线粒体荧光探针大全：TMRM，Mitotracker，JC-1（4）
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.136–138 The mitochondrial uptake of this metachromatic dye is reported not to depend on membrane potential. It is toxic at high concentrations 139 and apparently binds to cardiolipin in all mitochondria, regardless of their energetic state.140–143 This derivative has been used to analyze mitochondria by flow cytometry,144 to characterize multidrug resistance145 (Section 15.6) and to measure changes in mitochondrial mass during apoptosis in rat thymocytes.52
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; 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 (Figure 20.13), where it responds to changes in mitochondrial pH.146
CoroNa Red Chloride
As shown by colocalization with MitoTracker Green FM, the CoroNa Red Na+ indicator (C24430, C24431; Section 21.1) spontaneously localizes in the mitochondria (Figure 21.14) and may be useful for measuring intramitochondrial Na+ transients.
Lucigenin
The well-known chemiluminescent probe lucigenin (L6868) accumulates in mitochondria of alveolar macrophages.147 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.147 Lucigenin from Molecular Probes has been highly purified to remove a bright blue-fluorescent contaminant that is found in some commercial samples.
Mitochondrial Transition Pore Assays
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.148 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.149Cytochrome 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,150,151 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, 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
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,150,151 provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone (Figure 15.100). 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, 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:
Calcein AM
CoCl2
Ionomycin
Dimethylsulfoxide (DMSO)
A detailed protocol
Sufficient reagents are provided for 100 assays, based on labeling volumes of 1 mL.
Yeast Mitochondrial Stain Sampler Kit
Because fluorescence microscopy has been extensively used to study yeast,21,119 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 64,152–154
Rhodamine B hexyl ester 101 (Figure 12.25)
MitoTracker Green FM
SYTO 18 yeast mitochondrial stain 155
DiOC6(3) 21,118,119,156–162
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).
Avidin Conjugates for Staining Mitochondria
Endogenously biotinylated proteins in mammalian cells, bacteria, yeast and plants — biotin carboxylase enzymes — are present almost exclusively in mitochondria, where biotin synthesis occurs;163 consequently, mitochondria can be selectively stained by almost any fluorophore- or enzyme-labeled avidin or streptavidin derivative (Section 7.6; Table 7.22; Figure 12.28, Figure 12.29) 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, Section 7.6).
Antibodies to Mitochondrial Proteins
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