12.2 Probes for Mitochondria
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, cellcycle stage and intracellular metabolic state. The key function of
mitochondria is energy production through oxidative phosphorylation (OxPhos) and lipid oxidation.1 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 — a process by which unneeded cells are removed
during development, and defective cells are selectively destroyed
without surrounding organelle damage in somatic tissues 2–4 (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.
Visualizing Mitochondria in Cells and Tissues
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
most 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 (see Mitochondria in Diseases). Organelle morphology is also controlled by cytoskeletal elements, including actin
filaments and microtubules. In nondividing tissue, overall mitochondrial morphology is very cell dependent, ranging from spiraling around the acrosome in spermatozoa, to intercalated bands
of ovoid mitochondria between the 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,5 hepatocytes 6
and certain renal epithelial cells 7 tend to be rich in active mitochondria, whereas quiescent immune-system progenitor or precursor cells show little staining with mitochondrion-selective
dyes.8 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 9 (Chapter 19).
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.10,11 Mitochondrion-selective reagents enable researchers to probe mitochondrial activity, localization and abundance,12,13 as well as to
monitor the effects of some pharmacological agents, such as
anesthetics that alter mitochondrial function.14 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.
MitoTracker Probes: Fixable MitochondrionSelective Probes
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 15 — a series of patented mitochondrionselective stains that are concentrated by active mitochondria and
well retained during cell fixation.16 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 (Table
12.1). 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, M-7510;
Figure 12.3) and the red-fluorescent X-rosamine (MitoTracker
Red CMXRos, M-7512; Figure 12.4), as well as our newest
derivatives, the MitoTracker Red 580 and MitoTracker Deep Red
633 probes (M-22425, M-22426; Figure 12.5, Figure 12.6; see
Widefield Deconvolution Microscopy in Section 12.1). Because
the MitoTracker Red CMXRos, MitoTracker Red 580 and Mito-
Section 12.2
473
Figure 12.3 M-7510 MitoTracker Orange
CMTMRos.
Figure 12.4 M-7512 MitoTracker Red CMXRos.
Tracker 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 (Figure 1.41, Figure 8.7, Figure 12.7, Figure 12.8, Figure 12.9).
Also available are chemically reduced forms of the tetramethylrosamine (MitoTracker
Orange CM-H2TMRos, M-7511; Figure 12.10) and X-rosamine (MitoTracker Red
CM-H2XRos, M-7513; Figure 12.11) 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, Figure 15.13). The MitoTracker probes have proven useful for:
• Assaying the role of a kinesin-like protein on germ plasm aggregation in Xenopus
oocytes 17
• Detecting early apoptosis (Section 15.5), which is marked by a disruption of mitochondrial transmembrane potential in all cell types studied 18–20
• Determining the mechanism by which mitochondrial shape is established and maintained in yeast 21
• Examining the time course of cell swelling in a human collecting-duct cell line using
total internal reflection (TIR) microfluorimetry 22
• Localizing a novel kinesin motor protein involved in transport of mitochondria along
microtubules 23
• Simultaneously observing fluorescent signals from a green-fluorescent protein (GFP)
chimera and from the MitoTracker dye 24–27
• Studying the localization of mitochondria in fibroblasts transformed with cDNA of
wild-type and mutant kinesin heavy chains 28
• Visualizing mitochondria while characterizing the subcellular distribution of calcium
channel subtypes in Aplysia californica bag cell neurons 29 and of the verotoxin B
subunit in Vero cells 30
MitoTracker Orange CMTMRos and its reduced form CM-H2TMRos have also been
used to investigate the metabolic state of Pneumocystis carinii mitochondria.31 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 (A-6397, A-6399; Section 7.4).
Figure 12.5 Bovine pulmonary artery endothelial
cells labeled with MitoTracker Red 580 (M-22425)
to stain mitochondria. Following fixation and permeabilization, the Golgi apparatus was labeled with
our anti–golgin-97 antibody (A-21270) and detected using the Alexa Fluor 488 goat anti–mouse IgG
antibody (A-11001). The cells were counterstained
with DAPI (D-1306, D-3571, D-21490). The image
was deconvolved using Huygens software (Scientific Volume Imaging, www.svi.nl). 3-D reconstruction was performed using Imaris software (Bitplane AG).
MitoTracker Green FM Probe
Mitochondria in cells stained with nanomolar concentrations of our patented MitoTracker Green FM dye (M-7514) exhibit bright-green, fluorescein-like fluorescence (Figure
12.13, Figure 12.34, Figure 14.62, Figure 16.20). 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.
Table 12.1 Spectral characteristics of the MitoTracker probes.
Cat #
Figure 12.6 Mitochondria of live bovine pulmonary
artery endothelial cells stained with the MitoTracker Deep Red 633 dye (M-22426).
474
MitoTracker Probe
M-7514
MitoTracker Green FM †
M-7510
MitoTracker Orange CMTMRos
M-7511
MitoTracker Orange CM-H2TMRos
M-7512
MitoTracker Red CMXRos
M-7513
MitoTracker Red CM-H2XRos
M-22425
M-22426
Abs * (nm)
Em * (nm)
490
516
Oxidation State
NA
551
576
Oxidized
551 ‡
576 ‡
Reduced
578
599
Oxidized
578 ‡
599 ‡
Reduced
MitoTracker Red 580
581
644
NA
MitoTracker Deep Red 633
644
665
NA
* Absorption (Abs) and fluorescence emission (Em) maxima, determined in methanol; values may vary
somewhat in cellular environments. † MitoTracker Green FM is nonfluorescent in aqueous environments.
‡ These reduced MitoTracker probes are not fluorescent until oxidized. NA = Not applicable.
