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Mitochondria are found in eukaryotic cells, where they make up as much as 10% of the cell volume. They are pleomorphic organelles with structural variations depending on cell type, cell-cycle stage and intracellular metabolic state. The key function of mitochondria is energy production through oxidative phosphorylation (OxPhos) and lipid oxidation. Several other metabolic functions are performed by mitochondria, including urea production and heme, non-heme iron and steroid biogenesis, as well as intracellular Ca2+ homeostasis. Mitochondria also play a pivotal role in apoptosis—the genetically controlled ablation of cells during normal development (Assays for Apoptosis—Section 15.5). For many of these mitochondrial functions, there is only a partial understanding of the components involved, with even less information on mechanism and regulation.
The morphology of mitochondria is highly variable. In dividing cells, the organelle can switch between a fragmented morphology with many ovoid-shaped mitochondria, as is often shown in textbooks, and a reticulum in which the organelle is a single, many-branched structure. The cell cycle– and metabolic state–dependent changes in mitochondrial morphology are controlled by a set of proteins that cause fission and fusion of the organelle mass. Mutations in these proteins are the cause of several human diseases, indicating the importance of overall morphology for cell functioning ( Mitochondria in Diseases—Note 12.2). Organelle morphology is also controlled by cytoskeletal elements, including actin filaments and microtubules (Figure 12.2.1). In nondividing tissue, overall mitochondrial morphology is very cell dependent, with mitochondria spiraling around the axoneme in spermatozoa, and ovoid bands of mitochondria intercalating between actomyosin filaments. There is evidence of functionally significant heterogeneity of mitochondrial forms within individual cells.
The abundance of mitochondria varies with cellular energy level and is a function of cell type, cell-cycle stage and proliferative state. For example, brown adipose tissue cells, hepatocytes and certain renal epithelial cells tend to be rich in active mitochondria, whereas quiescent immune-system progenitor or precursor cells show little staining with mitochondrion-selective dyes. The number of mitochondria is reduced in Alzheimer disease and their proteins and nucleic acids are susceptible to damage by reactive oxygen species, including nitric oxide (Probes for Reactive Oxygen Species, Including Nitric Oxide—Chapter 18).
We provide a range of mitochondrion-selective fluorescent proteins and organic dyes with which to monitor mitochondrial morphology and organelle functioning. In contrast to the fluorescent protein–based CellLight probes, the uptake of most mitochondrion-selective organic dyes is dependent on the mitochondrial membrane potential. These dyes thereby enable researchers to probe mitochondrial activity, localization and abundance, as well as to monitor the effects of some pharmacological agents that alter mitochondrial function. The CellLight probes can be used in combination with these organic dyes to investigate relationships between mitochondrial morphology and membrane potential.
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Figure 12.2.2 Mitochondrial transport along microtubules. U2OS cells were transduced with CellLight® MAP4-GFP (C10598) and CellLight® Mito-RFP (C10601) reagents. Images were taken every 3 min for 30 min. A mitochondrion (red) can be seen transported along the microtubules (green).
CellLight Mitochondria-GFP (C10600, Figure 12.2.2) and CellLight Mitochondria-RFP (C10601) reagents combine the utility and selectivity of targeted fluorescent proteins with the efficiency of the BacMam gene delivery and expression technology. These BacMam expression vectors encode Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP) fused with the leader sequence of E1α pyruvate dehydrogenase, which targets the fluorescent proteins to the mitochondria of live cells. CellLight reagents ( CellLight reagents and their targeting sequences—Table 11.1) incorporate all the customary advantages of BacMam technology, including high transduction efficiency and long-lasting, titratable expression ( BacMam Gene Delivery and Expression Technology—Note 11.1). They are provided in a ready-to-use format—simply add, incubate and image—with highly efficient expression in cell lines, primary cells, stem cells and neurons.
Mitochondrial motility during mitosis can be easily observed in cells transduced with CellLight Mitochondria-GFP or CellLight Mitochondria-RFP (Figure 12.2.3). In contrast to MitoTracker Red CMXRos, TMRE, rhodamine 123 and other cationic dyes, mitochondrial localization of fluorescent protein–based markers is not driven by membrane potential. They can therefore be used in combination with cationic dye probes to investigate relationships between mitochondrial morphology and membrane potential.
