Quantitative Comparison of Long-wavelength Alexa Fluor Dyes to Cy Dyes : Fluorescence of the Dyes and Their Bioconjugates
Molecular Probes, Inc., Eugene, Oregon (JEB,AR,GB,JB,DRG,BJF,SY,JL,C-YC,WC,JDH,JMB,RPH,RPH) and Experimental Transplantation and Immunology Branch, NCI-NIH, Bethesda, Maryland (WGT)
Correspondence to: James D. Hirsch, Molecular Probes, Inc., 29851 Willow Creek Road, Eugene, OR 97402. E-mail: jim.hirsch{at}probes.com
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Summary |
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Key Words: Alexa Fluor dyes Cy dyes long-wavelength dyes fluorescent bioconjugates photostability immunofluorescence FRET flow cytometry microscopy
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Introduction |
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This work compares newly available, water-soluble, amine-reactive N-hydroxysuccinimidyl esters (SEs, also known as NHS esters) of Alexa Fluor dyes (Molecular Probes; Eugene, OR) with the SEs of the Cy series of dyes and some other currently available long-wavelength dyes. Except for Alexa Fluor 633, which is a sulfonated rhodamine derivative, all of these new Alexa Fluor dyes are sulfonated carbocyanine molecules. The dyes in both series can be grouped by absorption maxima and are referred to here as Cy3-like, Cy5-like, and so forth. In the Cy3-like group, the Alexa Fluor 555, DY-550, and Atto 565 dyes are spectrally comparable to Cy3 (Cy 3.18, 3.29). The Alexa Fluor 633, Alexa Fluor 647, DY-630, and DY-635 dyes can be compared to Cy5 (Cy 5.18, 5.29) (Cy5-like), and in the Cy5.5-like group, the Alexa Fluor 660, Alexa Fluor 680, and DY-680 dyes are comparable to Cy 5.5. In the last group, Cy7-like, the Alexa Fluor 700 and Alexa Fluor 750 dyes can be compared to Cy7. The dyes and protein conjugates of the dyes were compared spectrally, and the functionality of dyeprotein conjugates was examined in several applications. Flow cytometry and fluorescence microscopy were used to compare dye conjugates, which are composed of a dye covalently linked to a protein, such as goat anti-mouse IgG antibody (GAM), goat anti-rabbit IgG antibody (GAR), streptavidin (SA), concanavalin A (ConA), or transferrin (Tf), at similar degrees of labeling. The results indicate that Alexa Fluor dyes, whose absorption and emission spectra are spectrally similar to those of the Cy dyes, are more resistant to fluorescence quenching and absorption spectral artifacts on conjugation to proteins. At high degrees of labeling, the Alexa Fluor dyes undergo much less self-quenching than the other dyes tested. For protein labeling, the ability of Alexa Fluor dyes to retain their intense fluorescence even when the conjugates are heavily labeled suggests that the long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and spectrally similar long-wavelength dyes.
In addition to the conventional conjugates, tandem conjugates were also analyzed. These tandem conjugates consist of three components: an absorber [a phycobiliprotein, either R-phycoerythrin (R-PE) or allophycocyanin (APC)], an emitter (one of the long-wavelength Alexa Fluor or Cy dyes), and a protein (e.g., streptavidin) for conferring bioaffinity. Phycobiliproteins, which are fluorescent light-harvesting proteins from cyanobacteria and some eukaryotic algae, act first as an energy absorber, then as a donor transferring energy to the dye. Tandem fluorescent molecules result in an increase in the effective Stokes shift of the reagent. The fluorescence emission peak of the reagent is effectively extended, by means of fluorescence resonance energy transfer (FRET), to a longer wavelength than that of the phycobiliprotein alone (Glazer and Stryer 1983). The tandem conjugates of an Alexa Fluor dye, a phycobiliprotein, and an anti-IgG antibody or streptavidin were compared to tandem conjugates containing Cy dyes. The tandem constructs involving Alexa Fluor dyes displayed higher FRET efficiencies and were functionally brighter in flow cytometry applications than comparable tandem constructs of the Cy dyes.
