content analysis applications and are compatible with multicolor experiments. For example, our Mitochondrial Membrane Potential/Annexin V Apoptosis Kit (Figure 2) utilizes MitoTracker Red CMXRos in combination with Alexa Fluor 488 annexin V in a two-color flow cytometric assay of apoptotic cells. Also shown in Figure 3 is detection of MitoTracker Orange CMTMRos and CellEvent Green Caspase 3/7 reagent in 2 cell types with and without treatment to induce apoptosis. The mitochondria membrane potential (orange fluorescence) decreases as the dose of drug increases, with concomitant increase in caspase activation (green fluorescence).

Figure 2. MitoTracker CMXRos dye mitochondria membrane potential detection. Jurkat human T-cell leukemia cells in complete medium were (A) first exposed to 10 µM camptothecin for 4 hours or (B) left untreated. Both cell populations were then treated with the reagents in the Mitochondrial Membrane Potential/Annexin V Apoptosis Kit and analyzed by flow cytometry. Note that the apoptotic cells show higher reactivity for annexin V and lower MitoTracker Red dye fluorescence than do live cells.

High-content images of cells stained with orange fluorescent mitochondria dye and green fluorescent caspase dye in 2 cell types with and without treatment to induce apoptosis. Also shows quantitative dose response curves of treated cells with decreasing orange fluorescence and increasing green fluorescence at higher concentrations of drug.

Figure 3. Mitochondria membrane potential quantification using MitoTracker Orange CMXRos. A549 or HeLa cells were cultured in Nunclon Sphera plates in GIBCO minimal essential media (MEM) to form spheroids over 2 days. Samples were treated with either DMSO vehicle or an increasing concentration of niclosamide for 24 hours before labeling with 250 nM MitoTracker Orange and 2.5 uM CellEvent Caspase 3/7 Green Reagent for 30 minutes at 37°C under normal cell culture conditions. Confocal images were acquired using the Thermo Fisher Scientific CellInsight CX7 LZR High Content Imaging System, followed by image analysis using HCS Studio Software V 2.0. Results show a niclosamide dose-dependent loss of mitochondrial membrane potential (orange) and increase in apoptotic cell death (green), qualitatively (upper panels) and quantitatively as a dose response vs mean signal intensity in both channels (lower graphs).

Fixable dyes selection guide

  MitoTracker dyes
MitoTracker Orange CMTMRos MitoTracker Red CMXRos MitoTracker Red FM
Readout Active mitochondria exhibit brighter fluorescence compared to apoptotic mitochondria.
Ex/Em (nm) 550/580 579/599 581/644
Common filter TRITC Texas Red Cy5
Instrument platform Flow cytometry
Fluorescence microscopy
Microplate reader
Sample type Live cells Live cells Live cells
Compatibility with fixation Yes Yes No
Format 20 x 50 μg 20 x 50 μg 20 x 50 μg
Cat. No. M7510 M7512 M22425

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Nonfixable mitochondria membrane potential dyes

DiIC11(5) cationic dye

Cationic carbocyanine dyes have been shown to accumulate in cells in response to membrane potential. The MitoProbe DiIC1(5) Kit is designed for flow cytometry applications and provides the far-red–fluorescent DiIC1(5) carbocyanine dye, along with a mitochondrial membrane potential disrupter, CCCP, for the study of mitochondrial membrane potential The dye penetrates the cytosol of eukaryotic cells and, at concentrations below 100 nM, accumulates primarily in mitochondria with active membrane potentials (Figure 4). Unlike DiOC2(3), however, DiIC1(5) dye exhibits a single wavelength emission whose fluorescence signal increases with more active mitochondria.

Flow cytometry histogram showing cells with active mitochondria in control compared to cells with depolarized mitochondria with CCCP treatment

Figure 4. Detection of changes in mitochondrial membrane potential using the MitoProbe DiIC1(5) Assay Kit for Flow Cytometry. Decrease in mitochondrial membrane potential as demonstrated with DiIC1(5) fluorescence due to the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Jurkat cells (T-cell leukemia, human) were stained with 50 nM DiIC1(5) alone (blue) or in the presence of 50 μM CCCP (red).

TMRM

Tetramethylrhodamine, methyl ester (TMRM) is a small, cell-permeant dye that accumulates in active mitochondria. If the cells are healthy and have functioning mitochondria, the signal is bright. Upon loss of the mitochondrial membrane potential, TMRM accumulation ceases and the signal dims or disappears. This ability to dynamically monitor changes in mitochondria membrane potential is a distinct advantage of this probe. TMRM signal can be detected with fluorescence microscopy, flow cytometry, cell sorting, high throughput screening, and high content analysis (Figure 5).