Chapter 12 — Probes for Organelles
www.probes.com
Figure 12.10 M-7511 MitoTracker Orange CMH2TMRos.
Figure 12.7 A bovine pulmonary artery endothelial cell (BPAEC) stained with mouse monoclonal
anti–β-tubulin in conjunction with Oregon Green
514 goat anti–mouse IgG antibody (O-6383),
MitoTracker Red CMXRos (M-7512) and DAPI
(D-1306, D-3571, D-21490).
Figure 12.11 M-7513 MitoTracker Red CMH2XRos.
Figure 12.8 Four-panel composite image of mouse
fibroblasts that were incubated with MitoTracker
Red CMXRos (M-7512), and then formaldehydefixed, acetone-permeabilized and stained with the Factin–specific probe, BODIPY FL phallacidin (B-607)
and with DAPI (D-1306, D-3571, D-21490). Images
were obtained by taking single and multiple exposures through bandpass optical filter sets appropriate for fluorescein, the Texas Red dye and DAPI.
Figure 12.9 Live bovine pulmonary artery endothelial cells loaded with MitoTracker Red CMXRos
(M-7512) then fixed and permeabilized. The cells
were then treated with a cocktail containing two antibodies to cytochrome oxidase: the anti–OxPhos
Complex IV subunit VIc (A-6401) and anti–OxPhos
Complex IV subunit I (A-6403) antibodies. The mitochondria were then labeled with Alexa Fluor 350 goat
anti–mouse IgG antibody (A-11045). The image on
the bottom is an overlay of the first two images.
Figure 12.12 Intracellular reactions of our fixable, mitochondrion-selective
MitoTracker Orange CM-H2TMRos (M-7511). When this cell-permeant
probe enters an actively respiring cell, it is oxidized to MitoTracker Orange
Figure 12.13 Bull sperm prelabeled with MitoTracker Green FM (M-7514) and used for in vitro
fertilization of bovine oocytes (Biol Reprod 55,
1195 (1996)). After fertilization, eggs with bound
or incorporated sperm were fixed in 2% formaldehyde, made permeable with Triton X-100 and labeled with an anti-tubulin antibody followed by a
tetramethylrhodamine-labeled secondary antibody
and counterstained with DAPI (D-1306, D-3571,
D-21490). Image contributed by Peter Sutovsky,
Department of Zoology, University of Wisconsin.
CMTMRos and sequestered in the mitochondria, where it can react with
thiols on proteins and peptides to form aldehyde-fixable conjugates.
Section 12.2
475
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 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
antibodies 35
• 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
Figure 12.14 Live bull sperm stained simultaneously with MitoTracker Green FM (M-7514) and
Hoechst 33342 (H-1399, H-3570, H-21492), as reported in Mol Reprod Dev 47, 79 (1997). Image
contributed by Peter Sutovsky, Oregon Regional
Primate Resource Center, Oregon Health Sciences
University. Used with permission of Wiley-Liss,
Inc., a subsidiary of John Wiley & Sons, Inc.
Figure 12.15 M-7514 MitoTracker Green FM.
Figure 12.16 M-7502 MitoFluor Green.
476
MitoFluor Probes: Nonfixable Mitochondrion-Selective Probes
MitoFluor Green Probe
As a companion to the MitoTracker Green FM derivative, we have developed the
MitoFluor Green probe 10 (M-7502), 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 both live and fixed cells.10,42 The MitoFluor
Green probe is also substantially more photostable than rhodamine 123 (Figure 12.17);
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. Like MitoTracker Green FM, the MitoFluor Green probe does not appear to be retained after cell
permeabilization.
MitoFluor Red 589 Probe
The MitoFluor Red 589 probe (M-22424) appears to accumulate in mitochondria
regardless of the mitochondria’s membrane potential, making it a useful stain for mitochondria in both live (Figure 12.18) and fixed cells. 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. Like our other MitoFluor Red dyes, the MitoFluor Red 589
probe provides a clear spectral window below 600 nm for dual labeling with greenfluorescent probes.
MitoFluor Red 594 and MitoFluor Far Red 680 Probes
We offer two mitochondrial membrane potential–sensing dyes that have long-wavelength fluorescence emission: the MitoFluor Red 594 (M-22422, Figure 12.19) and
MitoFluor Far Red 680 (M-22423, Figure 12.20) probes. The MitoFluor Red 594 dye
is a mitochondrial potential sensor that has been designed for optimal excitation by the
594 nm spectral line of the He–Ne laser. This long-wavelength probe can be combined
with green-fluorescent labels, including other site-selective probes or GFP chimeras.
The MitoFluor Far Red 680 dye, also known as rhodamine 800, is a mitochondrial
potential sensor with absorption and fluorescence emission in the near-infrared spectral
region. Accumulation of the MitoFluor Far Red 680 dye by active mitochondria produces a slight red shift in the probe’s absorption and fluorescence emission peaks,
accompanied by a marked decrease in fluorescence intensity.43 Although the dye’s
response is not directly visible to the human eye and must be captured using an infrared light–sensitive detector such as a CCD camera, this long-wavelength probe is ad-
Chapter 12 — Probes for Organelles
www.probes.com
Figure 12.17 Photostability comparison of mitochondrial staining by MitoFluor Green (upper series,
M-7502) and rhodamine 123 (lower series; R-302, R-22420). HeLa cells were stained with 100 nM
MitoFluor Green or 500 nM rhodamine 123 in growth medium for 20 minutes at 37°C. Cells were then
rinsed in Hanks’ Balanced Salt Solution (HBSS) with 10% calf serum. Samples were continuously illuminated and viewed on a fluorescence microscope using an Omega Optical fluorescein longpass optical filter set and Image-1 software (Universal Imaging Corp.). Images were acquired 0, 10, 30 and 60 seconds
after the start of illumination (as indicated in the top left-hand corner of each panel) and clearly demonstrate the superior photostability of MitoFluor Green.
