Mitochondria in cells stained with nanomolar concentrations of our patented MitoTracker Green FM dye (M7514) exhibit bright green, fluorescein-like fluorescence (Figure 12.13, Figure 12.33, Figure 14.68, Figure 16.21). 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 32,33 (see Note 12.4 "Product Highlight: Estimating 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 MitoTracker Green FM probe has been used to:
Assay the differentiation state of Trypanosoma brucei bloodstream forms 34
Demonstrate mitochondrion-selective labeling by avidin, streptavidin and anti-biotin antibodies35
Identify mitochondria in immunolocalization experiments in CHO cells 36
Label sperm in order to determine the fate of sperm mitochondria during fertilization and subsequent embryo development 37–39 (Figure 12.13, Figure 12.14)
Monitor mitochondrial distribution and transport in Tau-expressing CHO cells 40
Study the regulation of calcium signaling by mitochondria in T lymphocytes 41
The mitochondrial proteins that are selectively labeled by the MitoTracker Green FM reagent have been separated by capillary electrophoresis.42
As a companion to the MitoTracker Green FM derivative, we have developed the MitoFluor Green probe 11 (M7502), which has a structure similar to MitoTracker Green FM (Figure 12.15) but lacks its reactive chloromethyl moieties (Figure 12.16) and is not as well retained following fixation. As with MitoTracker Green FM, the MitoFluor Green probe can selectively stain mitochondria in live cells.11,43 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.
We offer two mitochondria markers with long-wavelength fluorescence emission: MitoFluor Red 589 (M22424, Figure 12.17) and MitoFluor Red 594 44 (M22422, Figure 12.17). 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; those "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 living cells.
MitoSOX Red mitochondrial superoxide indicator (M36008) is a novel fluorogenic dye for highly selective detection of superoxide in the mitochondria of live cells (Figure 12.18). 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. 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.
RedoxSensor Red CC-1 (2,3,4,5,6-pentafluorotetramethyldihydrorosamine, R14060; Figure 12.19) stain is a unique probe whose fluorescence localization appears to be based on a cell's cytosolic redox potential. Once it passively enters live cells, the RedoxSensor Red CC-1 stain may be 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.45 In proliferating cells, mitochondrial staining predominates; whereas in contact-inhibited cells, the staining is primarily lysosomal (Figure 18.15). The best method we have found to quantitate the distribution of the oxidized product is to use the mitochondrion-selective MitoTracker Green FM stain (M7514) in conjunction with the RedoxSensor Red CC-1 stain.45
The green-fluorescent JC-1 probe (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide, T3168; Figure 22.13) 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 (Figure 12.20, Figure 12.21, Figure 22.14). 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 (Figure 22.15). 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 (Table 23.12) 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 46–48 (Figure 22.16).
JC-1 has also been used to:
Analyze the effects of drugs by flow cytometry 49
Detect human encephalomyopathy 50
Follow mitochondrial changes during apoptosis 51,52
Investigate mitochondrial poisoning, uncoupling and anoxia 53
Monitor effects of ellipticine on mitochondrial potential 54
JC-1 has been combined with the reagents in our LIVE/DEAD Sperm Viability Kit (L7011, Section 15.3) to permit simultaneous assessment of cellular integrity and mitochondrial function by flow cytometry.55 We also offer JC-1 as part of the MitoProbe JC-1 Assay Kit for flow cytometry (M34152, Section 22.3). We have discovered another mitochondrial marker, JC-9 (3,3'-dimethyl-
-naphthoxazolium iodide, D22421; Figure 22.18), with a very different chemical structure (Figure 22.17) 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.
首页