Microscopic view of cells stained with orange fluorescence (mitochondria) and green fluorescence (tubulin)
A
Dot plot showing healthy, apoptotic and dead cells with and without treatment.
B

Figure 5. TMRM as a mitochondrial membrane potential assay for apoptosis. (A) HeLa cells labeled using Tubulin Tracker Green and Image-iT TMRM Reagent show superb multiplexing capability and staining specificity. Image shows multiple live mitotic cells with microtubules assembled into a mitotic spindle with visible microtubule filaments, as well as cleavage furrow indicating completion of cytokinesis. (B) Jurkat cells, a human T-lymphocyte cell line, were treated with DMSO (control) or 500 nM staurosporine for 2 hours. Cells were subsequently stained with MitoProbe TMRM for 30 min at 37˚C, followed by a wash and additional stain with Annexin V Pacific Blue conjugate. Staurosporine induced apoptosis, resulting in a mixed population of cells containing a population of healthy MitoProbe TMRM-positive cells as well as a population of apoptotic, Annexin V Pacific Blue-positive, MitoProbe TMRM-low cells.

Nonfixable dyes selection guide

  TMRM MitoProbe TMRM Assay Kit MitoProbe DiIC1(5) Assay Kit
Readout Active mitochondria exhibit brighter fluorescence compared to apoptotic mitochondria.
Ex/Em (nm) 548/574 nm 638/658
Common filter TRITC ~585/16 nm Alexa Fluor 647/APC
Instrument platform Fluorescence microscopy Flow cytometry Flow cytometry
Sample type Live cells Live cells Live cells
Compatibility with fixation No No No
Format 25 mg 5 x 100 μL Kit contents:
TMRM
CCCP (a mitochondrial membrane potential disrupter in DMSO)
Kit contents:
DiIC1(5) in DMSO
CCCP (a mitochondrial membrane potential disrupter in DMSO)
Cat. No. T668 I34361 M20036 M34151

Increases in cellular superoxide production have been implicated in multiple disease states (1). This increase, which is generated as a byproduct of oxidative phosphorylation, provides another method to assess the cells’ apoptotic state.

MitoSOX Red Mitochondrial Superoxide Indicator

MitoSOX Red mitochondrial superoxide indicator is a cationic reagent designed for highly selective detection of superoxide in the mitochondria of live, healthy cells (Figure 6). The reagent is readily oxidized by superoxide but not by other reactive oxygen species (ROS)—or reactive nitrogen species (RNS)–generating systems, and oxidation of the probe is prevented by superoxide dismutase (Figure 7 and 8). Oxidation of the MitoSOX Red indicator by superoxide results in a fluorescence excitation peak at ~400 nm that is absent in the excitation spectrum generated by reactive oxygen species other than superoxide. Thus, fluorescence excitation at 400 nm with emission detection at ~590 nm provides optimum discrimination of superoxide from other reactive oxygen species.

Microscopic view of cells with red (mitochondria) and blue (nucleus) fluorescence in absence of treatment compared to just blue fluorescence when cells are treated with scavenger.
 Click image to enlarge

Figure 6. Imaging detection of superoxide in live cells using MitoSOX Red superoxide indicator. Live 3T3 cells were treated with FeTCPP, a superoxide scavenger, (left) or left untreated (right). Cells were then labeled with MitoSOX Red reagent, which fluoresces red when oxidized by superoxide, and nuclei were stained with blue-fluorescent Hoechst 33342. The mitochondria of untreated cells exhibited red fluorescence, indicating the presence of superoxide, whereas the mitochondria of treated cells showed minimal fluorescence.

Bar graph showing lack of mitosox red fluorescence detected in presence of other reactive oxygen and nitrogen species present

Figure 7. Selectivity of the MitoSOX Red mitochondrial superoxide indicator. Cell-free systems were used to generate a variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS); each oxidant was then added to a separate 10 µM solution of MitoSOX Red reagent and incubated at 37°C for 10 minutes. Excess DNA was added (unless otherwise noted) and the samples were incubated for an additional 15 minutes at 37°C before fluorescence was measured. The Griess Reagent Kit (for nitric oxide, peroxynitrite, and nitrite standards only; blue bars) and dihydrorhodamine 123 (DHR 123, green bars) were employed as positive controls for oxidant generation. Superoxide dismutase (SOD), a superoxide scavenger, was used as a negative control for superoxide. The results show that the MitoSOX Red probe (red bars) is readily oxidized by superoxide but not by the other oxidants.

Multi-panel figure with microscopic views of cells untreated with high glucose medium that are stained with red and blue fluorescence compared to low glucose with untreated cells and cells treated with the nitric oxide generator showing blue fluorescence only, and cells treated with superoxide generator stained with both red and blue fluorescence.
Figure 8. Visualizing glucose-mediated oxidative stress. Live human osteosarcoma (U2OS) cells were plated in Minimum Essential Medium (MEM) and incubated overnight at 37°C with CO2. To mitigate the effect of high glucose–mediated oxidative stress, samples B–D were washed in PBS and immersed in low-glucose Dulbecco’s Modified Eagle Medium (DMEM) and incubated overnight at 37°C with CO2. Samples were then washed in Hanks' Balanced Salt Solution (HBSS) and then treated as follows for the next 30 minutes: (A, B) cells were left in HBSS; (C) cells were incubated in HBSS + 100 μM antimycin A; (D) cells were incubated in HBSS + 100 μM DEANO. MitoSOX Red reagent at 5 µM was added to each sample, and cells were incubated for 30 minutes and imaged by confocal microscopy.

