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Quantitative Comparison of Long-wavelength Alexa Fluor Dyes to Cy Dyes: Fluorescence of the Dyes and Their Bioconjugates

Quantitative Comparison of Long-wavelength Alexa Fluor Dyes to Cy Dyes: Fluorescence of the Dyes and Their Bioconjugates

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Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins.
Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins.

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 © The Histochemical Society, Inc.
 Volume 51(12): 1699–1712, 2003The Journal of Histochemistry & Cytochemistry
 Quantitative Comparison of Long-wavelength Alexa Fluor Dyesto Cy Dyes: Fluorescence of the Dyes and Their Bioconjugates
 Judith E. Berlier, Anca Rothe, Gayle Buller, Jolene Bradford, Diane R. Gray, Brian J. Filanoski,William G. Telford, Stephen Yue, Jixiang Liu, Ching-Ying Cheung, Wesley Chang,James D. Hirsch, Joseph M. Beechem, Rosaria P. Haugland, and Richard P. Haugland
Molecular Probes, Inc., Eugene, Oregon (JEB,AR,GB,JB,DRG,BJF,SY,JL,C-YC,WC,JDH,JMB,RPH,RPH) and ExperimentalTransplantation and Immunology Branch, NCI-NIH, Bethesda, Maryland (WGT)
-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyeswith principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700nm, and 750 nm were conjugated to antibodies and other selected proteins. These conju-gates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7,DY-630, DY-635, DY-680, and Atto 565 dyes. As
-hydroxysuccinimidyl ester dyes, the AlexaFluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction co-efficients. However, both Alexa Fluor dyes were significantly more resistant to pho-tobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugatesprepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates ofthe Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. Theanomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presum-ably due to the formation of dye aggregates. Absorption of light by the dye aggregatesdoes not result in fluorescence, thereby diminishing the fluorescence of the conjugates.The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited signifi-cantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyeswere significantly more fluorescent than those of the Cy dyes, especially at high degrees oflabeling. The results from our flow cytometry, immunocytochemistry, and immunohisto-chemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluordyes have advantages compared to the Cy dyes and other long-wavelength dyes in typicalfluorescence-based cell labeling applications.
 J Histochem Cytochem 51:1699–1712, 2003
 of fluorescent dyes with emissionmaxima beyond 550 nm and into the near infraredhave become valuable tools in histochemical and cy-tochemical research because these long-wavelengthdyes expand the range of options for multicolor fluo-rescence detection. The sulfonated indocyanine dyesCy3, Cy5, Cy5.5, and Cy7 (Amersham Biosciences;Piscataway, NJ) are examples of long-wavelength dyescommonly used in fluorescence microscopy, flow cy-tometry, and nucleic acid-based detection (Mujumdaret al. 1993,1996; Brismar et al. 1995; Roederer et al.1996; Flanagan et al. 1997; Fradelizi et al. 1999;Toutchkine et al. 2002). Long-wavelength dyes arewidely used because they are optimally excited bylight sources typical of many fluorescence microscopesand flow cytometers (Sowell et al. 2002). In addition,these dyes fluoresce at wavelengths longer than theusual sources of cell autofluorescence, and the back-ground fluorescence of the dyes is generally low (Cul-lander 1994; Flanagan et al. 1997; Haugland 2002;Sowell et al. 2002). However, the loss of fluorescenceof the conjugated Cy dyes (Gruber et al. 2000; Ander-son and Nerurkar 2002) limits their usefulness.This work compares newly available, water-soluble, amine-reactive
 -hydroxysuccinimidyl esters (SEs, also
 Correspondence to: James D. Hirsch, Molecular Probes, Inc.,29851 Willow Creek Road, Eugene, OR 97402. E-mail: jim.hirsch@probes.comReceived for publication April 9, 2003; accepted July 30, 2003(3A6072).