Figure 12.18 Mitochondria of live bovine pulmonary artery endothelial cells stained with MitoFluor
Red 589 (M-22424).
TECHNICAL NOTE
Estimating Mitochondrial Mass
Accurate measurements of mitchondrial mass require a probe that will accumulate
in mitochondria regardless of the mitochondrial membrane potential, a property displayed by several of our MitoTracker and MitoFluor dyes — MitoFluor Green (M-7502),
MitoFluor Red 589 (M-22424), MitoTracker Red 580 (M-22425) and MitoTracker Deep
Red 633 (M-22426). Mitochondrial uptake of nonyl acridine orange (NAO, A-1372) and
MitoTracker Green FM (M-7514) has also been reported to be independent of mitochondrial membrane potential,1,2 although studies have shown that this may not true for all
cell types.3,4 Mitochondrial staining by
nonyl acridine orange is attributed to
binding of cardiolipin in the inner
mitochondrial membrane,5 suggesting
that the observed fluorescence signal
may not correlate with the “mass” of
the entire mitochondria but instead may
measure the amount of inner membrane present. Indeed, one study has
shown that mitochondrial mass can
increase while the mitochondrial volume remains unchanged.6
Mitochondrial mass varies with cell
type and can be useful is separating
populations of healthy cells (see FigPeripheral blood mononuclear cells (PBMCs)
ure). The mitochondrial mass a cell
stained with an R-phycoerythrin–labeled anti-CD3
does change with time, as demonstratantibody (A-21333) and the MitoTracker Green
ed by the loss of mitochondrial mass in
+
dye (M-7514). For comparison, CD3 PBMCs
studies of aging cells.7 Changes in
without MitoTracker Green staining are shown on
mitochondrial
mass are also observed
the same plot (lower right population). This figure
in
apoptotic
cells,
but are not necessardemonstates that the mitochondrial mass of
ily associated with the loss of mitoPBMCs is approximately the same for CD3+ and
CD3- individuals.
chondrial membrane potential.6,8
References
1. Histochemistry 82, 51 (1985); 2. Immunol Lett 61, 157 (1998); 3. Basic Appl Histochem 33, 71
(1989); 4. Cytometry 39, 203 (2000); 5. Eur J Biochem 209, 267 (1992); 6. Proc Natl Acad Sci U S A
98, 9505 (2001); 7. Mech Ageing Dev 77, 83 (1994); 8. Exp Cell Res 221, 281 (1995).
Figure 12.19 Viable bovine pulmonary artery endothelial cells incubated simultaneously with the
red-fluorescent mitochondrial stain MitoFluor Red
594 (M-22422) and the blue-fluorescent nuclear
stain Hoechst 33342 (H-1399, H-3570, H-21492).
The image is a composite of two micrographs, using appropriate filter sets.
Figure 12.20 Live bovine pulmonary artery endothelial cells were incubated simultaneously with
MitoFluor Far Red 680 (M-22423), LysoTracker
Green DND-26 (L-7526) and Hoechst 33342
(H-1399, H-3570, H-21492). MitoFluor Far Red 680
accumulated in mitochondria, and its fluorescence
(emission maximum ~700 nm) was pseudocolored
red. Green-fluorescent LysoTracker Green dye accumulated in the lysosomes, and blue-fluorescent Hoechst 33342 dye stained the nuclei.
Section 12.2
477
vantageous when working with tissue, blood and other biological fluids that give rise to high absorbance or autofluorescence
at shorter wavelengths.44
RedoxSensor Red CC-1 Stain
RedoxSensor Red CC-1 (R-14060) stain is a unique probe
whose fluorescence localization appears to be based on a cell’s
cytosolic redox potential. Scientists at Molecular Probes have
found that RedoxSensor Red CC-1 stain passively enters live
cells. Once inside, this nonfluorescent probe is either oxidized
in the cytosol to a red-fluorescent product (excitation/emission
maxima ~540/600 nm), which then accumulates in the mitochondria, or is 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, staining of mitochondria predominates; whereas in contact-inhibited cells the
staining is primarily lysosomal. The best method we have found
to quantitate the distribution of the oxidized product is to use the
mitochondrion-selective MitoTracker Green FM stain (M-7514)
in conjunction with the RedoxSensor Red CC-1 stain.
JC-1 and JC-9: Dual-Emission
Potential-Sensitive Probes
The green-fluorescent JC-1 probe (5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide, T-3168;
Figure 23.13) 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 redfluorescent “J-aggregates” that exhibit a broad excitation spectrum and an emission maximum at ~590 nm (Figure 12.21, Figure 12.22, Figure 23.14, Figure 23.15). Thus, the emission of this
Figure 12.21 Potential-dependent staining of mitochondria in CCL64 fibroblasts by JC-1 (T-3168). The mitochondria were visualized by epifluorescence
microscopy using a 520 nm longpass optical filter. Regions of high mitochondrial polarization are indicated by red fluorescence due to J-aggregate formation by the concentrated dye. Depolarized regions are indicated by the green
fluorescence of the JC-1 monomers. Image contributed by Lan Bo Chen, Dana
Farber Cancer Institute, Harvard Medical School.
