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Molecular Probes acidotropic reagents can be used to stain lysosomes and yeast vacuoles, as well as several other types of acidic compartments such as trans-Golgi vesicles, endosomes and subpopulations of coated vesicles in fibroblasts, secretory vesicles in insulin-secreting pancreatic β-cells, acrosomes of spermatozoa and plant vacuoles. Lysosomes contain glycosidases, acid phosphatases, elastase, cathepsins, carboxypeptidases and a variety of other proteases. Enzyme Substrates and Assays—Chapter 10 describes a number of substrates for detecting the activity of these hydrolytic enzymes. An excellent compendium of human diseases that affect intracellular transport processes through lysosomes, Golgi and endoplasmic reticulum (ER) has been published.
Like lysosomes, peroxisomes are single membrane–bound vesicles that contain digestive enzymes. The chief function of these basic organelles is to enzymatically oxidize fatty acids and to subsequently catalyze the breakdown of H2O2, a by-product of fatty acid degradation. Recently, interest in peroxisomes has increased, especially studies related to peroxisomal origin and maintenance. Morphological abnormalities in peroxisomes related to disease states and diet have also been the subject of current research. The SelectFX Alexa Fluor 488 Peroxisome Labeling Kit (S34201), described below, provides an antibody-based method for labeling peroxisomes in fixed cells.
CellLight reagents combine the utility and selectivity of targeted fluorescent proteins with the efficiency of the BacMam gene delivery and expression technology. These reagents incorporate all the customary advantages of BacMam technology, including high efficiency transduction of mammalian cells and long-lasting, titratable expression ( BacMam Gene Delivery and Expression Technology—Note 11.1). CellLight reagents are provided in a ready-to-use format—simply add, incubate and image—with highly efficient expression in cell lines, primary cells, stem cells and neurons. A complete list of CellLight reagents and their targeting sequences can be found in CellLight reagents and their targeting sequences—Table 11.1.
CellLight Lysosomes-GFP (C10596) and (C10597, Figure 12.3.1) are BacMam expression vectors encoding fusions of Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP) with the targeting sequence from Lamp1 (lysosomal-associated membrane protein 1). These CellLight reagents generate lysosomally localized fluorescent labeling in live cells that is retained after fixation and permeabilization procedures—procedures that will dissipate LysoTracker Red DND-99 staining. The titratable expression capacity of BacMam vectors is a particularly useful feature in the context of the Lamp1–GFP fusion, as high levels of overexpression have sometimes been found to induce aberrant aggregation of late-endocytic organelles.
CellLight Early Endosomes–GFP (C10586, Figure 12.3.2) and CellLight Early Endosomes–RFP (C10587) reagents provide BacMam expression vectors encoding fusions of GFP or RFP with the small GTPase Rab5a. Rab5a fusions with autofluorescent proteins are sensitive and precise early endosome markers for real-time imaging of endosomal transport along microtublues (Figure 12.3.3) and of clathrin-mediated endocytosis in live cells.
CellLight Peroxisome-GFP (C10604, Figure 12.3.4) is a BacMam expression vector encoding GFP C-terminally linked to a peroxisomal targeting sequence (GFP–PTS1). Live-cell imaging with the GFP–PTS1 fusion has provided many insights into normal and pathologically abnormal biogenesis and degradation of peroxisomes and the controlling influence of peroxisome proliferator–activated receptors (PPARs).
Figure 12.3.3 Endosomal movement along microtubules. U2OS cells were transduced with CellLight® Early Endosome–GFP (C10586) and CellLight® MAP4-RFP (C10599) reagents. Images were taken every minute for a period of 30 min. Nuclei were counterstained with 1 µg/mL Hoechst 33342 nucleic acid stain.
Weakly basic amines selectively accumulate in cellular compartments with low internal pH and can be used to investigate the biosynthesis and pathogenesis of lysosomes. One frequently used probe for acidic organelles, DAMP (D1552), is not fluorescent and therefore must be used in conjunction with anti-DNP antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4) directly or indirectly conjugated to a fluorophore or enzyme in order to visualize the staining pattern. The fluorescent probes neutral red (N3246) and acridine orange (A1301, A3568) are also commonly used for staining acidic organelles, though they lack specificity.
