The morphology of mitochondria is highly variable. In dividing cells, the organelle can switch between a fragmented morphology with many ovoid-shaped mitochondria, as 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. 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 emerging 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's disease and their protein and nucleic acids are affected by reactive oxygen species, including nitric oxide (Probes for Reactive Oxygen Species, Including Nitric Oxide - Chapter 18).

Molecular Probes has a range of mitochondrion-selective dyes with which to monitor mitochondrial morphology and organelle functioning. The uptake of most mitochondrion-selective dyes is dependent on the mitochondrial membrane potential; nonyl acridine orange and possibly our MitoTracker Green FM, MitoFluor Green and MitoFluor Red 589 probes are notable exceptions, although their membrane potential–independent uptake and fluorescence has been questioned in some cell types. Mitochondrion-selective reagents enable researchers to probe mitochondrial activity, localization and abundance, as well as to monitor the effects of some pharmacological agents, such as anesthetics that alter mitochondrial function. Molecular Probes offers a variety of cell-permeant stains for mitochondria, as well as subunit-specific monoclonal antibodies directed against proteins in the oxidative phosphorylation (OxPhos) system, all of which are discussed below.

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, Molecular Probes has developed MitoTracker probes — a series of Patented 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.

Molecular Probes offers seven MitoTracker reagents that differ in spectral characteristics, oxidation state and fixability (Spectral characteristics of the MitoTracker probes - Table 12.2, MitoTracker(R) and MitoFluor(R) Mitochondrion-Selective Probes). 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 our newest derivatives, the MitoTracker Red 580 and MitoTracker Deep Red 633 probes (M22425, M22426; , ). Because the MitoTracker Red CMXRos, MitoTracker Red 580 and MitoTracker Deep Red 633 probes produce longer-wavelength fluorescence that is well resolved from the fluorescence of green-fluorescent dyes, they are suitable for multicolor labeling experiments (, , , , ), including those that employ image deconvolution techniques (Wide Field Deconvolution Microscopy - Note 12.3). 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 12.12, , ). The MitoTracker probes have proven useful for:

• Assaying the role of a kinesin-like protein on germ plasm aggregation in Xenopus oocytes
• Detecting early apoptosis (Assays for Apoptosis - Section 15.5), which is marked by a disruption of mitochondrial transmembrane potential in all cell types studied
• Determining the mechanism by which mitochondrial shape is established and maintained in yeast
• Examining the time course of cell swelling in a human collecting-duct cell line using total internal reflection (TIR) microfluorimetry
• Localizing a novel kinesin motor protein involved in transport of mitochondria along microtubules
• Simultaneously observing fluorescent signals from a green-fluorescent protein (GFP) chimera and from the MitoTracker dye (Fluorescent Probes for Use with GFP - Note 12.1)
• Studying the localization of mitochondria in fibroblasts transformed with cDNA of wild-type and mutant kinesin heavy chains
• Visualizing mitochondria while characterizing the subcellular distribution of calcium channel subtypes in Aplysia californica bag cell neurons and of the verotoxin B subunit in Vero cells

Our Vybrant Apoptosis Assay Kit #11 (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 15.96). MitoTracker Orange CMTMRos and its reduced form CM-H₂TMRos have also been used to investigate the metabolic state of Pneumocystis carinii mitochondria. 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 or anti–Texas Red dye antibodies (A6397, A6399; Anti-Dye and Anti-Hapten Antibodies - Section 7.4).