Figure 12.2.3 Mitochondrial dynamics during mitosis. U2OS cells were transduced with CellLight® Mito-RFP reagent (C10601) and imaged every 5 min for 16 hr. Extensive mitochondrial motility is seen throughout mitosis and following mitosis, as the cell regains its pre-mitotic shape.
Although conventional fluorescent stains for mitochondria, such as rhodamine 123 and tetramethylrosamine, are readily sequestered by functioning mitochondria, they are subsequently washed out of the cells once the mitochondrion's membrane potential is lost. This characteristic limits their use in experiments in which cells must be treated with aldehyde-based fixatives or other agents that affect the energetic state of the mitochondria. To overcome this limitation, we have developed MitoTracker probes—a series of mitochondrion-selective stains that are concentrated by active mitochondria and well retained during cell fixation. Because the MitoTracker Orange, MitoTracker Red and MitoTracker Deep Red probes are also retained following permeabilization, the sample retains the fluorescent staining pattern characteristic of live cells during subsequent processing steps for immunocytochemistry, in situ hybridization or electron microscopy. In addition, MitoTracker reagents eliminate some of the difficulties of working with pathogenic cells because, once the mitochondria are stained, the cells can be treated with fixatives before the sample is analyzed.
Properties of MitoTracker Probes
MitoTracker probes are cell-permeant mitochondrion-selective dyes that contain a mildly thiol-reactive chloromethyl moiety. The chloromethyl group appears 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; with the exception of MitoTracker Green FM, subsequent permeabilization with cold acetone does not appear to disturb the staining pattern of the MitoTracker dyes.
We offer seven MitoTracker reagents that differ in spectral characteristics, oxidation state and fixability ( Spectral characteristics of the MitoTracker probes—Table 12.2). MitoTracker probes are provided in specially packaged sets of 20 vials, each containing 50 µg for reconstitution as required.
Orange-, Red- and Infrared-Fluorescent MitoTracker Dyes
We offer MitoTracker derivatives of the orange-fluorescent tetramethylrosamine (MitoTracker Orange CMTMRos, M7510; ) and the red-fluorescent X-rosamine (MitoTracker Red CMXRos, M7512; ), as well as MitoTracker Red FM (M22425; ) and MitoTracker Deep Red FM probes (M22426; ). Because the MitoTracker Red CMXRos, MitoTracker Red FM and MitoTracker Deep Red FM probes produce longer-wavelength fluorescence that is well resolved from the fluorescence of green-fluorescent dyes, they are suitable for multicolor labeling experiments (, ) ). Also available are chemically reduced forms of the tetramethylrosamine (MitoTracker Orange CM-H2TMRos, M7511; ) and X-rosamine (MitoTracker Red CM-H2XRos, M7513; ) MitoTracker probes. Unlike MitoTracker Orange CMTMRos and MitoTracker Red CMXRos, the reduced versions of these probes do not fluoresce until they enter an actively respiring cell, where they are oxidized to the fluorescent mitochondrion-selective probe and then sequestered in the mitochondria (Figure 12.2.4, , )
Our Mitochondrial Membrane Potential/Annexin V Apoptosis Kit (V35116, Assays for Apoptosis—Section 15.5) utilizes MitoTracker CMXRos in combination with Alexa Fluor 488 annexin V in a two-color assay of apoptotic cells (Figure 12.2.5). Following fixation, the oxidized forms of the tetramethylrosamine and X-rosamine MitoTracker dyes can be detected directly by fluorescence or indirectly with either anti-tetramethylrhodamine (A6397), or anti–Texas Red dye antibodies (A6399; Anti-Dye and Anti-Hapten Antibodies—Section 7.4).
Figure 12.2.4 Intracellular reactions of our fixable, mitochondrion-selective MitoTracker Orange CM-H2TMRos (M7511). When this cell-permeant probe enters an actively respiring cell, it is oxidized to MitoTracker Orange CMTMRos and sequestered in the mitochondria, where it can react with thiols on proteins and peptides to form aldehyde-fixable conjugates.
Figure 12.2.5 Flow cytometric analysis of Jurkat cells using the Mitochondrial Membrane Potential/Annexin V Apoptosis Kit (V35116). 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.
