Cartoon image of a mitochondria

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

Mitochondria function changes in apoptosis

A feature of the early stages of programmed cell death is the disruption of mitochondria function. This mitochondrial disruption includes changes in the membrane potential, a central feature of mitochondrial health, and alterations to the oxidation–reduction potential of the mitochondria. The inner mitochondrial membrane potential is essential in Ca2+ uptake and storage, reactive oxygen species generation and detoxification and, most importantly, the synthesis of ATP by oxidative phosphorylation (1). Therefore, the membrane’s depolarization is a good indicator of mitochondrial dysfunction, which is increasingly implicated in drug toxicity (2-6). Changes in the membrane potential, along with decreases ATP to ADP ratios, increased mitochondrial matrix calcium levels, oxidative stress, and release of cytochrome c into the cytosol are all presumed to be associated with the mitochondrial permeability transition, resulting in disruption of ions and small molecule homeostasis via the mitochondrial permeability transition pore (MPTP).
 

Overview of mitochondria function assays

The disruption of mitochondria function can be detected using a variety of fluorescence-based assays including measurements of mitochondrial calcium, superoxide, mitochondrial permeability transition, and membrane potential. A summary of these assays are listed in Table 1 below.

Although disruption of mitochondria function is associated with apoptosis, the various aspects listed above are not specific to apoptotic cell death. Because the aforementioned apoptosis-related mitochondria function parameters are not specific for apoptosis and may vary across cell types, it is often advantageous to consider multi-parametric assays, including the use of specific markers for the activation of particular caspase proteases.
 

Summary of mitochondria function assays

 Mitochondria function assays
 Membrane potentialSuperoxide productionCalciumMitochondrial Permeability Transition
What can be identified?

Cells with healthy, metabolically active mitochondria.

2 options available:

Detection of dynamic changes in relative mitochondria membrane potential (reversible detection).

End point assays for detecting relative mitochondria membrane potential at a specific timepoint (irreversible detection).

Irreversible detection of mitochondrial superoxide production in live cells.

 

Irreversible detection of mitochondrial calcium in live cells.Irreversible detection of the mitochondrial permeability transition (MPT) subsequent to dysregulation of the MPT pore.
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 that accumulates in mitochondria in a membrane potential-dependent fashion where it is selectively oxidized and subsequently binds nucleic acids.Measure the increasing fluorescence of a calcium indicator with increasing calcium concentration in the mitochondria.Loss of mitochondrial calcein fluorescence due to mitochondrial permeability transition.
 

Is the assay fixable?

Only a small selection of the end point assays for detecting mitochondria membrane potential are fixable. Please see the table of MitoTracker probes below.

None of the dynamic assays are fixable.

Not compatible with fixation.

Not compatible with fixation.

Not compatible with fixation.

 

 

Selection Guides

Detection of dynamic changes in mitochondrial membrane potential

 

JC-1MitoProbe JC-1 Assay KitMitoProbe DiOC2(3) Assay Kit
ReadoutActive 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)
Suggested filtersTRITCFITC and PEFITC and APC
Instrument platformImaging microscopyFlow cytometryFlow cytometry
Sample typeLive cellsLive cellsLive cells
Compatibility with fixationNoNoNo
Format5 mgKit 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.T3168M34152M34150

Want more information about these reagents? Please read more in the detailed information about mitochondria function assays below.

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 TMRMMitoProbe TMRM Assay KitMitoProbe DiIC1(5) Assay Kit
ReadoutActive mitochondria exhibit brighter fluorescence compared to apoptotic mitochondria.
Ex/Em (nm)548/574 nm638/658
Suggested filterTRITC~585/16 nmAlexa Fluor 647/APC
Instrument platformFluorescence microscopyFlow cytometryFlow cytometry
Sample typeLive cellsLive cellsLive cells
Compatibility with fixationNoNoNo
Format25 mg5 x 100 μLKit 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.T668I34361M20036M34151

Want more information about these reagents? Please read more in the detailed information about mitochondria function assays below.

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JC-1MitoProbe JC-1 Assay KitMitoProbe DiOC2(3) Assay Kit
ReadoutActive 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)
Suggested filtersTRITCFITC and PEFITC and APC
Instrument platformImaging microscopyFlow cytometryFlow cytometry
Sample typeLive cellsLive cellsLive cells
Compatibility with fixationNoNoNo
Format5 mgKit 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.T3168M34152M34150

Want more information about these reagents? Please read more in the detailed information about mitochondria function assays below.