Alexa Fluor dyesCy dyeslong-wavelength dyesfluorescent bioconjugatesphotostabilityimmunofluorescenceFRETflow cytometrymicroscopy
 Berlier, Rothe, Buller, Bradford, Gray, Filanoski, Telford, Yue, Liu, Cheung, Chang, Hirsch, Beecham,Haugland, Haugland 
 known as NHS esters) of Alexa Fluor dyes (MolecularProbes; Eugene, OR) with the SEs of the Cy series of dyes and some other currently available long-wave-length dyes. Except for Alexa Fluor 633, which is asulfonated rhodamine derivative, all of these new Al-exa Fluor dyes are sulfonated carbocyanine molecules.The dyes in both series can be grouped by absorptionmaxima and are referred to here as Cy3-like, Cy5-like,and so forth. In the Cy3-like group, the Alexa Fluor555, DY-550, and Atto 565 dyes are spectrally com-parable to Cy3 (Cy 3.18, 3.29). The Alexa Fluor 633,Alexa Fluor 647, DY-630, and DY-635 dyes can becompared to Cy5 (Cy 5.18, 5.29) (Cy5-like), and inthe Cy5.5-like group, the Alexa Fluor 660, AlexaFluor 680, and DY-680 dyes are comparable to Cy5.5. In the last group, Cy7-like, the Alexa Fluor 700and Alexa Fluor 750 dyes can be compared to Cy7.The dyes and protein conjugates of the dyes werecompared spectrally, and the functionality of dye–pro-tein conjugates was examined in several applications.Flow cytometry and fluorescence microscopy wereused to compare dye conjugates, which are composedof a dye covalently linked to a protein, such as goatanti-mouse IgG antibody (GAM), goat anti-rabbit IgGantibody (GAR), streptavidin (SA), concanavalin A(ConA), or transferrin (Tf), at similar degrees of label-ing. The results indicate that Alexa Fluor dyes, whoseabsorption and emission spectra are spectrally similarto those of the Cy dyes, are more resistant to fluores-cence quenching and absorption spectral artifacts onconjugation to proteins. At high degrees of labeling,the Alexa Fluor dyes undergo much less self-quench-ing than the other dyes tested. For protein labeling,the ability of Alexa Fluor dyes to retain their intensefluorescence even when the conjugates are heavily la-beled suggests that the long-wavelength Alexa Fluordyes have advantages compared to the Cy dyes andspectrally similar long-wavelength dyes.In addition to the conventional conjugates, tandemconjugates were also analyzed. These tandem conju-gates consist of three components: an absorber [a phy-cobiliprotein, either R-phycoerythrin (R-PE) or allo-phycocyanin (APC)], an emitter (one of the long-wavelength Alexa Fluor or Cy dyes), and a protein(e.g., streptavidin) for conferring bioaffinity. Phycobil-iproteins, which are fluorescent light-harvesting pro-teins from cyanobacteria and some eukaryotic algae,act first as an energy absorber, then as a donor trans-ferring energy to the dye. Tandem fluorescent mole-cules result in an increase in the effective Stokes shiftof the reagent. The fluorescence emission peak of thereagent is effectively extended, by means of fluores-cence resonance energy transfer (FRET), to a longerwavelength 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 tan-dem conjugates containing Cy dyes. The tandem con-structs involving Alexa Fluor dyes displayed higherFRET efficiencies and were functionally brighter inflow cytometry applications than comparable tandemconstructs of the Cy dyes.
Materials and Methods
 SE derivatives of Alexa Fluor dyes with absorption maximanear 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm,and 750 nm from Molecular Probes were compared to SEderivatives of Cy3, Cy5, Cy5.5, and Cy7 from AmershamBiosciences, SE derivatives of DY-630, DY-635, and DY-680 from Dyomics (MoBiTec; Göttingen, Germany), andthe SE derivative of Atto 565 from Fluka (Milwaukee, WI).Unlabeled GAM, GAR, and streptavidin (SA) starting mate-rials were from Molecular Probes. Concanavalin A (ConA)was purchased from EY Laboratories (San Mateo, CA), andhuman holo-transferrin was from Sigma/Aldrich (St Louis,MO). Alexa Fluor dye conjugates of GAM, GAR, or SAfrom Molecular Probes were compared to Cy3 and Cy5 dyeconjugates of GAM, GAR, or SA from several vendors, in-cluding Rockland Immunochemicals (Gilbertsville, PA),Amersham Biosciences, Kirkegaard & Perry Laboratories(Gaithersburg, MD), Chemicon International (Temecula,CA), Zymed Laboratories (South San Francisco, CA), Ab-cam (Cambridge, UK), Jackson ImmunoResearch Laborato-ries (West Grove, PA), and Caltag Laboratories (Burlin-game, CA). The degree of labeling (DOL
 the number of moles of dye incorporated per mole of protein) provided bythe manufacturers for each of the Cy3 and Cy5 conjugateswas compared to the DOL determined independently forthis study. The tandem conjugates analyzed were AlexaFluor 647 R-PE SA, Alexa Fluor 680 APC SA, Alexa Fluor700 APC SA, Alexa Fluor 750 APC SA (Molecular Probes),Cy5 R-PE SA (BD Biosciences; San Jose, CA), and Cy7 APCSA (Caltag Laboratories and BD Biosciences). All other lab-oratory reagents were the highest grade commercially avail-able and were used as received.