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Materials and Methods |
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Instrumentation
Absorption spectra were acquired with a U-2000 spectrophotometer (Hitachi Instruments; Boulder, CO). Fluorescence absorption and emission data were obtained with an AmincoBowman Series II Luminescence Spectrometer (Thermo Spectronic; Rochester, NY).
Flow cytometry experiments were performed using a Coulter Elite flow cytometer (Beckman Coulter; Miami Lakes, FL) equipped with a 488-nm argon ion laser and a 575-nm bandpass filter for detecting cells labeled with the Alexa Fluor 555 or Cy3 dye conjugates. To detect far-red fluorescence in cells labeled with the Alexa Fluor 647 or Cy5 dye conjugates, the Coulter Elite flow cytometer was equipped with a 633-nm HeNe laser, a 675-nm bandpass emission filter, and a 640 nm dichroic longpass filter. EXPO32 software (Beckman Coulter, version 1.0) was used for sample acquisition and analysis. The fluorescence intensity of cells labeled with Alexa Fluor 647 R-PE SA or Cy5 R-PE SA tandem conjugates was measured with a FACSCalibur benchtop flow cytometer (BD Biosciences) equipped with a 635-nm red diode laser. Data were acquired and analyzed with CellQuest v. 3.3 software (BD Biosciences).
The fluorescence microscopes (Meridian Instrument Company; Kent, WA) used were a Nikon Eclipse E400 for photobleaching and immunofluorescence brightness determination and a Nikon Eclipse E800 for cell and tissue imaging. Optical filters (Omega Optical; Brattleboro, VT) used to visualize Alexa Fluor 555 and Cy3 dye conjugates were the Omega XF32 and XF102 filters, and the filter used to detect Alexa Fluor 647 and Cy5 dye conjugates was the Omega XF110. Images were acquired with a MicroMAX digital camera (Princeton Scientific Instruments; Monmouth Junction, NJ) with a x1300 1030 charged-coupled device (CCD) array (Roper Scientific; Trenton, NJ), controlled by MetaMorph software (Universal Imaging; Downingtown, PA).
Fluorescence Spectral Profiles
Extinction coefficients for all unconjugated dyes in methanol were provided by the manufacturers. The relative quantum yield (RQY, the integrated photon emission relative to that of an appropriate dye standard) of each conjugate was calculated using the following standard dyes: 5-(and-6)-carboxytetramethylrhodamine (Molecular Probes) for the Cy3, Atto 565, and Alexa Fluor 555 dyes; DDAO (7-hydroxy-9H(1,3-dichloro-9,9-dimethylacridine-2-one)) (Molecular Probes) for the Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Cy5, Cy5.5, DY-630, DY-635, and DY-680 dyes; oxazine 1 perchlorate (Eastman Kodak; Rochester, NY) for the Alexa Fluor 700 dye; and IR125 (Lamda Physik; Ft Lauderdale, FL) for the Alexa Fluor 750 and Cy7 dyes. For fluorescence analysis, the conjugates were matched at identical absorbance to that of the standard dye at the appropriate excitation wavelength. The total fluorescence (TF, the product of the RQY and the DOL) was used to measure fluorescence output (brightness) of the conjugate (Haugland 2000).
Photobleaching
Glass capillary tubes filled with 0.5 µM solutions of Alexa Fluor 555, Alexa Fluor 647, Cy3, or Cy5 SE dye derivatives in PBS, pH 7.5, were excited with light emitted by the 100-W mercury arc lamp of the Nikon Eclipse E400 fluorescence microscope. Using the x40 objective, integrated fluorescence emission intensity under continuous illumination was measured initially and then every 5 sec for 95 sec, and the observed fluorescence intensities were normalized to the initial values.
Labeling Reactions
Protein conjugates containing the Alexa Fluor dyes were prepared, purified, and characterized as described previously (PanchukVoloshina et al. 1999; Haugland 2000
; Hahn et al. 2001
). Cy, Atto, and Dyomics dye conjugates were prepared according to manufacturers' instructions. The DOL of each conjugate was determined spectrophotometrically as previously described (PanchukVoloshina et al. 1999
; Haugland 2000
). To evaluate dye R-PE SA tandem conjugates by FRET, samples were matched for equal absorption by equalizing their optical density at the excitation wavelength of 488 nm.