These limitations have motivated us to search for alternative acidic organelle–selective probes, both for short-term and long-term tracking studies. The LysoTracker probes are fluorescent acidotropic probes for labeling and tracing acidic organelles in live cells. These probes have several important features, including high selectivity for acidic organelles and effective labeling of live cells at nanomolar concentrations. Furthermore, the LysoTracker probes are available in several fluorescent colors ( Summary of the LysoTracker and LysoSensor probes—Table 12.3, Figure 12.3.5), making them especially suitable for multicolor applications.
The LysoTracker probes, which comprise a fluorophore linked to a weak base that is only partially protonated at neutral pH, are freely permeant to cell membranes and typically concentrate in spherical organelles (). We have found that the fluorescent LysoTracker probes must be used at very low concentrations—usually about 50 nM—to achieve optimal selectivity. Their mechanism of retention has not been firmly established but is likely to involve protonation and retention in the organelles' membranes, although staining is generally not reversed by subsequent treatment of the cells with weakly basic cell-permeant compounds. Unfortunately, these lysosomal probes can exhibit an alkalinizing effect on the lysosomes, such that longer incubation with these probes can induce an increase in lysosomal pH. Therefore, we recommend incubating cells with these probes for only one to five minutes before imaging.
The larger acidic compartments of cells stained with LysoTracker Red DND-99 (L7528; , ) usually retain their staining pattern following fixation with aldehydes. Simultaneous staining of lysosomes by two LysoTracker dyes—LysoTracker Yellow HCK-123 (L12491) and LysoTracker Red DND-99 (L7528)—yields identical staining patterns when viewed through either the bandpass filter set appropriate for fluorescein or a longpass filter set appropriate for rhodamine (). The LysoTracker probes were principally developed for fluorescence microscopy applications. The lysosomal fluorescence in LysoTracker dye–stained cells may constitute only a portion of total cellular fluorescence due to cellular autofluorescence or nonspecific staining. Consequently, successful application of these probes for quantitating the number of lysosomes by flow cytometry or fluorometry will likely depend on the particular cell lines and staining protocols used.
LysoTracker Green DND-26 (L7526) was used to identify acidic compartments in a study of a membrane protein that facilitates vesicular sequestration of zinc, to visualize acidic organelles labeled with rhodamine B in denervated skeletal muscle and to assess acrosomal integrity in cryopreserved bovine spermatozoa. This LysoTracker probe also proved useful in a continuous assay for the secretion of pulmonary surfactant by exocytosis of lamellar bodies. LysoTracker Red DND-99 provided researchers with a probe for examining lysosome damage in Trypanosoma brucei after specific uptake of cytokine tumor necrosis factor-α, for studying apoptosis in organogenesis-stage mouse embryos and for determining the subcellular localization of receptor and channel proteins.
Image-iT LIVE Lysosomal and Nuclear Labeling Kit
The Image-iT LIVE Lysosomal and Nuclear Labeling Kit (I34202) provides two stains—red-fluorescent LysoTracker Red DND-99 dye (excitation/emission maxima ~577/590 nm) and blue-fluorescent Hoechst 33342 dye (excitation/emission maxima when bound to DNA ~350/461 nm)—for highly selective staining of lysosomes and the nucleus, respectively, in live, green-fluorescent protein (GFP)–transfected cells (). When used according to the sample protocol, cell-permeant LysoTracker Red DND-99 dye provides highly selective lysosomal staining with minimal background. A significant amount of specific staining is retained after formaldehyde fixation, although some cytoplasmic background staining may be seen. 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. This dye does not interfere with GFP fluorescence and is retained after fixation and permeabilization. It is not recommended that the dyes be combined into one staining solution; they should instead be used in separate labeling steps, with Hoechst 33342 staining first.
The Image-iT LIVE Lysosomal and Nuclear Labeling Kit contains:
- LysoTracker Red DND-99 dye
- Hoechst 33342 dye
- Labeling protocols (Image-iT LIVE Lysosomal 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.
For researchers studying the dynamic aspects of lysosome biogenesis and function in live cells, we have developed the LysoSensor probes—fluorescent pH indicators that partition into acidic organelles. The LysoSensor dyes are acidotropic probes that appear to accumulate in acidic organelles as the result of protonation. This protonation also relieves the fluorescence quenching of the dye by its weakly basic side chain, resulting in an increase in fluorescence intensity. Thus, the LysoSensor reagents exhibit a pH-dependent increase in fluorescence intensity upon acidification, in contrast to the LysoTracker probes, which exhibit fluorescence that is not substantially enhanced at acidic pH.