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 TMRMMitoProbe TMRM Assay KitMitoProbe DiIC1(5) Assay Kit
ReadoutActive mitochondria exhibit brighter fluorescence compared to apoptotic mitochondria.
Ex/Em (nm)548/574 nm638/658
Suggested filterTRITC~585/16 nmAlexa Fluor 647/APC
Instrument platformFluorescence microscopyFlow cytometryFlow cytometry
Sample typeLive cellsLive cellsLive cells
Compatibility with fixationNoNoNo
Format25 mg5 x 100 μLKit 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.T668I34361M20036M34151

Want more information about these reagents? Please read more in the detailed information about mitochondria function assays below.

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End point assays in mitochondrial membrane potential

 MitoTracker probes
MitoTracker Green FMMitoTracker Orange CMTMRosMitoTracker Red CMXRosMitoTracker Red FMMitoTracker Deep Red FM
ReadoutActive mitochondria exhibit brighter fluorescence compared to apoptotic mitochondria.
Ex/Em (nm)490/516550/580579/599581/644644/665
Suggested filterFITCTRITCTexas RedCy5Cy5
Instrument platformFlow cytometry
Fluorescence microscopy
Microplate reader
Sample typeLive cellsLive cellsLive cellsLive cellsLive cells
Compatibility with fixation *NoYesYesNoNo
Format20 x 50 μg20 x 50 μg20 x 50 μg20 x 50 μg20 x 50 μg
Cat. No.M7514M7510M7512M22425M22426

* Want more information about these reagents, or need to understand the impact of fixation on MitoTracker probes? Please read more about MitoTracker probes below.

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ProductApoptosis Reagent TypeReagentEx/EmAdditional reagents in kitSizeCat. No.
Mitochondrial Membrane Potential Apoptosis KitMitochondrial membrane potentialMitoTracker Red579/599 nmAnnexin binding buffer (5x)50 assaysV35116
Membrane Asymmetry StainAnnexin V, Alexa Fluor 488499/521 nm
HCS Mitochondrial Health KitMitochondrial membrane potentialMitoHealth Stain550/580 nmImage-iT DEAD Green viability stain (Ex/Em: 488/515 nm) DMSO2 x 96-well platesH10295
Condensed chromatin stainHoechst 33342350/461 nm

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 MitoTracker probes
MitoTracker Green FMMitoTracker Orange CMTMRosMitoTracker Red CMXRosMitoTracker Red FMMitoTracker Deep Red FM
ReadoutActive mitochondria exhibit brighter fluorescence compared to apoptotic mitochondria.
Ex/Em (nm)490/516550/580579/599581/644644/665
Suggested filterFITCTRITCTexas RedCy5Cy5
Instrument platformFlow cytometry
Fluorescence microscopy
Microplate reader
Sample typeLive cellsLive cellsLive cellsLive cellsLive cells
Compatibility with fixation *NoYesYesNoNo
Format20 x 50 μg20 x 50 μg20 x 50 μg20 x 50 μg20 x 50 μg
Cat. No.M7514M7510M7512M22425M22426

* Want more information about these reagents, or need to understand the impact of fixation on MitoTracker probes? Please read more about MitoTracker probes below.

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ProductApoptosis Reagent TypeReagentEx/EmAdditional reagents in kitSizeCat. No.
Mitochondrial Membrane Potential Apoptosis KitMitochondrial membrane potentialMitoTracker Red579/599 nmAnnexin binding buffer (5x)50 assaysV35116
Membrane Asymmetry StainAnnexin V, Alexa Fluor 488499/521 nm
HCS Mitochondrial Health KitMitochondrial membrane potentialMitoHealth Stain550/580 nmImage-iT DEAD Green viability stain (Ex/Em: 488/515 nm) DMSO2 x 96-well platesH10295
Condensed chromatin stainHoechst 33342350/461 nm

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Detection of mitochondrial superoxide production

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.

 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
Suggested filterRFP/TRITC
Instrument platformFluorescence microscopy
Flow cytometry
Microplate reader
High content analysis
Sample typeLive cells
Fixation compatibilityNot compatible with fixation or detergent
FormatSolid, 10 x 50 mg
Cat. No.M36008

Want more information about this reagent? Please read more about the MitoSOX Red Mitochondrial Superoxide Indicator below.

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Detection of mitochondrial calcium

 Rhod-2, AM reagent
Readout
  • Calcium indicator targeted to mitochondria
  • Increases fluorescence with Ca 2+ concentration
Ex/Em (nm)552/577
Suggested filterTRITC
Instrument platform
  • Fluorescence microscopy
  • Flow cytometry
Sample typeLive cells
Compatibility with fixationNot compatible with fixation or detergent
FormatSolid, 20 x 50 mg
Cat. No.R1245MP

Want more information about this reagent? Please read more about the Rhod-2, AM reagent below.