Figure 15.96 Flow cytometric analysis of Jurkat cells using the Vybrant Apoptosis Assay Kit #11 (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 Vybrant Apoptosis Assay Kit #11 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 Probe

Mitochondria in cells stained with nanomolar concentrations of our Patented MitoTracker Green FM dye (M7514, MitoTracker(R) and MitoFluor(R) Mitochondrion-Selective Probes) 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 (Estimating Mitochondrial Mass - Note 12.4). 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 MitoTracker Green FM probe has been used to:

• Assay the differentiation state of Trypanosoma brucei bloodstream forms
• Demonstrate mitochondrion-selective labeling by avidin, streptavidin and anti-biotin antibodies
• Identify mitochondria in immunolocalization experiments in CHO cells
• Label sperm in order to determine the fate of sperm mitochondria during fertilization and subsequent embryo development (, )
• Monitor mitochondrial distribution and transport in Tau-expressing CHO cells
• Study the regulation of calcium signaling by mitochondria in T lymphocytes

The mitochondrial proteins that are selectively labeled by the MitoTracker Green FM reagent have been separated by capillary electrophoresis.

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, green-fluorescent protein (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. Cell-permeant MitoTracker Red CMXRos dye provides highly selective mitochondrial staining with minimal background. Hoechst 33342 dye, a cell-permeant nucleic acid stain that is selective for DNA and spectrally similar to DAPI, is UV excitable and emits blue fluorescence when bound to DNA. These dyes should not interfere with GFP fluorescence, and 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(R) 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

MitoFluor Green Probe

As a companion to the MitoTracker Green FM derivative, we have developed the MitoFluor Green probe (M7502), which has a structure similar to MitoTracker Green FM () but lacks its reactive chloromethyl moieties () and is not as well retained following fixation. As with MitoTracker Green FM, the MitoFluor Green probe can selectively stain mitochondria in live cells. The MitoFluor Green probe is also substantially more photostable than rhodamine 123, produces a brighter, more mitochondrion-selective signal at lower concentrations, and exhibits a blue-shifted emission maximum relative to that of rhodamine 123 that is better resolved from that of red-fluorescent probes in double-labeling experiments. Neither MitoTracker Green FM, nor the MitoFluor Green probe, appears to be retained after cell permeabilization.

Long-Wavelength MitoFluor Red Probes

We offer two mitochondria markers with long-wavelength fluorescence emission: MitoFluor Red 589 (M22424, ) and MitoFluor Red 594 (M22422, ). The MitoFluor Red 589 probe appears to accumulate in mitochondria regardless of the mitochondria's membrane potential, making it a potentially useful stain for estimating mitochondrial mass. This probe has absorption and emission peaks at 588 nm and 622 nm, respectively, and can be viewed with filter sets appropriate for the Texas Red dye. The MitoFluor Red 594 probe is a mitochondrial membrane potential–sensing dye that has been designed for optimal excitation by the 594 nm spectral line of the He–Ne laser. Both of these MitoFluor Red dyes provide a clear spectral window below 600 nm for dual labeling with green-fluorescent probes, including other site-selective probes or GFP chimeras.

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 ROS 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's, Alzheimer's and amyotrophic lateral sclerosis (ALS).

The assumption that mitochondria serve as the major intracellular source of ROS has been based largely on experiments with isolated mitochondria rather than direct measurements in live cells. MitoSOX Red mitochondrial superoxide indicator (M36008) is a novel fluorogenic dye for highly selective detection of superoxide in the mitochondria of live cells (). MitoSOX Red reagent is live-cell permeant and is rapidly and selectively targeted to the mitochondria. Once in the mitochondria, MitoSOX Red reagent is oxidized by superoxide and exhibits bright red fluorescence upon binding to nucleic acids (excitation/emission maxima = 510/580 nm). MitoSOX Red reagent is readily oxidized by superoxide but not by other ROS- or reactive nitrogen species (RNS)–generating systems, and oxidation of the probe is prevented by superoxide dismutase (Figure 12.20). This reagent may enable researchers to distinguish artifacts of isolated mitochondrial preparations from direct measurements of superoxide generated in the mitochondria of live cells. It may also provide a valuable tool in the discovery of agents that modulate oxidative stress in various pathologies.

Figure 12.20 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 nitrite determination kit (for nitric oxide, peroxynitrite, and nitrite standards only; gray bars) and dihydrorhodamine 123 (DHR 123; white 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 (black bars) is readily oxidized by superoxide but not by the other oxidants.