478
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 ~510/527 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 (Table 24.8)
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
23.16). 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 (L-7011, Section 15.3) to permit simultaneous
assessment of cellular integrity and mitochondrial function by flow
cytometry.55 We have discovered another mitochondrial marker,
JC-9 (3,3′-dimethyl-β-naphthoxazolium iodide, D-22421; Figure
Figure 12.22 NIH 3T3 fibroblasts stained with JC-1 (T-3168), showing the progressive loss of red J-aggregate fluorescence and cytoplasmic diffusion of
green monomer fluorescence following exposure to hydrogen peroxide. Images
show the same field of cells viewed before H2O2 treatment and 5, 10 and 20
minutes after treatment. The images were contributed by Ildo Nicoletti, Perugia
University Medical School.
Chapter 12 — Probes for Organelles
www.probes.com
23.18), with a very different chemical structure (Figure 23.17) but
similar potential-dependent spectroscopic properties. However, the
green fluorescence of JC-9 is essentially invariant with membrane
potential while the red fluorescence is significantly increased at
hyperpolarized membrane potentials.
Mitochondrion-Selective Rhodamines and
Rosamines
Rhodamine 123
Rhodamine 123 (R-302; FluoroPure Grade, R-22420; Figure
12.23) is a cell-permeant, cationic, fluorescent dye that is readily
sequestered by active mitochondria without inducing cytotoxic
effects.56 Uptake and equilibration of rhodamine 123 is rapid (a
few minutes) compared to dyes such as DASPMI, which may
take 30 minutes or longer.13 Viewed through a fluorescein longpass optical filter (Table 24.8), 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,57 live bacteria,58,59 plants 60,61 and
human spermatozoa.62 Using flow cytometry, researchers employed rhodamine 123 to sort respiratory-deficient yeast cells 63,64
and to isolate those lymphocytes that are responsive to mitogen
stimulation.65 Rhodamine 123 has also been used to study:
Apoptosis 52,66
Axoplasmic transport of mitochondria 67
Bacterial viability and vitality 58
Mitochondrial enzymatic activities 68,69
Mitochondrial transmembrane potential and other membrane
activities 14,60,70–73
• Multidrug resistance 74–81 (Section 15.6)
• Mycobacterial drug susceptibility 82,83
• Oocyte maturation 84
•
•
•
•
•
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 85 (>24 hours), making rhodamine 123 a potential anticancer agent for photodynamic therapy.86–91 Rhodamine 123 is
Figure 12.23 R-302 rhodamine 123.
known to be preferentially taken up and retained by mitochondria
of carcinoma cells 92 and to inhibit their proliferation; 93,94 cardiac
muscle cells also retain rhodamine 123 for days.95
Tetrabromorhodamine 123
The brominated analog of rhodamine 123, tetrabromorhodamine 123 (T-7539), is also potentially useful for photodynamic
therapy. Rhodamine 123 is a relatively weak phototoxin 90 with
a quantum yield for singlet oxygen (1O2) generation of <0.01.
Continuous illumination of rhodamine 123–stained cells causes
mitochondrial fragmentation,96 possibly due to this release of
activated oxygen. Tetrabromorhodamine 123 is a much more
effective 1O2 generator,90 with a quantum yield of 0.7. Although
the dye is not localized strictly to mitochondria in cells, tetrabromorhodamine 123 is highly phototoxic to carcinoma cells 90 and
its photoproduct is well retained. Tetrabromorhodamine 123 does
not stain the nucleus in live cells; however, it binds to DNA in
solution, where it has been used as a probe for DNA internal
flexibility on the microsecond time scale.97
Rosamines and Other Rhodamine Derivatives, Including TMRM
and TMRE
Other mitochondrion-selective dyes include tetramethylrosamine (T-639, Figure 12.24), whose fluorescence contrasts
well with that of fluorescein for multicolor applications, and
rhodamine 6G 89,98–100 (R-634, Figure 12.25), 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 74 (Section
15.6). Rhodamine 6G has been employed to study microvascular
reperfusion injury 101 and the stimulation and inhibition of
F1-ATPase from the thermophilic bacterium PS3.102
At low concentrations, certain lipophilic rhodamine dyes
selectively stain mitochondria in live cells.103 Molecular Probes’
researchers have observed that low concentrations of the hexyl
ester of rhodamine B (R 6, R-648) accumulate selectively in
mitochondria and appear to be relatively nontoxic. We have
included this probe in our Yeast Mitochondrial Stain Sampler Kit
(Y-7530, see below for description). At higher concentrations,
rhodamine B hexyl ester and rhodamine 6G stain the endoplasmic
reticulum of animal cells 103 (Section 12.4).
The accumulation of tetramethylrhodamine methyl and ethyl
esters (TMRM, T-668; TMRE, T-669) in mitochondria and the
endoplasmic reticulum has also been shown to be driven by their
Figure 12.24 T-639 tetramethylrosamine chloride.
Figure 12.25 R-634 rhodamine 6G chloride.
Section 12.2
479
membrane potential 104,105 (Section 23.3). 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.106 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.107–109 TMRM and TMRE have been used to
measure mitochondrial depolarization related to cytosolic Ca2+
transients 110 and to image time-dependent mitochondrial membrane potentials.108 A high-throughput assay utilizes TMRE and
our low-affinity Ca2+ indicator fluo-5N AM (F-14204, Section
20.3) to screen inhibitors of the opening of the mitochondrial
transition pore.111 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.70
Reduced Rhodamines and Rosamines
Inside live cells, the colorless dihydrorhodamines and dihydrotetramethylrosamine are oxidized to fluorescent products that
stain mitochondria.112 However, the oxidation may occur in organelles other than the mitochondria. Dihydrorhodamine 123
(D-632, D-23806; Figure 12.26) reacts with hydrogen peroxide in
the presence of peroxidases,113 iron or cytochrome c 114 to form
rhodamine 123. This reduced rhodamine has been used to monitor reactive oxygen intermediates in rat mast cells 115 and to measure hydrogen peroxide in endothelial cells.114 Dihydrorhodamine
6G (D-633, Figure 12.27) is another reduced rhodamine that has
been shown to be taken up and oxidized by live cells.116–118
Chloromethyl derivatives of reduced rosamines (MitoTracker
Orange CM-H2TMRos, M-7511; MitoTracker Red CM-H2XRos,
M-7513), 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 (R-1244, R-1245; Section 20.3) has
Figure 12.26 D-632 dihydrorhodamine 123.
