Membrane-Permeant Reactive Tracers—Section 14.2

Page Contents

• Thiol-Reactive CellTracker Probes
• Other Thiol-Reactive Tracers
• Amine-Reactive Tracers
• Data Table
• Ordering Information

Molecular Probes CellTracker reagents are fluorescent chloromethyl derivatives that freely diffuse through the membranes of live cells (, , ). Once inside the cell, these mildly thiol-reactive probes undergo what is believed to be a glutathione S-transferase–mediated reaction to produce membrane-impermeant glutathione–fluorescent dye adducts, although our experiments suggest that they may also react with other intracellular components (Figure 14.2.1). Regardless of the mechanism, many cell types loaded with the CellTracker probes are both fluorescent and viable for at least 24 hours after loading and often through several cell divisions. Most other cell-permeant fluorescent dyes, including the acetoxymethyl (AM) esters of calcein and BCECF (Viability and Cytotoxicity Assay Reagents—Section 15.2), are retained in viable cells for no more than a few hours at physiological temperatures (Figure 14.2.2). Furthermore, unlike the free dye, the peptide–fluorescent dye adducts contain amino groups and can therefore be covalently linked to surrounding biomolecules by fixation with formaldehyde or glutaraldehyde. This property permits long-term storage of the labeled cells or tissue and, in cases where the cognate anti-dye antibody is available (see below), amplification of the dye hapten by standard immunohistochemical techniques. Fixation without loss of the tracer also facilitates the safe handling and analysis of cells containing pathogens.

Figure 14.2.1 Intracellular reactions of our fixable CellTracker Green CMFDA (5-chloromethylfluorescein diacetate; C2925, C7025). Once this membrane-permeant probe enters a cell, esterase hydrolysis converts nonfluorescent CMFDA to fluorescent 5-chloromethylfluorescein, which can then react with thiols on proteins and peptides to form aldehyde-fixable conjugates. This probe may also react with intracellular thiol-containing biomolecules first, but the conjugate is nonfluorescent until its acetates are removed.

Figure 14.2.2 Loading and retention characteristics of intracellular marker dyes. Cells of a human lymphoid line (GePa) were loaded with the following cell-permeant acetoxymethyl ester (AM) or acetate derivatives of fluorescein: 1) calcein AM (C1430, C3099, C3100MP), 2) BCECF AM (B1150), 3) fluorescein diacetate (FDA, F1303), 4) carboxyfluorescein diacetate (CFDA, C1354) and 5) CellTracker Green CMFDA (5-chloromethylfluorescein diacetate, C2925, C7025). Cells were incubated in 4 µM staining solutions in Dulbecco's modified eagle medium containing 10% fetal bovine serum (DMEM+) at 37°C. After incubation for 30 minutes, cell samples were immediately analyzed by flow cytometry to determine the average fluorescence per cell at time zero (0 hours). Retained cell samples were subsequently washed twice by centrifugation, resuspended in DMEM+, maintained at 37°C for 2 hours and then analyzed by flow cytometry. The decrease in the average fluorescence intensity per cell in these samples relative to the time zero samples indicates the extent of intracellular dye leakage during the 2-hour incubation period.

CellTracker Probes in a Variety of Fluorescent Colors

Molecular Probes CellTracker product line includes reactive chloromethyl derivatives of:

• Blue-fluorescent 7-aminocoumarin (CellTracker Blue CMAC, C2110)
• Blue-fluorescent 7-hydroxycoumarin (CellTracker Blue CMHC, C2111)
• Blue-fluorescent 6,8-difluoro-7-hydroxycoumarin (CellTracker Blue CMF₂HC, C12881)
• Violet-fluorescent 2,3,6,7-tetrahydro-9- bromomethyl-1H,5H-quinolizino( 9,1-gh)coumarin (CellTracker Violet BMQC, C10094)
• Green-fluorescent fluorescein diacetate (CellTracker Green CMFDA, C2925 and C7025, Figure 14.2.1)
• Green-fluorescent BODIPY derivative (CellTracker Green BODIPY, C2102)
• Orange-fluorescent tetramethylrhodamine (CellTracker Orange CMTMR, C2927)
• Orange-fluorescent CellTracker Orange CMRA (C34551)
• Red-fluorescent CellTracker Red CMTPX (C34552)