We offer four LysoSensor reagents that differ in color and pKa ( Summary of the LysoTracker and LysoSensor probes—Table 12.3). Because these probes may localize in the membranes of organelles, it is probable that the pKa values listed in Summary of the LysoTracker and LysoSensor probes—Table 12.3 will not be equivalent to those measured in cellular environments and that only qualitative and semiquantitative comparisons of organelle pH will be possible. The green-fluorescent LysoSensor probes are available with optimal pH sensitivity in either the acidic or neutral range (pKa ~5.2 or ~7.5 in aqueous buffers). With their low pKa values, LysoSensor Blue DND-167 (L7533) and LysoSensor Green DND-189 (L7535) are almost nonfluorescent except when inside acidic compartments, whereas LysoSensor Green DND-153 (L7534) is brightly fluorescent at neutral pH. LysoSensor Yellow/Blue DND-160 (PDMPO, L7545) is unique in that it exhibits both dual-excitation and dual-emission spectral peaks that are pH dependent (Figure 12.3.6, ) .
LysoSensor Yellow/Blue DND-160 exhibits predominantly yellow fluorescence in acidic organelles, and in less acidic organelles it exhibits blue fluorescence. Dual-emission measurements facilitate ratio imaging of the pH in acidic organelles such as lysosomes, myeloid leukemic cells and acidic vacuoles of plant cells. LysoSensor Yellow/Blue DND-160, frequently referred to by the acronym PDMPO, has been widely utilized as a tracer of silica deposition and transport in marine diatoms. Kinetic studies on the internalization of LysoSensor Yellow/Blue DND-160 indicate that the probe is taken up by live cells within seconds. Unfortunately, this lysosomal probe can exhibit an alkalinizing effect on the lysosomes, such that longer incubation with this probe can induce an increase in lysosomal pH. Therefore, it is a useful pH indicator only when incubation times are kept short; we recommend incubating cells for only one to five minutes before imaging.
The cell-permeant LysoSensor probes can be used singly or in combination to investigate the acidification of lysosomes and alterations of lysosomal function or trafficking that occur in cells. For example, lysosomes in some tumor cells have a lower pH than normal lysosomes, whereas other tumor cells contain lysosomes with higher pH. In addition, cystic fibrosis and other diseases result in defects in the acidification of some intracellular organelles, and the LysoSensor probes are useful in studying these aberrations. LysoSensor Green DND-189 has been used to selectively label acidic compartments within granule cell neurites and, along with LysoSensor Green DND-153, to examine the acidification of endosomes and lysosomes in a mutant CHO cell line. LysoSensor Yellow/Blue DND-160 was employed in a study that demonstrated the involvement of lysosomes in the acquired drug-resistance phenotype of a doxorubicin-selected variant of human U-937 myeloid leukemia cells.
As with the LysoTracker probes, the cell-permeant LysoSensor probes were originally developed for fluorescence microscopy applications. The lysosomal fluorescence in LysoSensor dye–stained cells may constitute only a portion of total cellular fluorescence due to cellular autofluorescence or nonspecific staining. Therefore, the successful application of these probes for quantitating the number of lysosomes or their pH by flow cytometry or fluorometry will likely depend on the particular cell lines and staining protocols used.
LysoSensor Yellow/Blue Dextran
We have prepared a 10,000 MW dextran conjugate of the LysoSensor Yellow/Blue dye (L22460). As this labeled dextran is taken up by the cells and moves through the endocytic pathway, the fluorescence of the LysoSensor dye changes from blue fluorescent in the near-neutral endosomes to longer-wavelength yellow fluorescent in the acidic lysosomes. The greatest change in fluorescence emission occurs near the pKa of the dye at pH ~3.9. Unlike the cell-permeant LysoSensor dyes, LysoSensor Yellow/Blue dextran allows measurement of pH in lysosomes using either fluorescence microscopy () or flow cytometry.