Figure 12.27 D-633 dihydrorhodamine 6G.
480
been extensively used to measure the relatively slow changes in
intramitochondrial Ca2+ (Figure 20.28, Figure 20.34).
Other Mitochondrion-Selective Probes
Carbocyanines
Most carbocyanine dyes with short (C1–C6) alkyl chains
(Section 23.3) 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) (D-273) stains mitochondria in live yeast 21,119–121 and
other eukaryotic cells,100,122 as well as sarcoplasmic reticulum in
beating heart cells.123 It has also been used to demonstrate mitochondria moving along microtubules.23 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.124 Several other potential-sensitive carbocyanine probes described in Section 23.3 also stain mitochondria in
live cultured cells.100
The carbocyanine DiOC7(3) (D-378), 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.125
Its other uses include:
• Distinguishing cycling and noncycling fibroblasts 126 and
viable and nonviable bacteria 127
• Following the reorganization of the endoplasmic reticulum
during fertilization in the ascidian egg 128
• Identifying functional vasculature in murine tumors 129,130
• Studying multidrug resistance 131 (Section 15.6)
• Visualizing the detailed morphology of neurites of Alzheimer’s disease neurons 132
Styryl Dyes
The styryl dyes DASPMI (4-Di-1-ASP, D-288) and DASPEI
(D-426) can be used to stain mitochondria in live cells.13 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.133 DASPMI
and DASPEI have been shown to be useful for:
• Determining the distribution of mitochondria in yeast mutants 63
• Long-term imaging of live mammalian nerve cells and their
connections 134–136
• Monitoring the metabolic state of Pneumocystis carinii mitochondria 31
• Screening aberrant mitochondrial distribution and morphology
in yeast 137
Nonyl Acridine Orange
Nonyl acridine orange (A-1372) is well retained in the mitochondria of live HeLa cells for up to ten days, making it a useful
probe for following mitochondria during isolation and after cell
fusion.138–140 The mitochondrial uptake of this metachromatic dye
is reported not to depend on membrane potential. It is toxic at
Chapter 12 — Probes for Organelles
www.probes.com
high concentrations 141 and apparently binds to cardiolipin in all
mitochondria, regardless of their energetic state.142–145 This derivative has been used to analyze mitochondria by flow cytometry,146
to characterize multidrug resistance 147 (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 (C-1271, C-1272; Section 21.2), followed by 4
hours of incubation at room temperature leads to highly selective
localization of the carboxy SNARF-1 dye in mitochondria (Figure 21.15), where it responds to changes in mitochondrial pH.148
CoroNa Red Chloride
As shown by colocalization with MitoTracker Green FM, our
newest Na+ indicator, CoroNa Red chloride (C-24430, C-24431;
Section 22.1), spontaneously localizes in the mitochondria (Figure 22.11) and may be useful for measuring intramitochondrial
Na+ transients.
Lucigenin
The well-known chemiluminescent probe lucigenin (L-6868)
accumulates in mitochondria of alveolar macrophages.149 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.149 Lucigenin from Molecular
Probes has been highly purified to remove a bright blue-fluorescent contaminant that is found in some commercial samples.
Figure 12.28 The intermediate filaments in bovine pulmonary artery endothelial
cells, localized using our anti-desmin antibody (A-21283), which was visualized
with the Alexa Fluor 647 goat anti–mouse IgG antibody (A-21235). Endogenous biotin in the mitochondria was labeled with Alexa Fluor 546 streptavidin
(S-11225) and DNA in the cell was stained with blue-fluorescent DAPI (D-1306,
D-3571, D-21490).
Yeast Mitochondrial Stain Sampler Kit
Fluorescence microscopy has been extensively used to study
yeast.21,121 Molecular Probes offers a Yeast Mitochondrial Stain
Sampler Kit (Y-7530). 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 64,150–152
Rhodamine B hexyl ester 103
MitoTracker Green FM
SYTO 18 yeast mitochondrial stain 153
DiOC6(3) 21,120,121,154–160
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 (S-7529).
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; 161 consequently, mitochondria can be selectively stained
by almost any fluorophore- or enzyme-labeled avidin or streptavidin derivative (Section 7.6; Table 7.17; Figure 12.28, Figure 12.29)
without applying any biotinylated ligand. This staining, which can
complicate the use of avidin–biotin techniques in sensitive cellbased assays, can be blocked by the reagents in our Endogenous
Biotin Blocking Kit (E-21390, Section 7.6).
Figure 12.29 The cytoskeleton of a fixed and permeabilized bovine pulmonary
artery endothelial cell detected using mouse monoclonal anti–α-tubulin antibody (A-11126), visualized with Alexa Fluor 647 goat anti–mouse IgG antibody
(A-21235) and pseudocolored magenta. Endogenous biotin in the mitochondria
was labeled with green-fluorescent Alexa Fluor 488 streptavidin (S-11223) and
DNA was stained with blue-fluorescent DAPI (D-1306, D-3571, D-21490).