MitoTracker Green FM Dye
Mitochondria in cells stained with nanomolar concentrations of MitoTracker Green FM dye (M7514, ) exhibit bright green, fluorescein-like fluorescence (, , ). The MitoTracker Green FM probe has the added advantage that it is essentially nonfluorescent in aqueous solutions and only becomes fluorescent once it accumulates in the lipid environment of mitochondria. Hence, background fluorescence is negligible, enabling researchers to clearly visualize mitochondria in live cells immediately following addition of the stain, without a wash step.
Unlike MitoTracker Orange CMTMRos and MitoTracker Red CMXRos, the MitoTracker Green FM probe appears to preferentially accumulate in mitochondria regardless of mitochondrial membrane potential in certain cell types, making it a possible tool for determining mitochondrial mass. Furthermore, the MitoTracker Green FM dye is substantially more photostable than the widely used rhodamine 123 fluorescent dye and produces a brighter, more mitochondrion-selective signal at lower concentrations. Because its emission maximum is blue-shifted approximately 10 nm relative to the emission maximum of rhodamine 123, the MitoTracker Green FM dye produces a fluorescent staining pattern that should be better resolved from that of red-fluorescent probes in double-labeling experiments. The mitochondrial proteins that are selectively labeled by the MitoTracker Green FM reagent have been separated by capillary electrophoresis.
Image-iT LIVE Mitochondrial and Nuclear Labeling Kit
The Image-iT LIVE Mitochondrial and Nuclear Labeling Kit (I34154) provides two stains—red-fluorescent MitoTracker Red CMXRos dye (excitation/emission maxima ~578/599 nm) and blue-fluorescent Hoechst 33342 dye (excitation/emission maxima when bound to DNA ~350/461 nm)—for highly selective mitochondrial and nuclear staining, respectively, in live, GFP–transfected cells. These dyes can be combined into one staining solution using the protocol provided, saving labeling time and wash steps while still providing optimal staining. Both dyes are retained after formaldehyde fixation and permeabilization. The Image-iT LIVE Mitochondrial and Nuclear Labeling Kit contains:
- MitoTracker CMXRos dye
- Hoechst 33342 dye
- Dimethylsulfoxide (DMSO)
- Labeling protocols (Image-iT LIVE Mitochondrial and Nuclear Labeling Kit)
Each kit provides enough staining solution for 500 assays using the protocol provided for labeling live, cultured cells that are adhering to coverslips.
Mitochondrial superoxide is generated as a by-product of oxidative phosphorylation. In an otherwise tightly coupled electron transport chain, approximately 1–3% of mitochondrial oxygen consumed is incompletely reduced; these "leaky" electrons can quickly interact with molecular oxygen to form superoxide anion, the predominant reactive oxygen species in mitochondria. Increases in cellular superoxide production have been implicated in cardiovascular diseases, including hypertension, atherosclerosis and diabetes-associated vascular injuries, as well as in neurodegenerative diseases such as Parkinson disease, Alzheimer disease and amyotrophic lateral sclerosis (ALS).
MitoSOX Red mitochondrial superoxide indicator (M36008) is a cationic derivative of dihydroethidum (also known as hydroethidine; see below) designed for highly selective detection of superoxide in the mitochondria of live cells (). The cationic triphenylphosphonium substituent of MitoSOX Red indicator is responsible for the electrophoretically driven uptake of the probe in actively respiring mitochondria. Oxidation of MitoSOX Red indicator (or dihydroethidium) by superoxide results in hydroxylation at the 2-position (Figure 12.2.6). 2-hydroxyethidium (and the corresponding derivative of MitoSOX Red indicator) exhibit a fluorescence excitation peak at ~400 nm that is absent in the excitation spectrum of the ethidium oxidation product 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 (Figure 12.2.7).