Flow Cytometry
Flow cytometry was used to compare the fluorescence intensity of cells labeled with dyeprotein conjugates prepared for this study as described above or labeled with commercially available conjugates. For comparison of dyeGAM conjugates, human peripheral blood was collected in a Vacutainer tube containing sodium heparin (BD Biosciences). An aliquot (100 µl) of the whole blood was added to a 3-ml plastic tube and blocked with 10% normal goat serum (NGS) in PBS on ice for 10 min. After addition of mouse anti-human CD3 antibody (1 µg) (Caltag Laboratories) to label T-cells, or PBS (5 µl) as a control, the tubes were incubated on ice for 30 min. Cells were washed, resuspended to 100 µl with PBS, and incubated with secondary reagent (dyeGAM conjugates; 0.5 µg) on ice for another 30 min. Erythrocytes in the sample were lysed by the addition of 2.5 ml ammonium chloride lysis buffer (0.15 M ammonium chloride, 0.01 M potassium bicarbonate, and 0.1 mM EDTA) (Stewart and Stewart 2001). The labeled cells were analyzed by flow cytometry with gating for lymphocytes.
The fluorescence intensity of cells labeled with Alexa Fluor 647 R-PE SA or Cy5 R-PE SA tandem conjugates was compared by flow cytometry as described previously (Telford et al. 2001a,b
). Briefly, washed EL4 lymphoma cells (ATCC; Manassas, VA) were incubated first with a biotinylated anti-CD44 mouse monoclonal antibody, then labeled with either the Alexa Fluor 647 R-PE SA tandem conjugate or the Cy5 R-PE SA tandem conjugate and analyzed.
Flow cytometry was also used to compare a Cy7 APC SA tandem conjugate to APC SA conjugates of Alexa Fluor 680, Alexa Fluor 700, or Alexa Fluor 750 dye. For tandem conjugates, human peripheral blood was collected in a Vacutainer tube as above and centrifuged at 3300 x g for 30 min at room temperature (RT). Mononuclear cells were harvested from the white-colored layer directly below the plasma layer. The cells were washed once with PBS (pH 7.2), counted with a Z1 Coulter particle counter (Beckman-Coulter), and resuspended to a concentration of 1 x 107 cells/ml. An aliquot (100 µl) of cell suspension was transferred to a reaction tube and blocked for 10 min at RT with nonspecific mouse IgG antibody (0.1 µg). Cells were washed, resuspended to 100 µl with PBS, and then incubated with 5 µl of either biotinylated mouse anti-human CD3 antibody (Caltag Laboratories) to label T-cells or with PBS as a control, at RT for 15 min. Cells were washed and secondary reagents containing streptavidin (tandem constructs) (0.11 µg per reaction) were added. Samples were protected from light, incubated at RT for 15 min, washed, and analyzed by flow cytometry with gating for lymphocytes.
Immunofluorescence Microscopy
Immunocytochemistry experiments with the bioconjugates were conducted on prepared slides with fixed HEp-2 human epithelial cells in wells (INOVA Diagnostics; San Diego, CA). The cells were incubated first with a human anti-nuclear antiserum (ANA) (INOVA Diagnostics) (30 µl/well) as the primary antibody for 30 min, then with biotinylated protein G (Molecular Probes) (0.2 µg/well) for 30 min, followed with a dye-labeled SA conjugate (0.2 µg/well) (PanchukVoloshina et al. 1999) for 30 min. Cells were extensively washed with PBS between incubations. Coverslips were applied with Prolong antifade mounting medium (Molecular Probes). Stained cell nuclei were visualized against a black or lightly stained cytoplasmic background. Five images were acquired for each bioconjugate. In each image, brightness values (mean ± SD) for the nucleus and cytoplasm of 10 representative cells were computed. The ratio of the fluorescence intensity of the nucleus to that of the cytoplasm was defined as the signal-to-noise ratio (S/N).