The reagent DAMP (N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl)methylamine, dihydrochloride; D1552; ) is a weakly basic amine that is taken up in acidic organelles of live cells. This cell-permeant acidotropic reagent can be detected with anti-DNP antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4), including those labeled with Alexa Fluor 488 dye, biotin, Qdot 655 nanocrystal or enzymes, making DAMP broadly applicable for detecting acidic organelles by electron and light microscopy. For example, DAMP has been used to investigate:
- Endocytic and secretory pathways
- Defective acidification of intracellular organelles in cells from cystic fibrosis patients
- Dependence on pH of the conversion of proinsulin to insulin in beta cells
- Development of autophagic vacuoles
- Location of intracellular acidic compartments during viral infection
As alternatives to DAMP, our cell-permeant fluorescent LysoTracker and LysoSensor probes described above have significant potential in many of these applications. Because they can be visualized directly without any secondary detection reagents, the LysoTracker and LysoSensor reagents enable researchers to study acidic organelles and follow their dynamic processes in live cells.
RedoxSensor Red CC-1 Stain
RedoxSensor Red CC-1 stain (2,3,4,5,6-pentafluorotetramethyldihydrorosamine, R14060) passively enters live cells and is subsequently oxidized in the cytosol to a red-fluorescent product (excitation/emission maxima ~540/600 nm), which then accumulates in the mitochondria. Alternatively, this nonfluorescent probe may be transported to the lysosomes where it is oxidized. The differential distribution of the oxidized product between mitochondria and lysosomes appears to depend on the redox potential of the cytosol. In proliferating cells, mitochondrial staining predominates; whereas in contact-inhibited cells, the staining is primarily lysosomal (). The 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.
Other Lysosomotropic Probes
BODIPY FL histamine (B22461) combines the pH-insensitive, bright green-fluorescent BODIPY FL dye with the weakly basic imidazole moiety of histamine. When used at low concentrations, this probe selectively stains lysosomes ().
As with the LysoTracker and LysoSensor probes, the weak basicity of the amine group in Dapoxyl (2-aminoethyl)sulfonamide (D10460) leads to its accumulation in acidic organelles. Dapoxyl (2-aminoethyl)sulfonamide () uptake by the acidic lumen of the intact acrosome of mouse sperm is accompanied by significant enhancement of this probe's fluorescence. The fluorescence of Dapoxyl (2-aminoethyl)sulfonamide is considerably reduced upon loss of the pH gradient at the onset of the acrosome reaction.
Our high-purity neutral red (N3246) is a common lysosomal probe that stains lysosomes a fluorescent red. It has also been used to determine the number of adherent and nonadherent cells in a microplate assay and to stain cells in brain tissue.
In addition, dansyl cadaverine (D113) and the DNA intercalator acridine orange (A1301, A3568) have been reported to be useful lysosomotropic reagents. Dansyl cadaverine has been shown to selectively label autophagic vacuoles, at least some of which had already fused with lysosomes; it did not, however, accumulate in early or late endosomes.
Biogenesis of the yeast vacuole has been extensively studied as a model system for eukaryotic organelle assembly. Using a combination of genetic and biochemical approaches, researchers have isolated a large collection of yeast vacuolar protein sorting (vps) mutants and characterized the vacuolar H+-ATPase (V-ATPase) responsible for compartment acidification. To facilitate the investigation of yeast vacuole structure and function, we offer membrane-permeant reagents and a Yeast Vacuole Marker Sampler Kit (Y7531).
FUN 1 Vital Cell Stain for Yeast
The FUN 1 () vital cell stain (F7030) exploits endogenous biochemical processing mechanisms that appear to be well conserved among different species of yeast and other fungi. When used at micromolar concentrations, the FUN 1 cell stain is freely taken up by several species of yeast and fungi and converted from a diffusely distributed pool of yellow-green–fluorescent intracellular stain into compact red-orange–fluorescent intravacuolar structures (). This conversion requires both plasma membrane integrity and metabolic capability. Only metabolically active cells are marked clearly with fluorescent intravacuolar structures, while dead cells exhibit extremely bright, diffuse, yellow-green fluorescence (Figure 12.3.7, ). FUN 1 staining has been used to detect antifungal activity against Candida species and to measure susceptibility of fungi to fungicides by flow cytometry. The FUN 1 cell stain is also available as a component in the LIVE/DEAD Yeast Viability Kit (L7009, Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3).
Figure 12.3.7 Fluorescence emission spectra of a Saccharomyces cerevisiae suspension that has been stained with the FUN 1 cell stain, which is available separately (F7030) or in the LIVE/DEAD Yeast Viability Kit (L7009). After the FUN 1 reagent was added to the medium, the fluorescence emission spectrum (excited at 480 nm) was recorded in a spectrofluorometer at the indicated times during a 30-minute incubation period. The shift from green (G) to red (R) fluorescence reflects the processing of FUN 1 by metabolically active yeast cells.