MitoSox Red Reagent specifications

  MitoSox Red Reagent
Readout
  • Fluorescence increases with increased superoxide concentration
  • Highly selective detection of superoxide in the mitochondria of live cells
Ex/Em (nm) 510/580
Common filter RFP/TRITC
Instrument platform Fluorescence microscopy
Flow cytometry
Microplate reader
High content analysis
Sample type Live cells
Fixation compatibility Not compatible with fixation or detergent
Format Solid, 10 x 50 mg
Cat. No. M36008

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Elevated mitochondrial Ca2+ plays an important role in initiation of programmed cell death (apoptosis) as well as in other cellular processes (1). Fluorescent probes that show a spectral response upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations using fluorescence microscopy, flow cytometry and fluorescence spectroscopy. As shown in Figure 9, rhod-2 is a fluorescent calcium indicator that can also be used to detect mitochondrial Ca2+ (1). The AM ester form of rhod-2 (rhod-2, AM) is used to easily load the dye into live cells. Upon entering the cell, intracellular esterases cleave the AM group, freeing the rhod-2 salt form to bind and fluoresce upon binding to mitochondrial Ca2+.

4 panels showing microscopic view of cells stained with green fluorescent mitochondria-targeting GFP and orange fluorescence of rhod-2 calcium indicator, also localized in mitochondria.

Figure 9. Multiplex imaging of mitochondrial calcium levels and dynamics. (A) HeLa cells were labeled with CellLight Mitochondria-GFP and 5 μM rhod-2 AM for 15 min at 37°C before imaging live over 100 sec. (B–D) The region outlined in (A) is enlarged to show individual mitochondria within a single cell over time. (C, D) Calcium is released from internal stores following application of 10 μM histamine. Mitochondria in close proximity to the calcium release are revealed by the increase in the orange-red fluorescence of rhod-2. The arrow in (C) denotes a mitochondrion that may have impaired calcium uptake, a detail that would have been missed using rhod-2 AM alone. The asterisk marks a mitochondrion that shows a transient elevation in calcium levels.

Rhod-2, AM product information

  Rhod-2 AM reagent
Readout
  • Calcium indicator targeted to mitochondria
  • Increases fluorescence with Ca 2+ concentration
Ex/Em (nm) 552/577
Common filter TRITC
Instrument platform
  • Fluorescence microscopy
  • Flow cytometry
Sample type Live cells
Compatibility with fixation Not compatible with fixation or detergent
Format Solid, 20 x 50 mg
Cat. No. R1245MP

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The mitochondrial permeability transition pore (MPTP) is a nonspecific channel formed by components from the inner and outer mitochondrial membranes, and appears to be involved in the release of mitochondrial components during cell death. This opening of the pore dramatically alters the permeability of mitochondria, as well as the mitochondria membrane potential. This continuous pore activation results from mitochondrial Ca2+ overload, oxidation of mitochondrial glutathione, increased levels of reactive oxygen species in mitochondria, and other pro-apoptotic conditions.

We have developed two kits for detecting mitochondrial transition pore opening, one for imaging microscopy (Image-iT LIVE Mitochondrial Transition Pore Assay Kit, Figure 10) and the other for flow cytometry (MitoProbe Transition Pore Assay Kit, Figure 11). Both kits provide a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. This assay employs the acetoxymethyl (AM) ester of calcein, a colorless and nonfluorescent esterase substrate, and CoCl2 (cobalt), a quencher of calcein fluorescence, to selectively label mitochondria. Cells are loaded with calcein AM, which passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochondria. Once inside cells, calcein AM is cleaved by intracellular esterases to liberate the very polar fluorescent dye calcein, which does not cross the mitochondrial or plasma membranes in appreciable amounts over relatively short periods of time. The fluorescence from cytosolic calcein is quenched by the addition of CoCl2, while the fluorescence from the mitochondrial calcein is maintained. As a control, cells that have been loaded with calcein AM and CoCl2 can also be treated with a Ca2+ ionophore such as ionomycin to allow entry of excess Ca2+ into the cells, which triggers mitochondrial pore activation and subsequent loss of mitochondrial calcein fluorescence. This ionomycin response can be blocked with cyclosporin A, a compound reported to prevent mitochondrial transition pore formation by binding cyclophilin D.