CellTracker Green CMFDA freely diffuses into the cell, where cytosolic esterases cleave the acetate groups, releasing the fluorescent product; CellTracker Orange CMRA also requires esterase cleavage to activate its fluorescence. The CellTracker Blue CMAC, CMHC and CMF₂HC, CellTracker Violet BMQC, CellTracker Green BODIPY, CellTracker Orange CMTMR and CellTracker Red CMTPX probes do not require enzymatic cleavage to activate their fluorescence. CellTracker Orange CMRA is a rhodol-based fluorophore with an overall net charge at neutral pH that is expected to be negative, in contrast with the overall net positive charge of the tetramethylrhodamine-based CellTracker Orange CMTMR. Thus, unlike CellTracker Orange CMTMR, CellTracker Orange CMRA should remain primarily in the cytoplasm instead of being sequestered inside actively respiring mitochondria. The long-wavelength CellTracker Red CMTPX exhibits bright red fluorescence that is easily distinguished from that of blue-, green- and far-red–fluorescent probes, including CellTracker Green CMFDA and CFSE (see below). We also offer the green-fluorescent BODIPY 493/503 methyl bromide (B2103), which has slightly greater thiol-reactivity than do chloromethyl derivatives.

Applications for CellTracker Probes

The thiol-reactive CellTracker Green CMFDA is suitable for long-term cell labeling and has been frequently used for in vitro labeling of cells prior to adoptive transfer to animals and tissues. Other prominent applications include the analysis of cell–cell fusion, cell adhesion and multidrug resistance transporter function.

CellTracker Green CMFDA was used to track wild-type and myosin II mutant Dictyostelium discoideum cells within aggregation streams during early multicellular morphogenesis; differentiation and morphogenesis pathways were reportedly unaffected in labeled cells imaged over several days by confocal laser-scanning microscopy. CellTracker Green BODIPY was used to label CD4⁺ T cells and follow their activation and proliferation in mice with the immunodeficiency syndrome MAIDS. CellTracker Green CMFDA and some other CellTracker dyes have been utilized to selectively label intracellular Chlamydia psittaci bacteria in infected cells.

CellTracker Orange CMTMR has been used to stain the cytoplasm of engrailed-expressing Drosophila cells in an in vitro reconstruction experiment, to follow T-cell differentiation and maturation and to trace implanted tumor cells for at least two weeks. Chlamydia labeled with CellTracker Orange CMTMR continued to grow and differentiate, and labeled chlamydia isolated from infected cells remained infectious.

Metabolic activity and drug-induced cytotoxicity were measured with CellTracker Blue CMAC in a fluorescence-based microplate assay. Peptidase substrates derived from CellTracker Blue CMAC yield blue-fluorescent products that are well retained in live cells (Detecting Peptidases and Proteases—Section 10.4). Our CellTracker Blue CMF₂HC (C12881) has a low pK_a that ensures that the dye's conjugates will be ionized and have bright blue fluorescence in the cytoplasm. CellTracker Blue CMAC was used in combination with calcein AM (C1430, C3099, C3100MP; Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6) and the lipophilic tracer DiI (D282, D3911, V22885; Tracers for Membrane Labeling—Section 14.4) to identify lipid mixing and cytoplasm mixing between labeled effector cells and labeled target cells.

The ability to fix the intracellular products of CellTracker Green CMFDA, CellTracker Green BODIPY and CellTracker Orange CMTMR in permeabilized cells permits the stained cells to be probed with our anti–fluorescein/Oregon Green, anti–BODIPY FL and anti-tetramethylrhodamine antibodies, respectively (Anti-Dye and Anti-Hapten Antibodies—Section 7.4, Anti-fluorophore and anti-hapten antibodies—Table 7.8).

Chloromethyl Derivatives of SNARF-1 and H₂DCFDA

Although they were designed for other purposes, the chloromethyl derivatives of our SNARF-1 pH indicator (C6826) and of 2',7'-dichlorodihydrofluorescein diacetate, acetate ester (CM-H₂DCFDA, C6827) possess some unique properties for tracking cells. As with the CellTracker probes, cytoplasmic enzymes hydrolytically remove the acetate groups from the membrane-permeant probes, and the chloromethyl moieties become conjugated to intracellular thiols. With its long Stokes shift, the SNARF-1 derivative has easily distinguished, red-orange fluorescence in the cytoplasm when excited at the same wavelengths used for the green-fluorescent CellTracker Green CMFDA ().

