Assays for Mitochondria Function

What can be identified?

Cells with healthy, metabolically active mitochondria. Dynamic changes detect relative mitochondria membrane potential (reversible detection). None of the dynamic assays are fixable.

What is the basis of the assay?

Measure fluorescence of reagents that accumulate in active (healthy) mitochondria with intact membrane potentials.

JC-1 is a cell-permeant dye widely used for detecting mitochondrial membrane potential in flow cytometry and microscopy platforms (Figure 1). JC-1 exhibits potential-dependent accumulate within the mitochondria, forming J-aggregates as the dye increases. This increase in potential will result in a shift from the green fluorescence (~529 nm) monomer to red fluorescence (~590 nm) J-aggregates. Alternatively, depolarization of the mitochondria is denoted by a decrease in the red/green fluorescence intensity ratio. 

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

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.

Another 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.

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Tetramethylrhodamine, methyl ester (TMRM) or the related TMRE (tetramethylrhodamine, ethyl ester) are small, cell-permeant dyes that accumulate in active mitochondria. If the cells are healthy and have functioning mitochondria, the signal is bright. Upon loss of the mitochondrial membrane potential, TMRM and TMRE accumulation ceases and the signal dims or is lost from the mitochondria. This ability to dynamically monitor changes in mitochondria membrane potential is a distinct advantage of this probe. TMRM and TMRE signals can be detected in live cells or isolate mitochondria with traditional and high content fluorescence microscopy, microplates, or by flow cytometry (Figure 2).

TMRM is available in different formats to fit your apoptosis assay needs. See the Selection Guide (Tab 2. Single-emission reagents) for detection of dynamic changes in mitochondria membrane potential using single-emission reagents. MitoProbe TMRM Assay Kit for Flow Cytometry includes TMRM along with CCCP, a mitochondrial membrane potential disrupter and detailed protocol for use in flow cytometry.

Figure 2. 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.

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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. At concentrations below 100 nm, the cationic DiIC₁(5) dye passively accumulates in the mitochondria with active membrane potentials in living cells (Figure 3). Unlike DiOC₂(3), however, DiIC₁(5) dye exhibits a single wavelength emission whose fluorescence signal increases with more active mitochondria.

What can be identified?

Cells with healthy, metabolically active mitochondria. End point assays detect relative mitochondria membrane potential at a specific timepoint (irreversible detection).

Only a small selection of the end point assays for detecting mitochondria membrane potential are fixable.

What is the basis of the assay?

Measure fluorescence of reagents that accumulate in active (healthy) mitochondria with intact membrane potentials.

MitoTracker probes are small (<1 kDa), cell-permeant mitochondrion-selective dyes that contain a thiol-reactive chloromethyl moiety that keeps the dye associated with the mitochondria after fixation. Because the probes form covalent bonds with the mitochondrial thiols, they can only be used as an end point assays to detect mitochondrial membrane potential at the time of loading in live cells, and should not be used as sensors of dynamic mitochondrial membrane changes over time.

While the MitoTracker signal can be fixed after labeling, in assays to study mitochondrial structure, if studying the mitochondrial membrane function, it is strongly suggested to use a few, select MitoProbe reagents. Only MitoTracker Orange CMTMRos and MitoTracker Red CMXRos have been found to maintain a consistent difference in signal loss when comparing mitochondrial membrane potential in control and treated samples after fixation. These single wavelength emission reagents can be used for flow cytometry, imaging microscopy, and high content analysis applications (Figures 4 and 5).

Learn more about MitoTracker probes for mitochondria structure

Figure 4. MitoTracker Red 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.

Figure 5. Mitochondria membrane potential quantification using MitoTracker Orange CMTMRos. 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 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).
 

The HCS Mitochondrial Health Kit was developed for simultaneous quantitative measurements of two cell health parameters by high content analysis in the same cell: mitotoxicity and cytotoxicity in a 96-well plate format. The MitoHealth stain accumulates in mitochondria in live cells proportional to the mitochondrial membrane potential (Figure 6). Cytotoxicity is measured with the Image-iT DEAD Green viability stain. The Image-iT DEAD Green viability stain has a high affinity for DNA and forms highly fluorescent and stable dye-nucleic acid complexes; it is non-fluorescent when not bound to DNA.

Learn more about HCS mitochondrial health and cell viability

Figure 6. Imaging of mitotoxicity and cytotoxicity of valinomycin in HeLa cells using the HCS Mitochondrial Health Kit.

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Increases in cellular superoxide production have been implicated in multiple disease states (7). This increase, which is generated as a byproduct of oxidative phosphorylation, provides another method to assess the cells’ apoptotic state.

What can be identified?

Irreversible detection of mitochondrial superoxide production in live cells.

What is the basis of the assay?

Measure the increasing fluorescence of a superoxide detection reagent that accumulates in mitochondria in a membrane potential-dependent fashion where it is selectively oxidized and subsequently binds nucleic acids.

MitoSOX Red and Green mitochondrial superoxide indicators are fluorogenic reagents designed for highly selective detection of superoxide in the mitochondria of live, healthy cells (Figure 7 and 8). The reagents are 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 (Figures 9 and 10). Oxidation of the MitoSOX Red indicators 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. MitoSOX Green indicator can be detected with traditional FITC/GFP filter set. For selective detection of superoxide occurs with 488 nm excitation (emission at 510 nm).

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Figure 9. Selectivity of MitoSOX Green (MSG) and MitoSOX Red (MSR) mitochondrial superoxide indicators in a cell free environment. Cell free systems were used to generate reactive oxygen species (ROS). Each ROS was added to separate solutions of 10 µM MSG and MSR and incubated for 30 min at ambient temperature. Excess DNA was added to the solutions with MSR. Superoxide dismutase was used as a negative control for superoxide. Superoxide was generated using potassium superoxide for MSG, and the xanthine/xanthine oxidase system was used to generate superoxide for MSR. The results show that both MSG and MSR are selective to superoxide and not other reactive oxygen species.

Figure 10. 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.

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What can be identified?

Irreversible detection of mitochondrial calcium in live cells.

What is the basis of the assay?

Measure the increasing fluorescence of a calcium indicator with increasing calcium concentration in the mitochondria.

Elevated mitochondrial Ca²+ plays an important role in initiation of programmed cell death (apoptosis) as well as in other cellular processes (8). 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 11, Rhod-2 is a fluorescent calcium indicator that can also be used to detect mitochondrial Ca²+ (8). 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+.

Figure 11. 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.

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What can be identified?

Irreversible detection of the mitochondrial permeability transition (MPT) subsequent to dysregulation of the MPT pore.

What is the basis of the assay?

Loss of mitochondrial calcein fluorescence due to mitochondrial permeability transition pore opening.

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 12) and the other for flow cytometry (MitoProbe Transition Pore Assay Kit, Figure 13). 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 13. 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.

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