Measurements of mitochondrial superoxide generation using MitoSOX Red indicator in mouse cortical neurons expressing caspase-cleaved tau microtubule-associated protein have been correlated with readouts from fluorescent indicators of cytosolic and mitochondrial calcium and mitochondrial membrane potential. The relationship of mitochondrial superoxide generation to dopamine transporter activity, measured using the aminostyryl dye substrate 4-Di-1-ASP (D288, see below), has been investigated in mouse brain astrocytes. MitoSOX Red indicator has been used for confocal microscopy analysis of reactive oxygen species (ROS) production by mitochondrial NO synthase (mtNOS) in permeabilized cat ventricular myocytes and, in combination with Amplex Red reagent, for measurement of mitochondrial superoxide and hydrogen peroxide production in rat vascular endothelial cells. In addition to imaging and microscope photometry measurements, several flow cytometry applications of MitoSOX Red have also been reported. Detailed protocols for simultaneous measurements of mitochondrial superoxide generation and apoptotic markers APC annexin V (A35110, Assays for Apoptosis—Section 15.5) and SYTOX Green (S7020, Nucleic Acid Stains—Section 8.1) in human coronary artery endothelial cells by flow cytometry have been published by Mukhopadhyay and co-workers.
Figure 12.2.6 Oxidation of MitoSox Red mitochondrial superoxide indicator to 2-hydroxy-5-(triphenylphosphonium)hexylethidium by superoxide (•O2–).
Figure 12.2.7 Selectivity of the MitoSOX Red mitochondrial superoxide indicator (M36008). 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 (G7921) (for nitric oxide, peroxynitrite, and nitrite standards only; blue bars) and dihydrorhodamine 123 (DHR 123, (D632); 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.
RedoxSensor Red CC-1 stain (2,3,4,5,6-pentafluorotetramethyldihydrorosamine, R14060; ) passively enters live cells and is subsequently oxidized in the cytosol to a red-fluorescent product (excitation/emission maxima ~540/600 nm), which then accumulates in the mitochondria. Alternatively, this nonfluorescent probe may be transported to the lysosomes where it is oxidized. The differential distribution of the oxidized product between mitochondria and lysosomes appears to depend on the redox potential of the cytosol. In proliferating cells, mitochondrial staining predominates; whereas in contact-inhibited cells, the staining is primarily lysosomal ().
The green-fluorescent JC-1 probe (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide, T3168; ) exists as a monomer at low concentrations or at low membrane potential. However, at higher concentrations (aqueous solutions above 0.1 µM) or higher potentials, JC-1 forms red-fluorescent "J-aggregates" that exhibit a broad excitation spectrum and an emission maximum at ~590 nm (, , ). Thus, the emission of this cyanine dye can be used as a sensitive measure of mitochondrial membrane potential. Various types of ratio measurements are possible by combining signals from the green-fluorescent JC-1 monomer (absorption/emission maxima ~514/529 nm in water) and the J-aggregate (emission maximum 590 nm), which can be effectively excited anywhere between 485 nm and its absorption maximum at 585 nm (). The ratio of red-to-green JC-1 fluorescence is dependent only on the membrane potential and not on other factors that may influence single-component fluorescence signals, such as mitochondrial size, shape and density. Optical filters designed for fluorescein and tetramethylrhodamine can be used to separately visualize the monomer and J-aggregate forms, respectively. Alternatively, both forms can be observed simultaneously using a standard fluorescein longpass optical filter set. Chen and colleagues have used JC-1 to investigate mitochondrial potentials in live cells by ratiometric techniques (Figure 12.2.8).
JC-1 has been combined with Alexa Fluor 647 annexin V (A23204, Assays for Apoptosis—Section 15.5) to permit simultaneous assessment of phosphatidylserine externalization and mitochondrial function by flow cytometry. We also offer JC-1 as part of the MitoProbe JC-1 Assay Kit for flow cytometry (M34152, Slow-Response Probes—Section 22.3). We have discovered another mitochondrial marker, JC-9 (3,3'-dimethyl-β-naphthoxazolium iodide, D22421; ), with a different chemical structure () but similar potential-dependent spectroscopic properties. However, the green fluorescence of JC-9 is essentially invariant with membrane potential, whereas the red fluorescence is significantly increased at hyperpolarized membrane potentials.
Figure 12.2.8 Bivariate JC-1 (T3168) analysis of mitochondrial membrane potential in HL60 cells by flow cytometry. The sensitivity of this technique is demonstrated by the response to K+/valinomycin (V1644, Fluorescent Na+ and K+ Indicators—Section 21.1)–induced depolarization (panels B and D). Distinct populations of cells with different extents of mitochondrial depolarization are detectable following apoptosis-inducing treatment with 5 µM staurosporine for two hours (panel C). Figure courtesy of Andrea Cossarizza, University of Modena and Reggio Emilia, Italy.