Section 12.2
481
Antibodies to Mitochondrial Proteins
Figure 12.30 Mitochondrial DNA labeled with DAPI
(D-1306, D-3571, D-21490) and colocalized with
MitoTracker Red (M-7512) in a bovine pulmonary
artery endothelial cell. MitoTracker Red was incubated with live cells, followed by fixation with 4%
formaldehyde. Staining of the mitochondrial DNA
by DAPI was performed after fixation.
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; Figure 12.30) 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 as yet 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.162–167
Antibodies against the various subunits of the OxPhos Complex are important tools
for investigating mitochondrial biogenesis and studying OxPhos-related diseases (see
Mitochondria in Diseases). Patient cell lines can now be screened for deficiencies in each
of the OxPhos Complexes by simple Western blotting.168,169 When compared to 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.170
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 171 and
for screening Complex IV–deficient patients.172
Molecular Probes offers a range of subunit-specific anti–OxPhos Complex mouse
monoclonal antibodies that recognize proteins in the oxidative phosphorylation system
(Table 12.2, Table 12.3, Table 12.4; Figure 12.31). 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
Figure 12.31. Major protein complexes of the oxidative phosphorylation system and antibodies that recognize them.
482
Chapter 12 — Probes for Organelles
www.probes.com
TECHNICAL NOTE
Mitochondria in Diseases
Given the multiple functions and numerous proteins present
in the mitochondria, it is not surprising that genetically inherited
defects of mitochondrial function are a major cause of morbidity
and mortality in humans. In particular, there are several human
diseases that have known defects in the proteins responsible for
oxidative phosphorylation (OxPhos) in cells. Typically, such defects
produce lactic acidemia, exercise intolerance or neurological disorders. Diseases of OxPhos are notoriously difficult to diagnose,
and it is even more difficult to correlate their phenotype–genotype
relationships. A subset of OxPhos defects is maternally inherited.
These defects result from mutations in mitochondrial DNA (mtDNA), a small, 16-kilobase genome present in hundreds to thousands of copies per cell. mtDNA, which encodes 13 polypeptides
of the OxPhos machinery, differs from the nuclear genome in its
absence of histones, poor repair mechanisms and very limited
recombination frequencies. As a result, mtDNA in somatic cells
builds up mutations over time due to errors in replication that are
not repaired and physical insult from a variety of toxins. Such
accumulated mutations are implicated in a number of neurodegenerative diseases — notably Parkinson’s and Alzheimer’s diseases
— where the mutation load triggers premature apoptotic or necrotic cell death. For example, a strong link has been established
between exposure to the pesticide rotenone, a well-defined and
specific inhibitor of OxPhos, and Parkinson’s disease. mtDNA
mutations function by reducing energy production within the cell
and are thought to contribute to cancer and to aging. Likewise,
mutations in the nuclear-encoded subunits of OxPhos have been
found to regulate the life span in flies and worms. Many of the
products listed in this section are useful tools for studying degenerative conditions.
A
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 Table 12.3. 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. 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 (Section 7.3, Table 7.3). The IgG1 antibodies in this group (Table 12.3) can be complexed with the reagents in our Zenon One
Mouse IgG1 Labeling Kits (Section 7.2, Table 7.1) for labeling mitochondrial proteins in
Western blots (Figure 12.32) and cells (Figure 15.64).
Table 12.2 Monoclonal antibodies to yeast oxidative phosphorylation complex IV (COX).
B
Figure 12.32 Western blot analysis of bovine heart
mitochondrial lysate probed with antibodies against
components of OxPhos Complex III and OxPhos
Complex V. Antibodies against OxPhos Complex III
core 2 subunit (A-11143) and OxPhos Complex V
alpha subunit (A-21350) were labeled with the
Zenon One Alexa Fluor 488 Mouse IgG1 Labeling Kit
(Z-25002, green fluorescence) and the Zenon One
Alexa Fluor 647 Mouse IgG1 Labeling Kit (Z-25008,
far-red fluorescence, pseudocolored magenta),
respectively, before probing the blot. The lanes in
panel A are 1) MW marker, 2) 8 µg, 3) 4 µg,
4) 2 µg, 5) 1 µg, 6) 0.5 µg, 7) 0.25 µg, 8) 0.125 µg,
9) 60 ng and 10) 30 ng of the bovine heart mitochondrial extract. Panel (B) shows lanes 6 through
10 imaged with a longer exposure time. The molecular weight standards are Precision Prestained
Protein Standards (Bio-Rad), which display far-red
fluorescence similar to that of Alexa Fluor 647.
Section 12.2
483
Monoclonal Antibodies Specific for OxPhos Complex IV
(Cytochrome Oxidase)
To facilitate the study of cytochrome oxidase (COX) structure
and mitochondrial biogenesis, Molecular Probes offers subunitspecific 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.173,174
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 175–179 (Table 12.2, Table 12.3, Table
12.4). Furthermore, because the antibodies to bovine COX also
Table 12.3 Monoclonal antibodies specific for proteins in the oxidative phosphorylation system.
484
Chapter 12 — Probes for Organelles
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recognize the corresponding human COX subunits, the antibodies
have proven valuable for analyzing human mitochondrial myopathies and related disorders.168,171,180,181 Alexa Fluor 488 and Alexa
Fluor 594 conjugates of anti–cytochrome oxidase subunit I are
also available for direct staining of mitochondria (A-21296,
A-21297; Figure 7.69).
Monoclonal Antibodies Specific for Complexes I, II, III and V
Molecular Probes supplies a large number of monoclonal
antibodies to the OxPhos Complex (Table 12.3, Figure 12.32).