Figure 10. BPAE cells stained with Image-iT LIVE Mitochondrial Transition Pore Assay Kit. BPAE cells are stained using components from the Image-iT LIVE Mitochondrial Transition Pore Assay Kit. Cells are counterstained with MitoTracker Red CMXRos to show mitochondria and with Hoechst 33342 to show nuclei. Panel A shows the uniform cellular fluorescence from unquenched calcein. Panel B shows the mitochondrial pattern after adding cobalt which quenches the cytoplasmic calcein fluorescence but not mitochondrial calcein. Panel C shows the loss of calcein fluorescence with the addition of ionomycin which opens the pore to allow cobalt in and calcein out (mitochondria still visible from MitoTracker Red). Cyclophilin D activity is necessary for MPTP formation, and is inhibited by cyclosporin A. Consequently, inhibition of pore formation by cyclosporin A (CspA) has been used as an argument that a function is MPTP-specific. Panel D shows that the calcein pattern is retained when cyclosporin A is added before ionomycin, indicating that the ionomycin-triggered change observed here is an MPTP-mediated event.

3 flow cytometry histograms showing shift in number of cells with green fluorescence, from high levels in untreated cells, mid levels when mitochondrial calcein is quenched, down to very low levels after fluorescence signal is quenched in both cytosol and mitochondria.

Figure 11. MitoProbe Transition Pore Assay Kit for flow cytometry. The flow cytometry histograms show the actions of the various kit components. Jurkat cells were incubated with the reagents in the MitoProbe Transition Pore Assay Kit and analyzed by flow cytometry. (A) In the absence of CoCl2 and ionomycin, fluorescent calcein is present in the cytosol as well as the mitochondria, resulting in a bright signal. (B) In the presence of CoCl2, calcein in the mitochondria emits a signal, but the cytosolic calcein fluorescence is quenched the overall fluorescence is reduced compared to calcein alone. (C) When ionomycin, a calcium ionophore, and CoCl2 are added to the cells at the same time as calcein AM, the fluorescence signals from both the cytosol and mitochondria are largely abolished.

Mitochondrial transition pore assay selection guide

  Image-IT LIVE Mitochondrial Transition Pore Assay MitoProbes Transition Pore Assay Kit
Application Method of measuring mitochondrial permeability transition pore opening
Readout In healthy cells the mitochondria remain brightly fluorescent until mitochondrial pore activation permits quenching of the fluorescence.
Ex/Em (nm) 494/517 (calcein) 494/517 (calcein)
579/599 (MitoTracker Red)
361/497 (Hoechst 33342)
Common filter TRITC FITC
Instrument platform Fluorescence microscopy Flow cytometry
Sample type Live cells Live cells
Compatibility with fixation Not compatible with fixation and detergent Not compatible with fixation and detergent
Kit components Kit contents:
calcein AM (calcium indicator)
ionomycin (ionophore)
CoCl2 (calcein quencher)
MitoTracker Red CMXRos stain
Hoechst 33342 (nuclear stain)
Kit contents:
Calcein AM (calcium indicator)
Ionomycin (ionophore)
CoCl2 (calcein quencher)
DMSO
Cat. No. I35103 M34153

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Healthy mitochondria membranes maintain a difference in electrical potential between the interior and exterior of the organelle, referred to as a membrane potential. Mitochondrion-selective stains that accumulate and are concentrated by active mitochondria with intact membrane potentials can be used to assay mitochondria health including detection of cells going through apoptosis. There are different options available to detect the mitochondria membrane potential state, including ratiometric versus single wavelength dyes, fixable versus nonfixable dyes, and dyes that are formatted for fluorescence imaging, flow cytometry or microplate reader instrumentation.

In this section you will find:

Ratiometric dyes

JC-1 dye

The most popular dye for detecting mitochondria membrane potential is the cell-permeant JC-1 dye which is used to detect apoptotic cells with flow cytometry and microscopy platforms (Figure 1). JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a concentration dependent–fluorescence emission shift from the monomer (green, ~529 nm) that is prominent at lower dye concentrations to aggregates (red, ~590 nm) which are formed as the dye concentration increases. Consequently, mitochondria depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.

Learn more about the JC-1 dye for mitochondria membrane potential

Microscopic view of mitochondria stained with green-red fluorescence at 4 different time course points.
A
Dot plot of JC-1 stained cells showing 2 cell populations-healthy cells colored red and apoptotic cells colored green.
B

Figure 1. Detection of mitochondria membrane potential using JC-1 dye. (A) NIH 3T3 fibroblasts stained with JC-1 dye show 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 min after treatment. The images were contributed by Ildo Nicoletti, Perugia University Medical School. (B) MitoProbe JC-1 Assay Kit was used to stain Jurkat cells (T-cell leukemia, human) which were then analyzed on a flow cytometer using 488 nm excitation with 530 nm and 585 nm bandpass emission filters. Green = apoptotic cells (reduced mitochondria membrane potential), red = normal cells.