As with other dihydrofluorescein derivatives (Generating and Detecting Reactive Oxygen Species—Section 18.2), CM-H₂DCFDA requires an additional oxidation step before becoming fluorescent. This probe is useful for following stimulation of oxidative activity by external agents or natural killer (NK) cells over extended periods, as well as for passively labeling cells that lack appropriate oxidative activity and then following their ingestion by scavengers such as neutrophils. CM-H₂DCFDA has been used to measure intracellular reactive oxygen species (ROS) in cardiac myocytes and in human embryonic kidney 293 (HEK 293) cells stably transfected with the human vanilloid receptor 1 (VR1) cation channel.

Bimanes: Blue-Fluorescent Reactive Tracers

The bimane derivatives, monobromobimane (mBBr, M1378, M20381) and monochlorobimane (mBCl, M1381MP), are important thiol-derivatization reagents (Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3). The essentially nonfluorescent mBBr and mBCl dyes are known to passively diffuse across the plasma membrane into the cytoplasm, where they form blue-fluorescent adducts with intracellular glutathione and thiol-containing proteins (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6).

CFSE and Its Derivatives

Carboxyfluorescein diacetate succinimidyl ester (5(6)-CFDA, SE; C1157) is commonly referred to as CFSE. Among its many applications, the two most prominent are ex vivo labeling of cells for adoptive transfer and flow cytometric tracking of cell division by label partition analysis. CFSE is colorless and nonfluorescent until its acetate groups are cleaved by intracellular esterases to yield highly fluorescent, amine-reactive 5(6)-carboxyfluorescein succinimidyl ester (equivalent to C1311, Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5). Upon reaction with amine-containing residues of intracellular proteins, the resulting dye–protein adducts are well retained in cells as they move and divide during embryonic development. In addition, these adducts survive subsequent fixation with formaldehyde or glutaraldehyde. Because it is intrinsically more reactive, the succinimidyl ester of CFDA is more likely to react at sites on the extracellular surface than is CMFDA.

Once incorporated into cells, CFDA SE remains there—even through cell division—thus preventing transfer to unlabeled cells. Labeled cells can subsequently be immunohistochemically detected post-mortem using our anti–fluorescein/Oregon Green antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4). The feasibility of using cell-permeant fluorescent tracers to follow asynchronous cell division of natural killer (NK) cells, B cells, T cells, thymocytes, lymphocytes, fibroblasts and hematopoietic cells has been demonstrated with CFDA SE (Figure 14.2.3). Lymphocytes labeled with CFDA SE were detected up to eight weeks after injection into mice during lymphocyte migration studies, and similarly labeled hepatocytes were easily located by fluorescence microscopy even 20 days after intrahepatic transplantation. CFDA SE has also been successfully employed to quantitate adhesion of neutrophils and leukocytes, to assay T-cadherin–mediated cell aggregation, to follow neurite growth in an in vitro bioassay and to trace fetal cells in culture. Different loading concentrations of CFDA SE can be used to produce cells that can be distinguished by their relative brightness. CFDA SE has been utilized for tracing the transport of viable bacterial cells in groundwater under no-growth conditions for periods of at least 28 days. CFDA SE is also available conveniently packaged for cell-tracing applications in our Vybrant CFDA SE Cell Tracer Kit (V12883) and for cell proliferation studies in our CellTrace Cell Proliferation Kit (C34554, Figure 14.2.4). The fluorescent CFDA SE product has excitation/emission maxima of ~492/517 nm and can be detected using a fluorescence microscope, flow cytometer or fluorescence microplate reader.

Carboxyeosin diacetate succinimidyl ester (C22803) has applications that are expected to be similar to CFDA SE; however, its fluorescence can be excited and detected at longer wavelengths, possibly permitting two-color tracing experiments of mixed-cell populations. Eosin derivatives are also effective reagents for photoablation of cells. Unlike CFDA SE, fluorescence of the intracellular hydrolysis products of the succinimidyl ester of 5-(and 6-)carboxy-2',7'-dichlorofluorescein diacetate (C1165) is relatively insensitive to fluctuations in pH. This amine-reactive tracer was reported to be more useful than the lipophilic marker DiI (D282, Tracers for Membrane Labeling—Section 14.4) in an investigation of palatal fusion in rodent embryos.