Immunofluorescent staining of the inhibitory protein of mitochondrial oxidative phosphorylation complex V [ATPase inhibitor protein (IF1)] and -tubulin in bovine pulmonary artery endothelial (BPAE) cells (ATCC) was performed as described previously (Hirsch et al. 2002
) using the appropriate primary antibodies (Molecular Probes). Briefly, BPAE cells were grown in Dulbecco's modified minimal essential Eagle's medium supplemented with 20% fetal bovine serum (FBS) (both from Invitrogen Life Technologies; Carlsbad, CA), plated onto 18-mm x 18-mm glass coverslips in 100-mm diameter Petri dishes, and cultured to 5060% confluency. Cultures were fixed in 4% formaldehyde (Polysciences; Warrington, PA) in PBS at 37C for 20 min. Cells were permeabilized with 0.1% Triton X-100 (Sigma/Aldrich)/PBS for 10 min, then incubated in 10% NGS/0.1% Triton X-100/PBS blocking buffer (BB1) for 30 min. Cells were incubated with monoclonal mouse anti-
-tubulin (2 µg/ml) or mouse anti-complex V inhibitory protein (5 µg/ml) antibodies in BB1 for 30 min with gentle rocking, then incubated with a secondary antibody GAM conjugate containing Alexa Fluor 555 dye (DOL = 6.3), Cy3 dye (DOL = 5.3), Alexa Fluor 647 dye (DOL = 5.7), or Cy5 dye (DOL = 5.5) (5 µg/ml in BB1) for 30 min, and then finally incubated with DAPI (Molecular Probes; 0.2 µg/ml in PBS) for 2 min. Cells were extensively washed with PBS between incubations. Coverslips were mounted on microscope slides as described above.
For immunohistochemical studies, a fluorescently labeled antibody against HuC/HuD, an RNA-binding protein specific to neuronal cells, was used to detect neuronal cell bodies in rat brain tissue sections. Perfused and frozen brain tissue from a postnatal day 24 rat (a generous donation from Woody Hopf; Ernst Gallo Clinic and Research Center, University of California, San Francisco) was transferred to Peel-Away molds (Polysciences), embedded in Sakura Finetek's Tissue-Tek OCT compound (VWR; West Chester, PA), and frozen in liquid nitrogen. Coronal sections (10 µm) were cut with a Leica CM3050S cryostat, collected on Superfrost Plus slides (VWR), air-dried, desiccated, and stored in slide boxes at -85C. For staining, slides were brought to RT and then rehydrated in PBS for 15 min. Tissue sections were permeabilized in 0.2% Triton X-100/0.2% bovine serum albumin (BSA) (Sigma/Aldrich)/PBS (PBT) for 15 min, then blocked with 5% NGS/PBT (BB2) for 30 min.
Sections were incubated with monoclonal mouse anti-HuC/HuD antibody (Molecular Probes; 5 µg/ml in BB2) overnight at 4C. Slides were washed four times for 15 min each in PBT and then incubated with dye-labeled GAM secondary antibodies (5 µg/ml in PBT) for 2 hr. After again washing four times for 15 min each in PBT, sections were counterstained with DAPI as described above, then washed in PBS and sealed with a coverslip as described above. For each pairwise dye comparison, optimized camera exposure times were obtained for the cell or tissue sample with the brighter labeling by identifying the exposure setting that produced a minimal level of pixel saturation in a 12-bit image. Once the sample with the brightest fluorescent signal intensity was identified, the identical camera setting was then used to acquire images from the other sample in the comparison. In addition, the preparations with the weaker signal intensities were imaged with a range of longer exposure times to identify exposure conditions that produced levels of pixel saturation comparable to those of the brightest samples. For the preparation of figures, the images were re-sized in Adobe Photoshop (San Jose, CA) with no adjustment to the level, brightness, and contrast values.