MitoProbe DiOC2(3) Assay Kit

Another cationic carbocyanine dye that can be used as a ratiometric probe for mitochondria membrane potential is DiOC2(3). The mechanism is similar to that of JC-1 dye in that the monomer exhibits green fluorescence at lower concentrations where mitochondria membrane potential is low and the aggregate of the dye in more active mitochondria causes the emission to shift toward the red. In the case of DiOC2(3), the shift is farther in the red than JC-1, above 650 nm. The MitoProbe DiOC2(3) Assay Kit has been specifically developed to work in flow cytometry applications and includes a mitochondrial membrane-potential disrupter, CCCP.

Ratiometric dye selection guide

  JC-1 MitoProbe JC-1 Assay Kit MitoProbe DiOC2(3) 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.
Ex/Em (nm) 514/529 (monomer, green) 485/497 (monomer, green)
514/590 (aggregate, red) 485/>650 (aggregate, far–red)
Common filters TRITC FITC and PE FITC and APC
Instrument platform Imaging microscopy Flow cytometry Flow cytometry
Sample type Live cells Live cells Live cells
Compatibility with fixation No No No
Format 5 mg Kit contents:
JC-1, DMSO
CCCP (a mitochondria membrane potential disrupter in DMSO)
10x PBS
Kit contents:
DiOC2(3) in DMSO
CCCP (a mitochondrial membrane potential disrupter in DMSO)
Cat. No. T3168 M34152 M34150

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Fixable mitochondria membrane potential dyes

MitoTracker probes are small (<1kDa), cell-permeant mitochondrion-selective dyes that contain a mildly thiol-reactive chloromethyl moiety that is thought to be responsible for keeping the dye associated with the mitochondria after fixation. To label mitochondria, cells are simply incubated in submicromolar concentrations of the MitoTracker probe, which passively diffuses across the plasma membrane and accumulates in active mitochondria. Once their mitochondria are labeled, the cells can be treated with aldehyde-based fixatives to allow further processing of the sample without losing the pattern of staining achieved in the live cells. (We should note that MitoTracker Red FM dye is not fixable and should only be used in live cells.) These single wavelength emission reagents can be used for flow cytometry, imaging microscopy, and high content analysis applications and are compatible with multicolor experiments. For example, our Mitochondrial Membrane Potential/Annexin V Apoptosis Kit (Figure 2) utilizes MitoTracker Red CMXRos in combination with Alexa Fluor 488 annexin V in a two-color flow cytometric assay of apoptotic cells. Also shown in Figure 3 is detection of MitoTracker Orange CMTMRos and CellEvent Green Caspase 3/7 reagent in 2 cell types with and without treatment to induce apoptosis. The mitochondria membrane potential (orange fluorescence) decreases as the dose of drug increases, with concomitant increase in caspase activation (green fluorescence).

Figure 2. MitoTracker CMXRos dye mitochondria membrane potential detection. Jurkat human T-cell leukemia cells in complete medium were (A) first exposed to 10 µM camptothecin for 4 hours or (B) left untreated. Both cell populations were then treated with the reagents in the Mitochondrial Membrane Potential/Annexin V Apoptosis Kit and analyzed by flow cytometry. Note that the apoptotic cells show higher reactivity for annexin V and lower MitoTracker Red dye fluorescence than do live cells.

High-content images of cells stained with orange fluorescent mitochondria dye and green fluorescent caspase dye in 2 cell types with and without treatment to induce apoptosis. Also shows quantitative dose response curves of treated cells with decreasing orange fluorescence and increasing green fluorescence at higher concentrations of drug.

Figure 3. Mitochondria membrane potential quantification using MitoTracker Orange CMXRos. A549 or HeLa cells were cultured in Nunclon Sphera plates in GIBCO minimal essential media (MEM) to form spheroids over 2 days. Samples were treated with either DMSO vehicle or an increasing concentration of niclosamide for 24 hours before labeling with 250 nM MitoTracker Orange and 2.5 uM CellEvent Caspase 3/7 Green Reagent for 30 minutes at 37°C under normal cell culture conditions. Confocal images were acquired using the Thermo Fisher Scientific CellInsight CX7 LZR High Content Imaging System, followed by image analysis using HCS Studio Software V 2.0. Results show a niclosamide dose-dependent loss of mitochondrial membrane potential (orange) and increase in apoptotic cell death (green), qualitatively (upper panels) and quantitatively as a dose response vs mean signal intensity in both channels (lower graphs).