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 anti–OxPhos Complex V subunit (bovine), mouse monoclonal 7H10 (anti–F1F0-ATPase subunit α, A-21350) and anti–
OxPhos Complex V subunit (bovine), mouse monoclonal 3D5
(anti–F1F0-ATPase subunit β, A-21299) antibodies have also been
shown to mimic angiostatin, a potent inhibitor of angiogenesis.182
Angiostatin protein (A-21351, 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 antibod-
Table 12.4 Molecular characteristics of components of the oxidative phosphorylation system.
Section 12.2
485
ies had similar inhibitory effects, implying that they also compromised ATP metabolism and may function as angiostatin analogs.
Antibodies Against Other Mitochondrial Proteins
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.183 This abundant transmembrane protein
forms a small pore (~3 nm) in the outer membrane, allowing
molecules less than ~10 kD to pass.184 Due to its abundance,
porin is often used as a standardization marker in Western blots
when assaying for other mitochondrial proteins 170,185 and serves
as an effective organelle marker for immunohistochemistry.119
Monoclonal antibodies against both human and yeast porin are
available from Molecular Probes (A-21317, A-6449; Table 12.5).
Pyruvate Dehydrogenase
Molecular Probes has available a series of antibodies against
the human pyruvate dehydrogenase (PDH) complex (Table 12.5),
a large, multienzyme assembly residing in the mitochondrial
matrix and consisting of three catalytic activities: pyruvate dehydrogenase, dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase (diaphorase).186 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; 187 severe cases
can lead to developmental defects such as congenital brain malformation.188
Mitochondrial Protein Extracts
For researchers seeking a source of mitochondrial protein
standards, Molecular Probes offers human heart mitochondrial
proteins for SDS-polyacrylamide gel electrophoresis (M-22430)
and 2-D gel electrophoresis (M-22431, Section 12.2). These
products are complete mitochondrial lysates that have tested
negative for hepatitis B and C, as well as HIV 1 and 2 in serology
tests. These mitochondrial protein extracts are useful for comparing new mitochondrial protein preparations in either 1-D or 2-D
gels and for testing mitochondrial antibodies.
Table 12.5 Monoclonal antibodies specific for mitochondrial proteins not associated with the oxidative phosphorylation system.
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Data Table — 12.2 Probes for Mitochondria
Cat #
A-1372
D-273
D-288
D-378
D-426
D-632
D-633
D-22421
D-23806
L-6868
M-7502
M-7510
M-7511
M-7512
M-7513
M-7514
M-22422
M-22423
M-22424
M-22425
M-22426
R-302
R-634
R-648
R-14060
R-22420
S-7529
T-639
T-668
T-669
T-3168
T-7539
MW
472.51
572.53
366.24
600.58
380.27
346.38
444.57
532.38
346.38
510.50
602.99
427.37
392.93
531.52
497.08
671.88
647.10
495.96
~550
724.00
543.58
380.83
479.02
627.18
434.41
380.83
~450
378.90
500.93
514.96
652.23
740.87
Storage
L
D,L
L
D,L
L
F,D,L,AA
F,D,L,AA
D,L
F,D,L,AA
L
L
F,D,L
F,D,L,AA
F,D,L
F,D,L,AA
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L
F,D,L,AA
F,D,L
F,D,L
L
F,D,L
F,D,L
D,L
F,D,L
Soluble
DMSO, EtOH
DMSO
DMF
DMSO
DMF
DMF, DMSO
DMF, DMSO
DMSO, DMF
DMSO
H2O
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
MeOH, DMF
EtOH
DMF, DMSO
DMSO
MeOH, DMF
DMSO
DMF, DMSO
DMSO, MeOH
DMSO, EtOH
DMSO, DMF
MeOH, DMF
Abs
495
484
475
482
461
289
296
522
289
455
489
551
235
578
245
490
604
682
588
588
640
507
528
556
239
507
483
550
549
549
514
524
EC
84,000
154,000
45,000
148,000
39,000
7,100
11,000
143,000
7,100
7,400
112,000
102,000
57,000
116,000
45,000
119,000
97,000
95,000
105,000
81,000
194,000
101,000
105,000
123,000
52,000
101,000
64,000
87,000
115,000
109,000
195,000
91,000
Em
519
501
605
504
589
none
none
535
none
505
517
576
none
599
none
516
637
702
622
645
662
529
551
578
none
529
none
574
573
574
529
550
Solvent
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
CHCl3
MeOH
H2O
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
EtOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
pH 7
MeOH
MeOH
MeOH
MeOH
MeOH
Notes
1
1
2, 3
2, 3
4
3, 5
6, 7
2, 3
2, 3
8
2, 9
10
8, 11, 12
13
For definitions of the contents of this data table, see “How to Use This Book” on page viii.
Notes
1. Abs and Em of styryl dyes are at shorter wavelengths in membrane environments than in reference solvents such as methanol. The difference is typically 20 nm for absorption and
80 nm for emission, but varies considerably from one dye to another.
2. This compound is susceptible to oxidation, especially in solution. Store solutions under argon or nitrogen. Oxidation appears to be catalyzed by illumination.
Section 12.2
487
Data Table — 12.2 Probes for Mitochondria — continued
3. These compounds are essentially colorless and nonfluorescent until oxidized. Oxidation products (in parentheses) are as follows: D-632 and D-23806 (R-302); D-633 (R-634);
M-7511 (M-7510); M-7513 (M-7512).