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Results |
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Fluorescence Microscopy
Dyes belonging to the Cy3-like, Cy5-like, and Cy5.5-like groups were conjugated to SA and evaluated as described in Materials and Methods for nuclear staining of HEp-2 cells (Table 2). Nine Cy3-like, ten Cy5-like, and two Cy5.5-like SA conjugates were compared. In the Cy3-like group, nuclei stained with Alexa Fluor 555 SA (DOL = 2.5) were the brightest (fluorescence intensity of the nucleus = 3.7 x 103) and had a high S/N ratio (8.8 in a range of 3.412.1). Nuclei stained with Cy3 SA ranged in fluorescence from 1.43.2 x 103 with high S/N ratios. The fluorescence intensity of nuclei stained with Atto 565 SA was the second highest in the Cy3-like group, whereas staining with DY-550 SA produced the lowest intensity. In the Cy5-like group, nuclei stained with SA conjugates of Alexa Fluor 633 SA and Alexa Fluor 647 SA had much greater fluorescence (2.63.5 x 103) and S/N ratios (6.518.3) than the nuclei stained with the SA conjugates of Cy5, DY-630, and DY-635 (fluorescence = 0.31.8 x 103 and S/N ratios = 1.54.6). In the Cy5.5-like group, nuclei stained with Alexa Fluor 680 SA (fluorescence = 1.1 x 103) were twice as bright as nuclei stained with DY-680 SA (fluorescence = 0.5 x 103), but both were less fluorescent than the most intensely fluorescent nuclei of the other two groups.
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Discussion |
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Although the Alexa Fluor 555 and Alexa Fluor 647 dyes have fluorescence emission properties similar to those of Cy3 and Cy5 dyes, respectively, quantitative analyses of photobleaching demonstrated that the Alexa Fluor 555 and Alexa Fluor 647 dyes were more photostable than their Cy3 and Cy5 counterparts (Figure 1). The decreased photobleaching seen with the Alexa Fluor dyes suggests that Alexa Fluor dyeprotein conjugates may be more photostable than the Cy3 and Cy5 dye conjugates, which would allow more time for repeated viewing of labeled samples and image capture.
To assess the fluorescence properties of dyes conjugated to proteins, dyes were conjugated to several proteins at various DOLs (Figure 2). In the Cy3-like comparison at DOL values higher than 2, the Alexa Fluor 555 dye conjugates were significantly brighter than the Cy3 dye conjugates and, at higher DOL values, the difference in fluorescence between the Alexa Fluor 555 GAR and the Cy3 GAR sharply increased (Figure 2A). This trend continued and was even more pronounced in comparisons of Alexa Fluor 647 GAR and Cy5 GAR (Figure 2B). In particular, the fluorescence of the Alexa Fluor 647 GAR remained high at all DOLs, whereas the Cy5 GAR conjugates at higher DOLs had significantly less fluorescence and some conjugates were virtually nonfluorescent. This difference in fluorescence intensities between the Alexa Fluor 647 and Cy5 dye conjugates was also observed with other proteins (GAM, SA, ConA, and Tf) (Figures 2C2F), particularly at higher DOLs. Quenching appeared to be an important factor in this difference in brightness. Conjugates containing Alexa Fluor 555 or Alexa Fluor 647 dyes were brighter, at least in part, because they exhibited much less of the self-quenching characteristic of Cy dye conjugates (Gruber et al. 2000; Hahn et al. 2001
; Anderson and Nerurkar 2002
) (Figure 3).
When commercially produced, dye conjugates are sold at a predetermined and presumably optimal DOL. However, by constructing their own conjugates, researchers have the flexibility to optimize DOLs for their particular needs. To compare commercially prepared conjugates with conjugates prepared for this study, the effectiveness of labeling GAM with the Alexa Fluor 647 or Cy5 dyes was determined by assaying the fluorescence of cells labeled with these conjugates. The Alexa Fluor 647 GAM resulted in greater labeling intensity than did the Cy5 GAM, regardless of the method of preparation (Figure 4). Even when compared to panels of commercially available conjugates with DOL values higher or lower than the Alexa Fluor samples (Figure 5), the fluorescence of cells labeled with the Alexa Fluor 555 or Alexa Fluor 647 dye conjugates was greater than that with the Cy3 or Cy5 dye conjugates, respectively.