Fixable dyes selection guide

  MitoTracker dyes
MitoTracker Orange CMTMRos MitoTracker Red CMXRos MitoTracker Red FM
Readout Active mitochondria exhibit brighter fluorescence compared to apoptotic mitochondria.
Ex/Em (nm) 550/580 579/599 581/644
Common filter TRITC Texas Red Cy5
Instrument platform Flow cytometry
Fluorescence microscopy
Microplate reader
Sample type Live cells Live cells Live cells
Compatibility with fixation Yes Yes No
Format 20 x 50 μg 20 x 50 μg 20 x 50 μg
Cat. No. M7510 M7512 M22425

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Nonfixable mitochondria membrane potential dyes

DiIC11(5) cationic dye

Cationic carbocyanine dyes have been shown to accumulate in cells in response to membrane potential. The MitoProbe DiIC1(5) Kit is designed for flow cytometry applications and provides the far-red–fluorescent DiIC1(5) carbocyanine dye, along with a mitochondrial membrane potential disrupter, CCCP, for the study of mitochondrial membrane potential The dye penetrates the cytosol of eukaryotic cells and, at concentrations below 100 nM, accumulates primarily in mitochondria with active membrane potentials (Figure 4). Unlike DiOC2(3), however, DiIC1(5) dye exhibits a single wavelength emission whose fluorescence signal increases with more active mitochondria.

Flow cytometry histogram showing cells with active mitochondria in control compared to cells with depolarized mitochondria with CCCP treatment

Figure 4. Detection of changes in mitochondrial membrane potential using the MitoProbe DiIC1(5) Assay Kit for Flow Cytometry. Decrease in mitochondrial membrane potential as demonstrated with DiIC1(5) fluorescence due to the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Jurkat cells (T-cell leukemia, human) were stained with 50 nM DiIC1(5) alone (blue) or in the presence of 50 μM CCCP (red).

TMRM

Tetramethylrhodamine, methyl ester (TMRM) is a small, cell-permeant dye that accumulates in active mitochondria. If the cells are healthy and have functioning mitochondria, the signal is bright. Upon loss of the mitochondrial membrane potential, TMRM accumulation ceases and the signal dims or disappears. This ability to dynamically monitor changes in mitochondria membrane potential is a distinct advantage of this probe. TMRM signal can be detected with fluorescence microscopy, flow cytometry, cell sorting, high throughput screening, and high content analysis (Figure 5).

Microscopic view of cells stained with orange fluorescence (mitochondria) and green fluorescence (tubulin)
A
Dot plot showing healthy, apoptotic and dead cells with and without treatment.
B

Figure 5. TMRM as a mitochondrial membrane potential assay for apoptosis. (A) HeLa cells labeled using Tubulin Tracker Green and Image-iT TMRM Reagent show superb multiplexing capability and staining specificity. Image shows multiple live mitotic cells with microtubules assembled into a mitotic spindle with visible microtubule filaments, as well as cleavage furrow indicating completion of cytokinesis. (B) Jurkat cells, a human T-lymphocyte cell line, were treated with DMSO (control) or 500 nM staurosporine for 2 hours. Cells were subsequently stained with MitoProbe TMRM for 30 min at 37˚C, followed by a wash and additional stain with Annexin V Pacific Blue conjugate. Staurosporine induced apoptosis, resulting in a mixed population of cells containing a population of healthy MitoProbe TMRM-positive cells as well as a population of apoptotic, Annexin V Pacific Blue-positive, MitoProbe TMRM-low cells.

Nonfixable dyes selection guide

  TMRM MitoProbe TMRM Assay Kit MitoProbe DiIC1(5) Assay Kit
Readout Active mitochondria exhibit brighter fluorescence compared to apoptotic mitochondria.
Ex/Em (nm) 548/574 nm 638/658
Common filter TRITC ~585/16 nm Alexa Fluor 647/APC
Instrument platform Fluorescence microscopy Flow cytometry Flow cytometry
Sample type Live cells Live cells Live cells
Compatibility with fixation No No No
Format 25 mg 5 x 100 μL Kit contents:
TMRM
CCCP (a mitochondrial membrane potential disrupter in DMSO)
Kit contents:
DiIC1(5) in DMSO
CCCP (a mitochondrial membrane potential disrupter in DMSO)
Cat. No. T668 I34361 M20036 M34151

Increases in cellular superoxide production have been implicated in multiple disease states (1). This increase, which is generated as a byproduct of oxidative phosphorylation, provides another method to assess the cells’ apoptotic state.

MitoSOX Red Mitochondrial Superoxide Indicator

MitoSOX Red mitochondrial superoxide indicator is a cationic reagent designed for highly selective detection of superoxide in the mitochondria of live, healthy cells (Figure 6). The reagent is readily oxidized by superoxide but not by other reactive oxygen species (ROS)—or reactive nitrogen species (RNS)–generating systems, and oxidation of the probe is prevented by superoxide dismutase (Figure 7 and 8). Oxidation of the MitoSOX Red indicator by superoxide results in a fluorescence excitation peak at ~400 nm that is absent in the excitation spectrum generated by reactive oxygen species other than superoxide. Thus, fluorescence excitation at 400 nm with emission detection at ~590 nm provides optimum discrimination of superoxide from other reactive oxygen species.

