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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.ref 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.ref

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.ref Morphological abnormalities in peroxisomes related to disease states and diet have also been the subject of current research.ref The SelectFX Alexa Fluor 488 Peroxisome Labeling Kit (S34201), described below, provides an antibody-based method for labeling peroxisomes in fixed cells.

CellLight Fluorescent Protein–Based Markers for Lysosomes, Peroxisomes and Endosomes

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 (C10507, C10596) and CellLight Lysosomes-RFP (C10504, C10597; Figure 12.3.1) are BacMam expression vectors encoding fusions of Green Fluorescent Protein (GFP) or Red Fluorescent Protein ref (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.ref 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.ref

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.ref We also offer CellLight Late Endosomes–GFP (C10588) and CellLight Late Endosomes–RFP (C10589) reagents, which are BacMam expression vectors encoding fusions of GFP or RFP with the late-endosomal protein Rab7a.

CellLight Peroxisome-GFP (C10604, Figure 12.3.4) and CellLight Peroxisome-RFP (C10605, Figure 12.3.4) are BacMam expression vectors encoding GFP or RFP linked on the C-terminal to a peroxisomal targeting sequence ref (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).

 

CellLight Lysosomes-RFP 

 

Figure 12.3.1 Human aortic smooth muscle cell (HASMC) labeled with CellLight Lysosomes-RFP (C10504, C10597) and CellLight MAP4-GFP (C10598) reagents and with Hoechst 33342 nucleic acid stain.

 

CellLight Early Endosomes-GFP 

 

Figure 12.3.2 Human aortic smooth muscle cell (HASMC) labeled with CellLight Early Endosomes-GFP (C10586) and Organelle Lights Golgi-OFP reagents and with Hoechst 33342 nucleic acid stain.

 

Endosomal movement along microtubules  
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.

 

CellLight Peroxisomes-GFP 

 

Figure 12.3.4 HEK 293 cell labeled with CellLight Peroxisomes-GFP (C10604) and CellLight Plasma Membrane-CFP (C10606) reagents.

LysoTracker Probes: Acidic Organelle–Selective Cell-Permeant Probes

LysoTracker Probes

Weakly basic amines selectively accumulate in cellular compartments with low internal pH and can be used to investigate the biosynthesis and pathogenesis of lysosomes.ref 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.ref The fluorescent probes neutral red (N3246) and acridine orange (A1301, A3568) are also commonly used for staining acidic organelles, though they lack specificity.ref

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 (photo). 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; photo, photo) 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 (photo). 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,ref to visualize acidic organelles labeled with rhodamine B in denervated skeletal muscle ref and to assess acrosomal integrity in cryopreserved bovine spermatozoa.ref This LysoTracker probe also proved useful in a continuous assay for the secretion of pulmonary surfactant by exocytosis of lamellar bodies.ref 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,ref for studying apoptosis in organogenesis-stage mouse embryos ref and for determining the subcellular localization of receptor and channel proteins.ref

 

Fluorescence Emission Spectra 

 

Figure 12.3.5 Normalized fluorescence emission spectra of 1) LysoTracker Blue DND-22 (L7525), 2) LysoTracker Green DND-26 (L7526) and 3) LysoTracker Red DND-99 (L7528) in aqueous solutions, pH 6.0.

 

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 (photo). 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:

Each kit provides enough staining solution for 500 assays using the protocol provided for labeling live, cultured cells that are adhering to coverslips.

LysoSensor Probes: Acidic Organelle–Selective pH Indicators

LysoSensor Probes

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, photo) .

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,ref myeloid leukemic cells ref and acidic vacuoles of plant cells.ref 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.ref 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,ref whereas other tumor cells contain lysosomes with higher pH.ref 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.ref LysoSensor Green DND-189 has been used to selectively label acidic compartments within granule cell neurites ref and, along with LysoSensor Green DND-153, to examine the acidification of endosomes and lysosomes in a mutant CHO cell line.ref 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.ref

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

Figure 12.3.6 The pH-dependent spectral response of LysoSensor Yellow/Blue DND-160 (L7545): A) fluorescence excitation spectra and B) fluorescence emission spectra.


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.ref 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 (photo) or flow cytometry.

DAMP and Other Lysosomotropic Probes

DAMP

The reagent DAMP (N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl)methylamine, dihydrochloride; D1552; structure) 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,ref 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 ref
  • Defective acidification of intracellular organelles in cells from cystic fibrosis patients ref
  • Dependence on pH of the conversion of proinsulin to insulin in beta cells ref
  • Development of autophagic vacuoles ref
  • Location of intracellular acidic compartments during viral infection ref 

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.ref In proliferating cells, mitochondrial staining predominates; whereas in contact-inhibited cells, the staining is primarily lysosomal (photo). 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.ref


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 (photo).