These results are consistent with other studies using Cy dye conjugates. DNA probes labeled with multiple Cy3 dyes were found to be less stable in probe:DNA duplexes than unlabeled probes, due in part to dyedye interactions (Randolph and Waggoner 1997). Gruber et al. (2000)
found that the fluorescence of Cy3, Cy5, and other Cy dyes conjugated to antibodies and other proteins was significantly quenched relative to the unconjugated dye, particularly at high DOL, an observation confirmed in this study (Figure 2). Some heavily labeled Cy5 conjugates were reported to be essentially nonfluorescent (Gruber et al. 2000
; Hahn et al. 2001
; Anderson and Nerurkar 2002
), an observation also confirmed in this study (Figures 2B, 2E, 2F, and 5).
In addition to flow cytometry, fluorescence microscopy was used to compare SA conjugates of a variety of long-wavelength dyes. Staining of HEp-2 nuclei showed significant differences in fluorescence intensity and S/N ratios between the Alexa Fluor dye SA conjugates and the SA conjugates of other dyes (Table 2). In each of the groups, Cy3-like, Cy5-like, or Cy5.5-like, the nuclei stained with the Alexa Fluor dye SA conjugate had the highest fluorescence intensity in the group. Regardless of DOL, nuclei stained with Alexa Fluor dye SA conjugates consistently had fluorescence intensities that were higher than those of most conjugates in the group. Nuclei stained with the Alexa Fluor 555 SA conjugates or the Alexa Fluor 647 SA conjugates had high S/N ratios, and Alexa Fluor 647 SA had the highest S/N value of all conjugates tested. These quantitative comparisons were reinforced by immunodetection with fluorescently labeled GAM secondary reagents (Figures 68). Target nuclear, mitochondrial, or cytoskeletal proteins detected with the appropriate primary antibody and labeled with an Alexa Fluor 555 or Alexa Fluor 647 dye conjugate as a secondary reagent gave a stronger fluorescent signal than proteins labeled with the corresponding Cy3 or Cy5 dye conjugate. Quantitatively, cell and tissue targets labeled with the Cy3 or Cy5 dye conjugate counterparts required as much as 1.3- to 2.5-fold longer exposure times to produce the same pixel saturation as targets labeled with the Alexa Fluor 555 or Alexa Fluor 647 dye conjugates.
In tandem conjugates, a phycobiliprotein, such as R-PE, can be covalently coupled to a longer-wavelength acceptor dye, such as the Alexa Fluor 647 dye. Excitation of the R-PE donor at a single wavelength, such as with the argon ion laser at 488 nm, results in energy transfer (FRET) to the Alexa Fluor 647 acceptor dye and in an effective Stokes shift of over 100 nm. Such constructs enable simultaneous detection of multiple targets (Glazer and Stryer 1983). When the dye partner in a tandem conjugate is a long-wavelength dye, the trend of greater fluorescence in Alexa Fluor dyes is particularly important. Not only is there little change in the absorbance and fluorescence spectra of Alexa Fluor dyes when conjugated to proteins but there is also greater TF than there is with the Cy dyes at the same DOL. In contrast, the self-quenching characteristic of the Cy5 and Cy7 dyes results in decreased FRET efficiencies in tandem constructs containing these dyes. The functional difference between tandem constructs made with Alexa Fluor dyes and those made with Cy dyes is readily apparent in the comparison of cells labeled with the Alexa Fluor 647 R-PE SA tandem conjugate to cells labeled with the Cy5 R-PE SA tandem conjugate [eightfold more fluorescence in a flow cytometry application (Figure 10) and threefold more fluorescence in FRET analysis (Figure 9)]. The Alexa Fluor 680 APC SA tandem conjugate (
fivefold brighter labeling than with Cy7 APC SA tandem conjugate) (Figure 11A), the Alexa Fluor 700 APC SA tandem conjugate (
fivefold brighter) (Figure 11B), and the Alexa Fluor 750 APC SA tandem conjugate (
1.4-fold brighter) (Figure 11C) also overcome the limitation, caused by the quenching, of the Cy7 fluorophore. Thus, either as conventional dyeprotein conjugates or as dyephycobiliproteinprotein tandem conjugates, the long-wavelength Alexa Fluor dyes were brighter than the corresponding Cy dyes and other long-wavelength dyes in each of the applications we evaluated.
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Acknowledgments |
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Alexa Fluor is a registered trademark of Molecular Probes, Inc. Cy is a trademark of Amersham Biosciences.
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Footnotes |
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