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 (Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11) 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 22.24). JC-1 has also been used to:

• Analyze the effects of drugs by flow cytometry
• Detect human encephalomyopathy
• Follow mitochondrial changes during apoptosis
• Investigate mitochondrial poisoning, uncoupling and anoxia
• Monitor effects of ellipticine on mitochondrial potential

JC-1 has been combined with the reagents in our LIVE/DEAD Sperm Viability Kit (L7011, Viability and Cytotoxicity Assay Kits for Diverse Cell Types - Section 15.3) to permit simultaneous assessment of cellular integrity 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 very 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.

Rhodamine 123

Rhodamine 123 (R302; FluoroPure Grade - Note 19.2, 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 (Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11), 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 presynaptic nerve terminals, live bacteria, plants and human spermatozoa. Using flow cytometry, researchers employed rhodamine 123 to sort respiratory-deficient yeast cells and to isolate those lymphocytes that are responsive to mitogen stimulation. Rhodamine 123 has also been used to study:

• Apoptosis
• Axoplasmic transport of mitochondria
• Bacterial viability and vitality
• Mitochondrial enzymatic activities
• Mitochondrial transmembrane potential and other membrane activities
• Multidrug resistance (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione - Section 15.6)
• Mycobacterial drug susceptibility
• Oocyte maturation

Although rhodamine 123 is usually not retained by cells when they are washed, a variety of human carcinoma cell lines (but not sarcomas or leukemic cells) retain the dye for unusually long periods (>24 hours), making rhodamine 123 a potential anticancer agent for photodynamic therapy. Rhodamine 123 is known to be preferentially taken up and retained by mitochondria of carcinoma cells and to inhibit their proliferation; cardiac muscle cells also retain rhodamine 123 for days.

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. Molecular Probes' researchers have observed that low concentrations of the hexyl ester of rhodamine B (R 6, 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 Ca{2+} Indicators Excited with Visible Light - Section 19.3) to screen inhibitors of the opening of the mitochondrial transition pore. Researchers have also taken advantage of the red shift exhibited by TMRM, TMRE and rhodamine 123 upon membrane potential–driven mitochondrial uptake to develop a ratiometric method for quantitating membrane potential.

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. The acetoxymethyl (AM) ester of dihydrorhod-2, which is prepared by chemical reduction of the calcium indicator rhod-2 AM (R1244, R1245MP; Fluorescent Ca{2+} Indicators Excited with Visible Light - Section 19.3; Preparation of Dihydrorhod-2 AM) has been extensively used to measure the relatively slow changes in intramitochondrial Ca²⁺ (, ).

Carbocyanines

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 (~0.5 µM or ~0.1 µg/mL); those with pentyl or hexyl substituents also stain the endoplasmic reticulum when used at higher concentrations (~5–50 µM or ~1–10 µg/mL). 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. Its other uses include:

• Distinguishing cycling and noncycling fibroblasts and viable and nonviable bacteria
• Following the reorganization of the endoplasmic reticulum during fertilization in the ascidian egg
• Identifying functional vasculature in murine tumors
• Studying multidrug resistance (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione - Section 15.6)
• Visualizing the detailed morphology of neurites of Alzheimer's disease neurons

Styryl Dyes

The styryl dyes DASPMI (4-Di-1-ASP, D288) and DASPEI (D426) can be used to stain mitochondria in live cells. 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. DASPMI and DASPEI have been shown to be useful for:

• Determining the distribution of mitochondria in yeast mutants
• Long-term imaging of live mammalian nerve cells and their connections
• Monitoring the metabolic state of Pneumocystis carinii mitochondria
• Screening aberrant mitochondrial distribution and morphology in yeast

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.

CoroNa Red Chloride

As shown by colocalization with MitoTracker Green FM, the CoroNa Red Na⁺ indicator (C24430, C24431; Fluorescent Na{+} and K{+} Indicators - Section 21.1) spontaneously localizes in the mitochondria () and may be useful for measuring intramitochondrial Na⁺ transients.