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Detection of changes in mitochondrial permeability transition pore

 Image-IT LIVE Mitochondrial Transition Pore AssayMitoProbes Transition Pore Assay Kit
ApplicationMethod of measuring mitochondrial permeability transition pore opening
ReadoutIn 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 stain)
361/497 (Hoechst 33342)
Suggested filterTRITCFITC
Instrument platformFluorescence microscopyFlow cytometry
Sample typeLive cellsLive cells
Compatibility with fixationNot compatible with fixation and detergentNot compatible with fixation and detergent
Kit componentsKit 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.I35103M34153

Want more information about these reagents? Please read more about the detection of mitochondrial permeability transition pore below.

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Detailed information about mitochondria function assays

JC-1 Dye and the MitoProbe JC-1 Assay Kit

A commonly used 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. When the dye accumulation within the mitochondria is high enough in concentration,
J-aggregates form, and there is a fluorescence emission shift from the monomer (green, ~529 nm) that is prominent at lower dye concentrations to J- aggregates (red, ~590 nm) which are formed as the dye concentration increases. Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.

JC-1 Dye is available as a standalone reagent (Cat. No. T3168) or in the MitoProbe JC-1 Assay Kit which contains the JC-1 Dye, CCCP (a mitochondria membrane potential disrupter in DMSO), 10x PBS, and DMSO.

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

A

Microscopic view of mitochondria stained with green-red fluorescence at 4 different time course points.

B

Dot plot of JC-1 stained cells showing 2 cell populations-healthy cells colored red and apoptotic cells colored green.

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.

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MitoProbe DiOC2(3) Assay Kit

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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TMRM and MitoProbe TMRM Assay Kit

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.

A

Microscopic view of cells stained with orange fluorescence (mitochondria) and green fluorescence (tubulin)

B

Dot plot showing healthy, apoptotic and dead cells with and without treatment.

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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MitoProbe DiIC1(5) 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. 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.

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

Figure 3. 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).

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MitoTracker probes

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.
 

Impact of fixation on MitoTracker probes

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).

See the MitoTracker Selection Guide above to identify which MitoTracker would best fit your experiment.

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 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).
 

High content mitochondrial health assay

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.

9 panel image showing  HeLa cells (untreated, treated with 165 nm or 120 μM valinomycin) stained with one of the following: Hoechst 33342, a cell membrane permeability stain or a mitochondrial membrane potential stain.

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

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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 7). 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 8 and 9). 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.

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

Figure 8. 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 9. 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.

Rhod-2, AM Reagent

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 10, 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, Cat. No. R1245MP) 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 10. 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.
 

Mitochondrial permeability transition pore assays

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 11) and the other for flow cytometry (MitoProbe Transition Pore Assay Kit, Figure 12). 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 11. 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 12. 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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References
  1. Nicholls, DG (2004) Mitochondrial Membrane Potential and Aging. Aging Cell 3: 35-40.
  2. Tirmenstein, MA, Hu, CX, Gales TL, et al. (2002) Effects of Troglitazone on HepG2 Viability and Mitochondrial Function Toxicol. Sci.69: 131-8.
  3. O'Brien PJ, Irwin W, Diaz D, et al. (2006) High Concordance of Drug-Induced Human Hepatotoxicity With in Vitro Cytotoxicity Measured in a Novel Cell-Based Model Using High Content Screening. Arch Toxicol 80: 580-604.
  4. Dykens JA, Will Y. (2007) The Significance of Mitochondrial Toxicity Testing in Drug Development. Drug Discovery Today 12:777-85.
  5. Dykens JA, Jamieson JD, Marroquin LD, et al. (2008) In Vitro Assessment of Mitochondrial Dysfunction and Cytotoxicity of Nefazodone, Trazodone, and Buspirone. Toxicol Sci 103: 335-45.
  6. Abraham VC, Towne DL, Waring JF, et al. (2008) Application of a High-Content Multiparameter Cytotoxicity Assay to Prioritize Compounds Based on Toxicity Potential in Humans. J Biomol Screen 13: 527-37.
  7. He L, He T, Farrar S et al. (2017) Antioxidants Maintain Cellular Redox Homeostasis by Elimination of Reactive Oxygen Species. Cell Physiol Biochem 44: 532-553.
  8. Giorgi C, Romagnoli A, Pinton P et al. (2008) Ca2+ Signaling, Mitochondria and Cell Death. Curr Mol Med 8: 119-130.
  9.  

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