FM 4-64 and FM 5-95
One of our FM styryl dyes, FM 4-64, has been reported to selectively stain yeast vacuolar membranes with red fluorescence (excitation/emission maxima ~515/640 nm). This styryl dye is proving to be an important tool for visualizing vacuolar organelle morphology and dynamics, for studying the endocytic pathway and for screening and characterizing yeast endocytosis mutants. We offer FM 4-64 in 1 mg vials (T3166) or specially packaged in 10 vials of 100 µg each (T13320). The increasing number of successful applications for our FM dyes has prompted us to synthesize FM 5-95 (T23360), a slightly less lipophilic analog of FM 4-64 with essentially identical spectroscopic properties.
Yeast Vacuole Marker Sampler Kit
Our experiments have demonstrated that several cell-permeant derivatives of 7-amino-4-chloromethylcoumarin (CMAC) are largely sequestered within yeast vacuoles. The corresponding 7-amino-4-methylcoumarin derivatives are known to be substrates for yeast vacuolar enzymes. This sampler kit's three coumarin-based vacuole markers selectively stain the lumen of the yeast vacuole. To complement the blue-fluorescent staining of the lumen, we provide a novel green-fluorescent membrane marker MDY-64 for staining the yeast vacuole membrane. Membrane staining can also be accomplished using the red-fluorescent probe FM 4-64, as described above. The commonly used vacuole marker 5-(and 6-)carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA) is supplied for use as a standard. Three of the components in the Yeast Vacuole Marker Sampler Kit—CellTracker Blue CMAC (C2110, Membrane-Permeant Reactive Tracers—Section 14.2), the proprietary yeast vacuole membrane marker MDY-64 (Y7536) and carboxy-DCFDA (C369, Viability and Cytotoxicity Assay Reagents—Section 15.2)—are also available separately for those researchers who find that one of these dyes is well suited for their application.
Peroxisomes, single membrane–bound vesicles found in most eukaryotic cells, function to enzymatically oxidize fatty acids and to subsequently catalyze the breakdown of H2O2, a by-product of fatty acid degradation. Peroxisomes are similar in size to lysosomes (0.5–1.5 µm). The SelectFX Alexa Fluor 488 Peroxisome Labeling Kit (S34201) provides all the reagents required for labeling peroxisomes in fixed cells, including cell fixation and permeabilization reagents. To specifically detect peroxisomes, this kit uses an antibody directed against peroxisomal membrane protein 70 (PMP 70), which is a high-abundance integral membrane protein in peroxisomes, and an Alexa Fluor 488 dye–labeled secondary antibody (). The Alexa Fluor 488 dye exhibits bright green fluorescence that is compatible with filters and instrument settings appropriate for fluorescein. PMP 70 is significantly induced by administration of hypolipidemic agents, in parallel with peroxisome proliferation and the induction of peroxisomal fatty acid β-oxidation enzymes.
Each SelectFX Alexa Fluor 488 Peroxisome Labeling Kit contains:
- Rabbit IgG anti–peroxisomal membrane protein 70 (PMP 70) antibody
- Highly cross-adsorbed Alexa Fluor 488 goat anti–rabbit 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 Alexa Fluor 488 Peroxisome Labeling Kit)
|D1552||384.26||F,D,L||pH <7, DMF||349||16,000||none||MeOH|
|F7030||528.84||F,D,L||DMSO||508||71,000||none||pH 7||1, 2|
|L7525||524.40||F,D,L||DMSO||373||9600||422||pH 7||1, 3|
|L7533||376.50||F,D,L||DMSO||373||11,000||425||pH 5||1, 5|
|L7534||356.43||F,D,L||DMSO||442||17,000||505||pH 5||1, 5|
|L7535||398.46||F,D,L||DMSO||443||16,000||505||pH 5||1, 5|
|L7545||366.42||F,D,L||DMSO||384||21,000||540||pH 3||1, 6|
|L22460||see Notes||F,D,L||H2O||384||ND||540||pH 3||6, 7, 8|
|N3246||288.78||D,L||H2O, EtOH||541||39,000||640||see Notes||9|
|T3166||607.51||D,L||H2O, DMSO||505||47,000||725||see Notes||10, 11|
|T13320||607.51||D,L||H2O, DMSO||505||47,000||725||see Notes||10, 11|