Lucigenin

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. Lucigenin from Molecular Probes 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 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 (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:

• 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)
• Ionomycin
• CoCl₂
• Dimethylsulfoxide (DMSO)
• A detailed protocol (Image-iT(R) LIVE Mitochondrial Transition Pore Assay Kit)

Sufficient reagents are provided for 100 assays, based on labeling volumes of 1 mL.

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 15.101). 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 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 (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.

Figure 15.101 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.

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:

• Calcein AM
• CoCl₂
• Ionomycin
• Dimethylsulfoxide (DMSO)
• A detailed protocol (MitoProbe Transition Pore Assay Kit)

Sufficient reagents are provided for 100 assays, based on labeling volumes of 1 mL.

Because fluorescence microscopy has been extensively used to study yeast, Molecular Probes offers 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:

• Rhodamine 123
• Rhodamine B hexyl ester ()
• MitoTracker Green FM
• SYTO 18 yeast mitochondrial stain
• DiOC₆(3)

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).

Monoclonal Antibodies Specific for Proteins in the Oxidative Phosphorylation System

Oxidative phosphorylation (OxPhos) activity occurs in the mitochondria and, in mammals, is catalyzed by five large membrane-bound protein complexes, namely NADH–ubiquinol oxidoreductase (Complex I), succinate–ubiquinol oxidoreductase (Complex II), ubiquinol–cytochrome c oxidoreductase (Complex III), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V). The complexes are composed of multiple subunits, some of which are encoded in the mitochondrion and some in the nucleus. For example, mammalian cytochrome oxidase (COX) is composed of 13 subunits, three encoded by mitochondrial DNA (subunits I, II and III; ) and ten encoded by nuclear DNA. Assembly of each complex involves a coordinated association of prosthetic groups (hemes, non-heme irons, flavins and copper atoms) with some polypeptides made in the mitochondrion and others made in the cytosol and then translocated to the organelle. This complicated process is poorly defined but known to require various assembly factors, each of which is specific for a particular complex. Defects in assembly of one or more of these complexes contribute to several described mitochondrial diseases and possibly Alzheimer's and Parkinson's diseases.

Antibodies against the various subunits of the OxPhos Complex are important tools for investigating mitochondrial biogenesis and studying OxPhos-related diseases (Mitochondria in Diseases - Note 12.2). Patient cell lines can now be screened for deficiencies in each of the OxPhos Complexes by simple Western blotting. When compared with control cell lines, this screen provides information about relative subunit expression levels and can be combined with native gel electrophoresis or sucrose gradient centrifugation to gather additional information regarding the assembly state of the OxPhos Complex. Many of our antibodies against subunits of the OxPhos Complex may also be used for immunohistochemical analysis. Image analysis of the antibody's staining pattern can reveal the relative expression and localization of a subunit. This approach has been particularly useful for studying OxPhos subunit expression in diseased muscle fibers and for screening Complex IV–deficient patients.

Molecular Probes offers a range of subunit-specific anti–OxPhos Complex mouse monoclonal antibodies that recognize proteins in the oxidative phosphorylation system (Monoclonal antibodies to yeast oxidative phosphorylation complex IV (COX) - Table 12.3, Monoclonal antibodies specific for proteins in the oxidative phosphorylation system - Table 12.4, Molecular characteristics of components of the oxidative phosphorylation system - Table 12.5; Figure 12.33) and have proven useful in the characterization and diagnosis of mitochondrial disease. One set of antibodies is against the Complex IV subunits of yeast, as this is the organism of choice for studying biogenesis of cytochrome oxidase. The remaining antibodies were generated against bovine or human material and were selected because they react with high specificity for the human form of the various proteins. All of our antibodies work well in Western blots and a majority can be used for immunohistochemistry, as listed in Monoclonal antibodies specific for proteins in the oxidative phosphorylation system - Table 12.4. These antibodies may also be employed to test other subcellular preparations for mitochondrial contamination. Stringent selection criteria were applied during the development of these monoclonal antibodies, including:

• Ability of the antibodies to detect native protein in solid-phase binding assays such as particle-concentration fluorescence immunoassays (PCFIAs) and enzyme-linked immunosorbent assays (ELISAs)
• Specificity for the appropriate denatured subunit in Western blots of whole-cell extracts and isolated mitochondria
• Where appropriate, specific mitochondrial subcellular localization of immunohistochemical reactivity in fixed cultured human cells

Detailed information regarding the IgG isotype and recommended working concentration is provided with each product (Monoclonal Antibodies Specific for Oxidative Phosphorylation, Monoclonal Antibodies Specific for Yeast Cytochrome Oxidase Subunits). For detection of these monoclonal antibodies, Molecular Probes offers anti–mouse IgG secondary antibodies labeled with biotin, enzymes, NANOGOLD and Alexa Fluor FluoroNanogold 1.4 nm gold clusters, Captivate ferrofluid or a wide range of fluorophores (Secondary Immunoreagents - Section 7.2, Summary of Molecular Probes' secondary antibody conjugates - Table 7.1). The antibodies in this group (Molecular Probes' mouse IgG{2a} monoclonal antibodies - Table 7.16, Molecular Probes' mouse IgG{2b} monoclonal antibodies - Table 7.17, Molecular Probes' rabbit antibodies - Table 7.18, Monoclonal antibodies specific for proteins in the oxidative phosphorylation system - Table 12.4) can also be complexed with the Zenon labeling reagents in the corresponding Zenon Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14) for detecting mitochondrial targets in cells (, ).

Monoclonal Antibodies Specific for OxPhos Complex IV (Cytochrome Oxidase)

To facilitate the study of cytochrome oxidase (COX) structure and mitochondrial biogenesis, Molecular Probes offers subunit-specific mouse anti–OxPhos Complex IV monoclonal antibodies that have been derived from the human, bovine and yeast forms of COX. COX catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen, with a concomitant translocation of protons across the mitochondrial inner membrane. This mitochondrial membrane–bound enzyme is composed of subunits that are encoded in both the mitochondria (COX subunits I, II and III) and the nucleus (all others), with a total of 13 subunits for mammalian COX and 11 subunits for yeast COX. The binding specificity exhibited by our anti–OxPhos Complex IV monoclonal antibody preparations allows researchers to investigate the regulation, assembly and orientation of COX subunits from a variety of organisms (Monoclonal antibodies to yeast oxidative phosphorylation complex IV (COX) - Table 12.3, Monoclonal antibodies specific for proteins in the oxidative phosphorylation system - Table 12.4, Molecular characteristics of components of the oxidative phosphorylation system - Table 12.5). Furthermore, because the antibodies to bovine COX also recognize the corresponding human COX subunits, the antibodies have proven valuable for analyzing human mitochondrial myopathies and related disorders. Alexa Fluor 488 and Alexa Fluor 594 conjugates of anti–COX subunit I are also available for direct staining of mitochondria (A21296, A21297; ). Mouse monoclonal 1D6 anti–COX subunit 1 antibody (A6403), which recognizes the mitochondrial DNA–encoded COX subunit I, has been shown to be an effective tool for following mitochondrial DNA depletion in cultured fibroblasts treated with nucleoside reverse transcriptase inhibitors (NRTIs) and potentially for monitoring patients on a regimen of NRTIs for the treatment of HIV.

Monoclonal Antibodies Specific for Complexes I, II, III and V

Molecular Probes supplies a large number of monoclonal antibodies to the OxPhos Complex (Monoclonal antibodies specific for proteins in the oxidative phosphorylation system - Table 12.4). These include antibodies specific for individual subunits of Complexes I, II, III and V, as well as the Complex V inhibitor protein. When these monoclonal antibodies are used in combination with the set of antibodies to cytochrome oxidase (Complex IV), the relative levels of all OxPhos enzyme complexes in normal and diseased tissues can be evaluated.