Figure 14.2.3 Tracking of asynchronous cell division using 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester (5(6)-CFDA SE or CFSE; C1157; V12883) labeling and flow cytometry. Cell division results in sequential halving of the initial fluorescence, resulting in a multipartite cellular fluorescence histogram. The peaks labeled 0, 1, 2, 3, 4 and 5 represent successive generations.

Figure 14.2.4 Following cell proliferation in human peripheral blood lymphocytes using the CellTrace CFSE Cell Proliferation Kit C34554). Human peripheral blood lymphocytes were harvested and stained with CellTrace CFSE (carboxyfluorescein diacetate, succinimidyl ester; 5(6)-CFDA, SE) on Day 0. A portion of the population was arrested at the parent generation using mitomycin C (red peak). The remainder of the sample was stimulated with phytohemagglutinin and allowed to proliferate for 5 days. Solid green peaks represent successive generations.

CellTrace Violet Cell Proliferation Kit

CellTrace Violet stain is an esterase-activated phenolic fluorophore with a succinimidyl ester substituent for coupling to cell surface and intracellular amines. It is functionally analogous to CFSE, equally partitioning between daughter cells during division resulting in successive two-fold reductions in cell-associated fluorescence intensity. When analyzed by flow cytometry, this progressive label partitioning provides a direct indication of cell proliferation status (Figure 14.2.5). In contrast to CFSE, CellTrace Violet stain is optimally excited by 405 nm violet diode lasers and generates blue fluorescence (emission peak ~455 nm). Consequently, it can be used in combination with CFSE to track cells from different origins after mixing or to analyze proliferation of GFP-expressing cells. The CellTrace Violet Cell Proliferation Kit (C34557) includes the CellTrace Violet stain together with dimethylsulfoxide (DMSO) for preparation of a stock solution.

Figure 14.2.5 Human peripheral blood lymphocytes were harvested and stained with CellTrace Violet stain. The violet peaks represent successive generations of cells stimulated with mouse anti–human CD3 and Interleukin-2 and grown in culture for 7 days. The peak outlined in black represents cells that were grown in culture for 7 days with no stimulus.

CellTrace Oregon Green 488 Carboxylic Acid Diacetate Succinimidyl Ester

The succinimidyl ester of Oregon Green 488 carboxylic acid diacetate (carboxy-DFFDA SE) offers several important advantages over CFDA SE as a fluorescent cell tracer. This Oregon Green 488 probe passively diffuses into cells, where it is colorless and nonfluorescent until its acetate groups are removed by intracellular esterases to yield a highly green-fluorescent, amine-reactive dye. Upon reaction with intracellular amines, the probe forms Oregon Green 488 conjugates that are well-retained by cells. Unlike fluorescein derivatives, however, Oregon Green 488 derivatives exhibit bright green fluorescence that is not pH dependent at typical cellular pH values. Moreover, Oregon Green 488 probes are usually brighter and more photostable than fluorescein probes. We offer carboxy-DFFDA SE in a 1 mg unit size (O34550) and specially packaged in a set of 20 vials, each containing 50 µg (CellTrace Oregon Green 488 carboxylic acid diacetate succinimidyl ester, C34555).

CellTrace Far Red DDAO-SE

CellTrace Far Red DDAO-SE (C34553) is a fixable, far-red–fluorescent tracer for long-term cell labeling. The succinimidyl ester (SE) reactive group forms a strong covalent attachment to primary amines that occur in proteins and other biomolecules inside and outside of cells. With its far-red fluorescence, CellTrace Far Red DDAO-SE has minimal spectral overlap with most other fluorophores (Figure 14.2.6) and thus can be used simultaneously with almost any blue, green or orange fluorophores including Green Fluorescent Protein (GFP).

Figure 14.2.6 Normalized absorption and fluorescence emission spectra of DDAO.

Amine-Reactive SNARF-1 Carboxylic Acid Acetate

To permit simultaneous long-term tracing of mixed-cell populations using different fluorescent colors, we have developed an amine-reactive probe whose applications are similar to those of CFDA SE. Cells labeled with our succinimidyl ester of SNARF-1 carboxylic acid acetate (S22801) have a red-orange fluorescence that can easily be distinguished from that of cells loaded with green-fluorescent tracers such as CFDA SE. However, the fluorescence intensity of cells loaded with this SNARF-1 derivative will not be as high as that of cells loaded with the same concentration of CFDA SE. Thus, it is necessary to adjust the reagent concentration and/or select optical filters to appropriately balance the fluorescence intensities when doing two-color experiments.