4. JC-9 exhibits long-wavelength J-aggregate emission at ~635 nm in aqueous solutions and polarized mitochondria.
5. This product is supplied as a ready-made solution in DMSO with sodium borohydride added to inhibit oxidation.
6. L-6868 has much stronger absorption at shorter wavelengths (Abs = 368 nm (EC = 36,000 cm-1M-1)).
7. This compound emits chemiluminescence (Em = 470 nm) upon oxidation in basic aqueous solutions.
8. MW: The preceding ~ symbol indicates an approximate value, not including counterions.
9. R-14060 is colorless and nonfluorescent until oxidized. The spectral characteristics of the oxidation product (2,3,4,5,6-pentafluorotetramethylrosamine) are similar to those of T-639.
10. This product is specified to equal or exceed 98% analytical purity by HPLC.
11. This product is supplied as a ready-made solution in the solvent indicated under Soluble.
12. S-7529 is fluorescent when bound to DNA (Abs = 490 nm, Em = 507 nm).
13. JC-1 forms J-aggregates with Abs/Em = 585/590 nm at concentrations above 0.1 µM in aqueous solutions (pH 8.0) (Biochemistry 30, 4480 (1991)).
Product List — 12.2 Probes for Mitochondria
Monoclonal antibody products are listed in Tables 12.2, 12.3 and 12.5.
Cat #
Product Name
A-1372
A-21296
acridine orange 10-nonyl bromide (nonyl acridine orange) ..................................................................................................................................
anti-OxPhos Complex IV subunit I, mouse IgG2a, monoclonal 1D6, Alexa Fluor® 488 conjugate
(anti-cytochrome oxidase subunit I, Alexa Fluor® 488 conjugate) *1 mg/mL* *human reactivity* .....................................................................
anti-OxPhos Complex IV subunit I, mouse IgG2a, monoclonal 1D6, Alexa Fluor® 594 conjugate
(anti-cytochrome oxidase subunit I, Alexa Fluor® 594 conjugate) *1 mg/mL* *human reactivity* .....................................................................
3,3′-diheptyloxacarbocyanine iodide (DiOC7(3)) ...................................................................................................................................................
3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)) ....................................................................................................................................................
dihydrorhodamine 123 .........................................................................................................................................................................................
dihydrorhodamine 123 *5 mM stabilized solution in DMSO* ..............................................................................................................................
dihydrorhodamine 6G ...........................................................................................................................................................................................
2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI) .........................................................................................................................
4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) ................................................................................................................
3,3′-dimethyl-α-naphthoxacarbocyanine iodide (JC-9; DiNOC1(3)) ......................................................................................................................
Endogenous Biotin-Blocking Kit *100 assays* .....................................................................................................................................................
lucigenin (bis-N-methylacridinium nitrate) *high purity* .....................................................................................................................................
mitochondrial proteins (human heart) for 2-D gel electrophoresis *5 mg/mL* ...................................................................................................
mitochondrial proteins (human heart) for SDS-polyacrylamide gel electrophoresis *2 mg/mL* .........................................................................
MitoFluor™ Far Red 680 .......................................................................................................................................................................................
MitoFluor™ Green .................................................................................................................................................................................................
MitoFluor™ Red 589 .............................................................................................................................................................................................
MitoFluor™ Red 594 .............................................................................................................................................................................................
MitoTracker® Deep Red 633 *special packaging* ................................................................................................................................................
MitoTracker® Green FM *special packaging* .......................................................................................................................................................
MitoTracker® Orange CM-H2TMRos *special packaging* ....................................................................................................................................
MitoTracker® Orange CMTMRos *special packaging* .........................................................................................................................................
MitoTracker® Red 580 *special packaging* .........................................................................................................................................................
MitoTracker® Red CM-H2XRos *special packaging* ............................................................................................................................................
MitoTracker® Red CMXRos *special packaging* .................................................................................................................................................
RedoxSensor™ Red CC-1 *special packaging* ....................................................................................................................................................
rhodamine 123 .....................................................................................................................................................................................................
rhodamine 123 *FluoroPure™ grade* ..................................................................................................................................................................
rhodamine 6G chloride .........................................................................................................................................................................................
rhodamine B, hexyl ester, perchlorate (R 6) .........................................................................................................................................................
SYTO® 18 yeast mitochondrial stain *5 mM solution in DMSO* ..........................................................................................................................
2′,4′,5′,7′-tetrabromorhodamine 123 bromide .....................................................................................................................................................
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; CBIC2(3)) ..............................................................................
tetramethylrhodamine, ethyl ester, perchlorate (TMRE) .......................................................................................................................................
tetramethylrhodamine, methyl ester, perchlorate (TMRM) ...................................................................................................................................
tetramethylrosamine chloride ...............................................................................................................................................................................
Yeast Mitochondrial Stain Sampler Kit .................................................................................................................................................................
A-21297
D-378
D-273
D-632
D-23806
D-633
D-426
D-288
D-22421
E-21390
L-6868
M-22431
M-22430
M-22423
M-7502
M-22424
M-22422
M-22426
M-7514
M-7511
M-7510
M-22425
M-7513
M-7512
R-14060
R-302
R-22420
R-634
R-648
S-7529
T-7539
T-3168
T-669
T-668
T-639
Y-7530
Unit Size
100 mg
100 µL
100 µL
100 mg
100 mg
10 mg
1 mL
25 mg
1g
1g
5 mg
1 kit
10 mg
100 µL
100 µL
10 mg
1 mg
1 mg
1 mg
20 x 50 µg
20 x 50 µg
20 x 50 µg
20 x 50 µg
20 x 50 µg
20 x 50 µg
20 x 50 µg
10 x 50 µg
25 mg
25 mg
1g
10 mg
250 µL
5 mg
5 mg
25 mg
25 mg
25 mg
1 kit
The full citations and, in most cases, links to PubMed for all references in this
Handbook are available at our Web site (www.probes.com/search).
488
Chapter 12 — Probes for Organelles
www.probes.com