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 ref (structure) uptake by the acidic lumen of the intact acrosome of mouse sperm is accompanied by significant enhancement of this probe's fluorescence.ref The fluorescence of Dapoxyl (2-aminoethyl)sulfonamide is considerably reduced upon loss of the pH gradient at the onset of the acrosome reaction.ref

Our high-purity neutral red (N3246) is a common lysosomal probe that stains lysosomes a fluorescent red.ref It has also been used to determine the number of adherent and nonadherent cells in a microplate assay ref and to stain cells in brain tissue.ref

In addition, dansyl cadaverine ref (D113) and the DNA intercalator acridine orange ref (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.ref

Cell-Permeant Probes for Yeast Vacuoles

Biogenesis of the yeast vacuole has been extensively studied as a model system for eukaryotic organelle assembly.ref Using a combination of genetic and biochemical approaches, researchers have isolated a large collection of yeast vacuolar protein sorting (vps) mutants ref and characterized the vacuolar H+-ATPase (V-ATPase) responsible for compartment acidification.ref 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 (structure) vital cell stain (F7030) exploits endogenous biochemical processing mechanisms that appear to be well conserved among different species of yeast and other fungi.ref 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 (photo). 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 ref (Figure 12.3.7, photo). FUN 1 staining has been used to detect antifungal activity against Candida species ref and to measure susceptibility of fungi to fungicides by flow cytometry.ref 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).

 

Saccharomyces cerevisiae suspension  

 

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 ref (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.ref 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

The Yeast Vacuole Marker Sampler Kit (Y7531) contains sample quantities of a series of both novel and well-established vacuole marker probes that show promise for the study of yeast cell biology:

  • 5-(and 6-)Carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA) ref
  • CellTracker Blue CMAC ref
  • Aminopeptidase substrate Arg-CMAC (photo)
  • Dipeptidyl peptidase substrate Ala-Pro-CMAC
  • Yeast vacuole membrane marker MDY-64 ref (photo)

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.ref 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.ref 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 ref (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.

SelectFX Alexa Fluor 488 Peroxisome Labeling Kit

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,ref and an Alexa Fluor 488 dye–labeled secondary antibody (photo). 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.ref

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)

Spectral and Chemical Data Table

Cat # Links MW Storage Soluble Abs EC Em Solvent Notes
A1301 icon icon 301.82 L H2O, EtOH 489 65,000 520 MeOH  
A3568 icon icon 301.82 RR,L H2O 489 65,000 520 MeOH 1
B22461 icon 385.22 F,D,L DMSO 503 82,000 511 MeOH  
D113 icon icon 335.46 L EtOH, DMF 335 4600 518 MeOH  
D1552 icon 384.26 F,D,L pH <7, DMF 349 16,000 none MeOH  
D10460 icon icon 386.47 L DMF, DMSO 373 23,000 571 MeOH  
F7030 icon 528.84 F,D,L DMSO 508 71,000 none pH 7 1, 2
L7525 icon icon 524.40 F,D,L DMSO 373 9600 422 pH 7 1, 3
L7526 icon icon 398.69 F,D,L DMSO 504 80,000 511 MeOH 1
L7528 icon icon 399.25 F,D,L DMSO 577 78,000 590 MeOH 1, 4
L7533 icon icon 376.50 F,D,L DMSO 373 11,000 425 pH 5 1, 5
L7534 icon icon 356.43 F,D,L DMSO 442 17,000 505 pH 5 1, 5
L7535 icon icon 398.46 F,D,L DMSO 443 16,000 505 pH 5 1, 5
L7545 icon icon 366.42 F,D,L DMSO 384 21,000 540 pH 3 1, 6
L12491 icon 364.40 F,D,L DMSO 466 22,000 536 MeOH 1
L22460 icon see Notes F,D,L H2O 384 ND 540 pH 3 6, 7, 8
N3246 icon 288.78 D,L H2O, EtOH 541 39,000 640 see Notes 9
R14060 icon 434.41 F,D,L,AA DMSO 239 52,000 none MeOH  
T3166 icon icon 607.51 D,L H2O, DMSO 505 47,000 725 see Notes 10, 11
T13320 icon icon 607.51 D,L H2O, DMSO 505 47,000 725 see Notes 10, 11
T23360 icon icon 565.43 D,L H2O, DMSO 560 43,000 734 CHCl3 10
Y7536 icon icon 384.48 F,L DMSO, DMF 456 27,000 505 MeOH  
  1. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."
  2. F7030 is fluorescent when bound to DNA (Em = 538 nm). Uptake and processing of the dye by live yeast results in red-shifted fluorescence (Em ~590 nm).
  3. L7525 has structured absorption and fluorescence spectra with additional peaks at Abs = 394 nm and Em = 401 nm.
  4. The pKa of the dimethylamino substituent of LysoTracker Red DND-99 is 7.5.ref The absorption and fluorescence spectra of the dye are insensitive to protonation of this substituent.
  5. This LysoSensor dye exhibits increasing fluorescence as pH decreases with no spectral shift. L7533 has additional absorption and fluorescence emission peaks at Abs = 394 nm and Em = 401 nm.
  6. LysoSensor Yellow/Blue spectra are pH dependent. Abs and Em shift to shorter wavelengths at pH >5.
  7. The molecular weight is nominally as specified in the product name but may have a broad distribution.
  8. ND = not determined.
  9. Spectra of N3246 are pH dependent (pKa ~6.7). Data reported are for 1:1 (v/v) EtOH/1% acetic acid.
  10. FM 4-64 and FM 5-95 are nonfluorescent in water. For two-color imaging in GFP-expressing cells, these dyes can be excited at 568 nm with emission detection at 690–730 nm.ref
  11. Abs, EC and Em determined for dye bound to detergent micelles (20 mg/mL CHAPS in H2O). These dyes are essentially nonfluorescent in pure water.

For Research Use Only. Not for use in diagnostic procedures.