Microscopic view of cells with red (mitochondria) and blue (nucleus) fluorescence in absence of treatment compared to just blue fluorescence when cells are treated with scavenger.
 Click image to enlarge

Figure 6. Imaging detection of superoxide in live cells using MitoSOX Red superoxide indicator. Live 3T3 cells were treated with FeTCPP, a superoxide scavenger, (left) or left untreated (right). Cells were then labeled with MitoSOX Red reagent, which fluoresces red when oxidized by superoxide, and nuclei were stained with blue-fluorescent Hoechst 33342. The mitochondria of untreated cells exhibited red fluorescence, indicating the presence of superoxide, whereas the mitochondria of treated cells showed minimal fluorescence.

Bar graph showing lack of mitosox red fluorescence detected in presence of other reactive oxygen and nitrogen species present

Figure 7. Selectivity of the MitoSOX Red mitochondrial superoxide indicator. Cell-free systems were used to generate a variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS); each oxidant was then added to a separate 10 µM solution of MitoSOX Red reagent and incubated at 37°C for 10 minutes. Excess DNA was added (unless otherwise noted) and the samples were incubated for an additional 15 minutes at 37°C before fluorescence was measured. The Griess Reagent Kit (for nitric oxide, peroxynitrite, and nitrite standards only; blue bars) and dihydrorhodamine 123 (DHR 123, green bars) were employed as positive controls for oxidant generation. Superoxide dismutase (SOD), a superoxide scavenger, was used as a negative control for superoxide. The results show that the MitoSOX Red probe (red bars) is readily oxidized by superoxide but not by the other oxidants.

Multi-panel figure with microscopic views of cells untreated with high glucose medium that are stained with red and blue fluorescence compared to low glucose with untreated cells and cells treated with the nitric oxide generator showing blue fluorescence only, and cells treated with superoxide generator stained with both red and blue fluorescence.
Figure 8. Visualizing glucose-mediated oxidative stress. Live human osteosarcoma (U2OS) cells were plated in Minimum Essential Medium (MEM) and incubated overnight at 37°C with CO2. To mitigate the effect of high glucose–mediated oxidative stress, samples B–D were washed in PBS and immersed in low-glucose Dulbecco’s Modified Eagle Medium (DMEM) and incubated overnight at 37°C with CO2. Samples were then washed in Hanks' Balanced Salt Solution (HBSS) and then treated as follows for the next 30 minutes: (A, B) cells were left in HBSS; (C) cells were incubated in HBSS + 100 μM antimycin A; (D) cells were incubated in HBSS + 100 μM DEANO. MitoSOX Red reagent at 5 µM was added to each sample, and cells were incubated for 30 minutes and imaged by confocal microscopy.

MitoSox Red Reagent specifications

  MitoSox Red Reagent
Readout
  • Fluorescence increases with increased superoxide concentration
  • Highly selective detection of superoxide in the mitochondria of live cells
Ex/Em (nm) 510/580
Common filter RFP/TRITC
Instrument platform Fluorescence microscopy
Flow cytometry
Microplate reader
High content analysis
Sample type Live cells
Fixation compatibility Not compatible with fixation or detergent
Format Solid, 10 x 50 mg
Cat. No. M36008

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Elevated mitochondrial Ca2+ plays an important role in initiation of programmed cell death (apoptosis) as well as in other cellular processes (1). Fluorescent probes that show a spectral response upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations using fluorescence microscopy, flow cytometry and fluorescence spectroscopy. As shown in Figure 9, rhod-2 is a fluorescent calcium indicator that can also be used to detect mitochondrial Ca2+ (1). The AM ester form of rhod-2 (rhod-2, AM) is used to easily load the dye into live cells. Upon entering the cell, intracellular esterases cleave the AM group, freeing the rhod-2 salt form to bind and fluoresce upon binding to mitochondrial Ca2+.

4 panels showing microscopic view of cells stained with green fluorescent mitochondria-targeting GFP and orange fluorescence of rhod-2 calcium indicator, also localized in mitochondria.

Figure 9. Multiplex imaging of mitochondrial calcium levels and dynamics. (A) HeLa cells were labeled with CellLight Mitochondria-GFP and 5 μM rhod-2 AM for 15 min at 37°C before imaging live over 100 sec. (B–D) The region outlined in (A) is enlarged to show individual mitochondria within a single cell over time. (C, D) Calcium is released from internal stores following application of 10 μM histamine. Mitochondria in close proximity to the calcium release are revealed by the increase in the orange-red fluorescence of rhod-2. The arrow in (C) denotes a mitochondrion that may have impaired calcium uptake, a detail that would have been missed using rhod-2 AM alone. The asterisk marks a mitochondrion that shows a transient elevation in calcium levels.