Rhodamine 123 (R302, R22420; ) is a cell-permeant, cationic, fluorescent dye that is readily sequestered by active mitochondria without inducing cytotoxic effects. Uptake and equilibration of rhodamine 123 is rapid (a few minutes) compared with dyes such as DASPMI (4-Di-1-ASP, D288), which may take 30 minutes or longer. Viewed through a fluorescein longpass optical filter, fluorescence of the mitochondria of cells stained by rhodamine 123 appears yellow-green. Viewed through a tetramethylrhodamine longpass optical filter, however, these same mitochondria appear red. Unlike the lipophilic rhodamine and carbocyanine dyes, rhodamine 123 apparently does not stain the endoplasmic reticulum.
Rhodamine 123 has been used with a variety of cell types such as astrocytes, neurons, live bacteria, plants and human spermatozoa. Using flow cytometry, researchers employed rhodamine 123 in combination with Hoechst 33342 (H1399, H3570, H21492; Probes for the Nucleus—Section 12.5) for the characterization of hematopoietic stem cells. Rhodamine 123 is widely used as a substrate for functional assays of ATP-binding cassette (ABC) drug transporters (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6).
Rosamines and Other Rhodamine Derivatives, Including TMRM and TMRE
Other mitochondrion-selective dyes include tetramethylrosamine (T639, ), whose fluorescence contrasts well with that of fluorescein for multicolor applications, and rhodamine 6G (R634, ), which has an absorption maximum between that of rhodamine 123 and tetramethylrosamine. Tetramethylrosamine and rhodamine 6G have both been used to examine the efficiency of P-glycoprotein–mediated exclusion from multidrug-resistant cells (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6). Rhodamine 6G has been employed to study microvascular reperfusion injury and the stimulation and inhibition of F1-ATPase from the thermophilic bacterium PS3.
At low concentrations, certain lipophilic rhodamine dyes selectively stain mitochondria in live cells. We have observed that low concentrations of the hexyl ester of rhodamine B (R648MP) accumulate selectively in mitochondria () and appear to be relatively nontoxic. We have included this probe in our Yeast Mitochondrial Stain Sampler Kit (Y7530, see below for description). At higher concentrations, rhodamine B hexyl ester and rhodamine 6G stain the endoplasmic reticulum of animal cells (Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4).
The accumulation of tetramethylrhodamine methyl and ethyl esters (TMRM, T668; TMRE, T669) in mitochondria and the endoplasmic reticulum has also been shown to be driven by their membrane potential (Slow-Response Probes—Section 22.3). Moreover, because of their reduced hydrophobic character, these probes exhibit potential-independent binding to cells that is 10 to 20 times lower than that seen with rhodamine 6G. Tetramethylrhodamine ethyl ester has been described as one of the best fluorescent dyes for dynamic and in situ quantitative measurements—better than rhodamine 123—because it is rapidly and reversibly taken up by live cells. TMRM and TMRE have been used to measure mitochondrial depolarization related to cytosolic Ca2+ transients and to image time-dependent mitochondrial membrane potentials. A high-throughput assay utilizes TMRE and our low-affinity Ca2+ indicator fluo-5N AM (F14204, Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3) to screen inhibitors of the opening of the mitochondrial transition pore.
Reduced Rhodamines and Rosamines
Inside live cells, the colorless dihydrorhodamines and dihydrotetramethylrosamine are oxidized to fluorescent products that stain mitochondria. However, the oxidation may occur in organelles other than the mitochondria. Dihydrorhodamine 123 (D632, D23806; ) reacts with hydrogen peroxide in the presence of peroxidases, iron or cytochrome c to form rhodamine 123. This reduced rhodamine has been used to monitor reactive oxygen intermediates in rat mast cells and to measure hydrogen peroxide in endothelial cells. Dihydrorhodamine 6G (D633, ) is another reduced rhodamine that has been shown to be taken up and oxidized by live cells. Chloromethyl derivatives of reduced rosamines (MitoTracker Orange CM-H2TMRos, M7511; MitoTracker Red CM-H2XRos, M7513), which can be fixed in cells by aldehyde-based fixatives, have been described above.