The mouse monoclonal 7H10 anti–OxPhos Complex V subunit (bovine) (anti–F₁F₀-ATPase subunit α, A21350) and mouse monoclonal 3D5 anti–OxPhos Complex V subunit (bovine) (anti–F₁F₀-ATPase subunit β, A21351) antibodies have also been shown to mimic angiostatin, a potent inhibitor of angiogenesis. Angiostatin protein (A23375, Assays for Cell Enumeration, Cell Proliferation and Cell Cycle - Section 15.4), a recombinant form of natural angiostatin, targets the F₁F₀-ATP synthase and inhibits cell-surface ATP metabolism of endothelial cells, thereby blocking cell migration and proliferation that is essential for angiogenesis. This research demonstrated that these anti-ATPase antibodies had similar inhibitory effects, implying that they also compromised ATP metabolism and may function as angiostatin analogs.

SelectFX Alexa Fluor 488 Cytochrome c Apoptosis Detection Kit

A distinctive feature of the early stages of programmed cell death is the disruption of active mitochondria. This mitochondria disruption includes changes in the membrane potential, presumably due to the opening of the mitochondrial permeability transition pore, which allows passage of ions and small molecules. The resulting equilibration of ions leads in turn to the decoupling of the respiratory chain and then the release of cytochrome c into the cytosol. The SelectFX Alexa Fluor 488 Cytochrome c Apoptosis Detection Kit (S35115) provides all the reagents required to detect cytochrome c in fixed cells. The Alexa Fluor 488 dye exhibits bright green fluorescence that is compatible with filters and instrument settings appropriate for fluorescein. Each kit contains:

• Mouse IgG₁ anti–cytochrome c antibody
• Highly cross-adsorbed Alexa Fluor 488 goat anti–mouse IgG antibody
• Concentrated fixative solution
• Concentrated phosphate-buffered saline (PBS)
• Concentrated permeabilization solution
• Concentrated blocking solution
• Detailed protocols for mammalian cell preparation and staining (SelectFX(R) Alexa Fluor(R) 488 Cytochrome c Apoptosis Detection Kit)

The SelectFX Alexa Fluor 488 Cytochrome c Apoptosis Detection Kit can be used in conjunction with probes for other cell targets to achieve multicolor cell staining.

Mitochondrial Porin

Mitochondrial porin is an outer-membrane protein that forms regulated channels (referred to as voltage-dependent anionic channels, or VDACs) between the cytosol and the mitochondrial inter-membrane space. This abundant transmembrane protein forms a small pore (~3 nm) in the outer membrane, allowing molecules less than ~10,000 daltons to pass. Due to its abundance, porin is often used as a standardization marker in Western blots when assaying for other mitochondrial proteins and serves as an effective organelle marker for immunohistochemistry. Monoclonal antibodies against both human and yeast porin are available from Molecular Probes (A21317, A31855, A6449; Monoclonal antibodies specific for mitochondrial proteins not associated with the oxidative phosphorylation system - Table 12.6).

Pyruvate Dehydrogenase

Molecular Probes has available a series of antibodies against the human pyruvate dehydrogenase (PDH) complex (Monoclonal antibodies specific for mitochondrial proteins not associated with the oxidative phosphorylation system - Table 12.6), a large, multienzyme assembly residing in the mitochondrial matrix and consisting of three catalytic activities: pyruvate dehydrogenase, dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase (diaphorase). The PDH complex is responsible for the oxidative decarboxylation of pyruvate to form acetyl coenzyme A, which is in turn fed into the citric acid cycle. Deficiencies in the PDH complex lead to lactic acidosis; severe cases can lead to developmental defects such as congenital brain malformation. In addition to unlabeled subunit-specific anti-PDH antibodies, we offer the red-fluorescent Alexa Fluor 594 conjugate of anti–PDH E1α subunit antibody (A31853), as well as the green-fluorescent Alexa Fluor 488 conjugate of anti–PDH E2 subunit antibody (A31854).