Rhod-2, AM product information

  Rhod-2 AM reagent
Readout
  • Calcium indicator targeted to mitochondria
  • Increases fluorescence with Ca 2+ concentration
Ex/Em (nm) 552/577
Common filter TRITC
Instrument platform
  • Fluorescence microscopy
  • Flow cytometry
Sample type Live cells
Compatibility with fixation Not compatible with fixation or detergent
Format Solid, 20 x 50 mg
Cat. No. R1245MP

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The mitochondrial permeability transition pore (MPTP) is a nonspecific channel formed by components from the inner and outer mitochondrial membranes, and appears to be involved in the release of mitochondrial components during cell death. This opening of the pore dramatically alters the permeability of mitochondria, as well as the mitochondria membrane potential. This continuous pore activation results from mitochondrial Ca2+ overload, oxidation of mitochondrial glutathione, increased levels of reactive oxygen species in mitochondria, and other pro-apoptotic conditions.

We have developed two kits for detecting mitochondrial transition pore opening, one for imaging microscopy (Image-iT LIVE Mitochondrial Transition Pore Assay Kit, Figure 10) and the other for flow cytometry (MitoProbe Transition Pore Assay Kit, Figure 11). Both kits provide a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. This assay employs the acetoxymethyl (AM) ester of calcein, a colorless and nonfluorescent esterase substrate, and CoCl2 (cobalt), a quencher of calcein fluorescence, to selectively label mitochondria. Cells are loaded with calcein AM, which passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochondria. Once inside cells, calcein AM is cleaved by intracellular esterases to liberate the very polar fluorescent dye calcein, which does not cross the mitochondrial or plasma membranes in appreciable amounts over relatively short periods of time. The fluorescence from cytosolic calcein is quenched by the addition of CoCl2, while the fluorescence from the mitochondrial calcein is maintained. As a control, cells that have been loaded with calcein AM and CoCl2 can also be treated with a Ca2+ ionophore such as ionomycin to allow entry of excess Ca2+ into the cells, which triggers mitochondrial pore activation and subsequent loss of mitochondrial calcein fluorescence. This ionomycin response can be blocked with cyclosporin A, a compound reported to prevent mitochondrial transition pore formation by binding cyclophilin D.

Figure 10. BPAE cells stained with Image-iT LIVE Mitochondrial Transition Pore Assay Kit. BPAE cells are stained using components from the Image-iT LIVE Mitochondrial Transition Pore Assay Kit. Cells are counterstained with MitoTracker Red CMXRos to show mitochondria and with Hoechst 33342 to show nuclei. Panel A shows the uniform cellular fluorescence from unquenched calcein. Panel B shows the mitochondrial pattern after adding cobalt which quenches the cytoplasmic calcein fluorescence but not mitochondrial calcein. Panel C shows the loss of calcein fluorescence with the addition of ionomycin which opens the pore to allow cobalt in and calcein out (mitochondria still visible from MitoTracker Red). Cyclophilin D activity is necessary for MPTP formation, and is inhibited by cyclosporin A. Consequently, inhibition of pore formation by cyclosporin A (CspA) has been used as an argument that a function is MPTP-specific. Panel D shows that the calcein pattern is retained when cyclosporin A is added before ionomycin, indicating that the ionomycin-triggered change observed here is an MPTP-mediated event.

3 flow cytometry histograms showing shift in number of cells with green fluorescence, from high levels in untreated cells, mid levels when mitochondrial calcein is quenched, down to very low levels after fluorescence signal is quenched in both cytosol and mitochondria.

Figure 11. MitoProbe Transition Pore Assay Kit for flow cytometry. The flow cytometry histograms show the actions of the various kit components. Jurkat cells were incubated with the reagents in the MitoProbe Transition Pore Assay Kit and analyzed by flow cytometry. (A) In the absence of CoCl2 and ionomycin, fluorescent calcein is present in the cytosol as well as the mitochondria, resulting in a bright signal. (B) In the presence of CoCl2, calcein in the mitochondria emits a signal, but the cytosolic calcein fluorescence is quenched the overall fluorescence is reduced compared to calcein alone. (C) When ionomycin, a calcium ionophore, and CoCl2 are added to the cells at the same time as calcein AM, the fluorescence signals from both the cytosol and mitochondria are largely abolished.

Mitochondrial transition pore assay selection guide

  Image-IT LIVE Mitochondrial Transition Pore Assay MitoProbes Transition Pore Assay Kit
Application Method of measuring mitochondrial permeability transition pore opening
Readout In healthy cells the mitochondria remain brightly fluorescent until mitochondrial pore activation permits quenching of the fluorescence.
Ex/Em (nm) 494/517 (calcein) 494/517 (calcein)
579/599 (MitoTracker Red)
361/497 (Hoechst 33342)
Common filter TRITC FITC
Instrument platform Fluorescence microscopy Flow cytometry
Sample type Live cells Live cells
Compatibility with fixation Not compatible with fixation and detergent Not compatible with fixation and detergent
Kit components Kit contents:
calcein AM (calcium indicator)
ionomycin (ionophore)
CoCl2 (calcein quencher)
MitoTracker Red CMXRos stain
Hoechst 33342 (nuclear stain)
Kit contents:
Calcein AM (calcium indicator)
Ionomycin (ionophore)
CoCl2 (calcein quencher)
DMSO
Cat. No. I35103 M34153

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