Most carbocyanine dyes with short (C1–C6) alkyl chains (Slow-Response Probes—Section 22.3) stain mitochondria of live cells when used at low concentrations (<100 nM); those with pentyl or hexyl substituents also stain the endoplasmic reticulum when used at higher concentrations (>1 µM). DiOC6(3) (D273) stains mitochondria in live yeast and other eukaryotic cells, as well as sarcoplasmic reticulum in beating heart cells. It has also been used to demonstrate mitochondria moving along microtubules. Photolysis of mitochondrion- or endoplasmic reticulum–bound DiOC6(3) specifically destroys the microtubules of cells without affecting actin stress fibers, producing a highly localized inhibition of intracellular organelle motility. We have included DiIC1(5) and DiOC2(3) in two of our MitoProbe Assay Kits for flow cytometry (M34151, M34150; Slow-Response Probes—Section 22.3). Several other potential-sensitive carbocyanine probes described in Slow-Response Probes—Section 22.3 also stain mitochondria in live cultured cells. The carbocyanine DiOC7(3) (D378), which exhibits spectra similar to those of fluorescein, is a versatile dye that has been reported to be a sensitive probe for mitochondria in plant cells.
The styryl dyes DASPMI (4-Di-1-ASP, D288) and DASPEI (D426) can be used to stain mitochondria in live cells and tissues. These dyes have large fluorescence Stokes shifts and are taken up relatively slowly as a function of membrane potential. The kinetics of mitochondrial staining with styrylpyridinium dyes has been investigated using the concentration jump method.
Nonyl Acridine Orange
Nonyl acridine orange (A1372) is well retained in the mitochondria of live HeLa cells for up to 10 days, making it a useful probe for following mitochondria during isolation and after cell fusion. The mitochondrial uptake of this metachromatic dye is reported not to depend on membrane potential. It is toxic at high concentrations and apparently binds to cardiolipin in all mitochondria, regardless of their energetic state. This derivative has been used to analyze mitochondria by flow cytometry, to characterize multidrug resistance (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6) and to measure changes in mitochondrial mass during apoptosis in rat thymocytes.
Carboxy SNARF-1 pH Indicator
A special cell-loading technique permits ratiometric measurement of intramitochondrial pH with our SNARF dyes. Cell loading with 10 µM 5-(and 6-)carboxy SNARF-1, acetoxymethyl ester, acetate (C1271, C1272; Probes Useful at Near-Neutral pH—Section 20.2), followed by 4 hours of incubation at room temperature leads to highly selective localization of the carboxy SNARF-1 dye in mitochondria (), where it responds to changes in mitochondrial pH.
The well-known chemiluminescent probe lucigenin (L6868) accumulates in mitochondria of alveolar macrophages. Relatively high concentrations of the dye (~100 µM) are required to obtain fluorescent staining; however, low concentrations reportedly yield a chemiluminescent response to stimulated superoxide generation within the mitochondria. Molecular Probes lucigenin has been highly purified to remove a bright blue-fluorescent contaminant that is found in some commercial samples.
Image-iT LIVE Mitochondrial Transition Pore Assay Kit for Fluorescence Microscopy
The mitochondrial permeability transition pore, a nonspecific channel formed by components from the inner and outer mitochondrial membranes, appears to be involved in the release of mitochondrial components during apoptotic and necrotic cell death. In a healthy cell, the inner mitochondrial membrane is responsible for maintaining the electrochemical gradient that is essential for respiration and energy production. As Ca2+ is taken up and released by mitochondria, a low-conductance permeability transition pore appears to flicker between open and closed states. During cell death, the opening of the mitochondrial permeability transition pore dramatically alters the permeability of mitochondria. 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. Cytochrome c release from mitochondria and loss of mitochondrial membrane potential are observed subsequent to continuous pore activation.
The Image-iT LIVE Mitochondrial Transition Pore Assay Kit (I35103), based on published experimentation for mitochondrial transition pore opening, provides 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, 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 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 (I24222, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8) 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 cyclosporine A, a compound reported to prevent mitochondrial transition pore formation by binding cyclophilin D.
The Image-iT LIVE Mitochondrial Transition Pore Assay Kit has been tested with HeLa cells and bovine pulmonary artery endothelial cells (BPAEC). Each Image-iT LIVE Mitochondrial Transition Pore Assay Kit provides sufficient reagents for 100 assays (based on 1 mL labeling volumes), including:
- Calcein AM
- MitoTracker Red CMXRos, a red-fluorescent mitochondrial stain (excitation/emission maxima ~579/599 nm)
- Hoechst 33342, a blue-fluorescent nuclear stain (excitation/emission maxima ~350/461 nm)
- Dimethylsulfoxide (DMSO)
- Detailed protocols (Image-iT LIVE Mitochondrial Transition Pore Assay Kit)
MitoProbe Transition Pore Assay Kit for Flow Cytometry
The MitoProbe Transition Pore Assay Kit (M34153), based on published experimentation for mitochondrial transition pore opening, provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone (Figure 12.2.9). As with the Image-iT LIVE mitochondrial transition pore assay described above, this assay employs the acetoxymethyl (AM) ester of calcein, a colorless and nonfluorescent esterase substrate, and CoCl2, 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 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 (I24222, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8) 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 cyclosporine A, a compound reported to prevent mitochondrial transition pore formation by binding cyclophilin D.
The MitoProbe Transition Pore Assay Kit has been tested with Jurkat cells, MH1C1 cells and bovine pulmonary artery endothelial cells (BPAEC). Each MitoProbe Transition Pore Assay Kit provides sufficient reagents for 100 assays (based on 1 mL labeling volumes), including:
- Calcein AM
- Dimethylsulfoxide (DMSO)
- Detailed protocols (MitoProbe Transition Pore Assay Kit)
Figure 12.2.9 Flow cytometric analysis of Jurkat cells using the MitoProbe Transition Pore Assay Kit (M34153). Jurkat cells were incubated with the reagents in the MitoProbe Transition Pore Assay Kit and analyzed by flow cytometry. In the absence of CoCl2 and ionomycin, fluorescent calcein is present in the cytosol as well as the mitochondria, resulting in a bright signal (panel A). In the presence of CoCl2, calcein in the mitochondria emits a signal, but the cytosolic calcein fluorescence is quenched; the overall fluorescence is reduced, as compared with calcein alone (panel B). When ionomycin, a Ca2+ ionophore, and CoCl2 are added to the cells at the same time that calcein AM is added, the fluorescent signals from both the cytosol and mitochondria are largely abolished (panel C). The change in fluorescence between panels B and C indicates the continuous activation of mitochondrial permeability transition pores.
Because fluorescence microscopy has been extensively used to study yeast, we offer a Yeast Mitochondrial Stain Sampler Kit (Y7530). This kit contains sample quantities of five different probes that have been found to selectively label yeast mitochondria. Both well-characterized and proprietary mitochondrion-selective probes are provided:
The mitochondrion-selective nucleic acid stain included in this kit—SYTO 18 yeast mitochondrial stain—exhibits a pronounced fluorescence enhancement upon binding to nucleic acids, resulting in very low background fluorescence even in the presence of dye. SYTO 18 is an effective mitochondrial stain in live yeast but neither penetrates nor stains the mitochondria of higher eukaryotic cells. Each of the components of the Yeast Mitochondrial Stain Sampler Kit is also available separately, including the SYTO 18 yeast mitochondrial stain (S7529).
Endogenously biotinylated proteins in mammalian cells, bacteria, yeast and plants—biotin carboxylase enzymes—are present almost exclusively in mitochondria, where biotin synthesis occurs; consequently, mitochondria can be selectively stained by almost any fluorophore- or enzyme-labeled avidin or streptavidin derivative (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6; Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.23; , ) without applying any biotinylated ligand. This staining, which can complicate the use of avidin–biotin techniques in sensitive cell-based assays, can be blocked by the reagents in our Endogenous Biotin-Blocking Kit (E21390, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6).
|D632||346.38||F,D,L,AA||DMF, DMSO||289||7100||none||MeOH||2, 3|
|D633||444.57||F,D,L,AA||DMF, DMSO||296||11,000||none||MeOH||2, 3|