 Absorption spectra were acquired with a U-2000 spectro-photometer (Hitachi Instruments; Boulder, CO). Fluores-cence absorption and emission data were obtained withan Aminco–Bowman Series II Luminescence Spectrometer(Thermo Spectronic; Rochester, NY).Flow cytometry experiments were performed using aCoulter Elite flow cytometer (Beckman Coulter; MiamiLakes, FL) equipped with a 488-nm argon ion laser and a575-nm bandpass filter for detecting cells labeled with theAlexa Fluor 555 or Cy3 dye conjugates. To detect far-redfluorescence in cells labeled with the Alexa Fluor 647 or Cy5dye conjugates, the Coulter Elite flow cytometer wasequipped with a 633-nm He–Ne laser, a 675-nm bandpassemission filter, and a 640 nm dichroic longpass filter.
Comparison of Long-wavelength Dye Bioconjugates
 EXPO32 software (Beckman Coulter, version 1.0) was usedfor sample acquisition and analysis. The fluorescence in-tensity of cells labeled with Alexa Fluor 647 R-PE SA or Cy5R-PE SA tandem conjugates was measured with a FACSCal-ibur benchtop flow cytometer (BD Biosciences) equippedwith a 635-nm red diode laser. Data were acquired and ana-lyzed with CellQuest v. 3.3 software (BD Biosciences).The fluorescence microscopes (Meridian Instrument Com-pany; Kent, WA) used were a Nikon Eclipse E400 for pho-tobleaching and immunofluorescence brightness determina-tion and a Nikon Eclipse E800 for cell and tissue imaging.Optical filters (Omega Optical; Brattleboro, VT) used to vi-sualize Alexa Fluor 555 and Cy3 dye conjugates were theOmega XF32 and XF102 filters, and the filter used to detectAlexa Fluor 647 and Cy5 dye conjugates was the OmegaXF110. Images were acquired with a MicroMAX digitalcamera (Princeton Scientific Instruments; Monmouth Junc-tion, NJ) with a
 1300 1030 charged-coupled device (CCD)array (Roper Scientific; Trenton, NJ), controlled by Meta-Morph software (Universal Imaging; Downingtown, PA).
Fluorescence Spectral Profiles
 Extinction coefficients for all unconjugated dyes in methanolwere provided by the manufacturers. The relative quantumyield (RQY, the integrated photon emission relative to thatof an appropriate dye standard) of each conjugate was cal-culated using the following standard dyes: 5-(and-6)-carboxy-tetramethylrhodamine (Molecular Probes) for the Cy3, Atto565, and Alexa Fluor 555 dyes; DDAO (7-hydroxy-9
 (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-680dyes; oxazine 1 perchlorate (Eastman Kodak; Rochester, NY)for the Alexa Fluor 700 dye; and IR125 (Lamda Physik; FtLauderdale, FL) for the Alexa Fluor 750 and Cy7 dyes. Forfluorescence analysis, the conjugates were matched at identi-cal absorbance to that of the standard dye at the appropriateexcitation wavelength. The total fluorescence (TF, the prod-uct of the RQY and the DOL) was used to measure fluores-cence output (brightness) of the conjugate (Haugland 2000).
 Glass capillary tubes filled with 0.5
 M solutions of AlexaFluor 555, Alexa Fluor 647, Cy3, or Cy5 SE dye derivatives inPBS, pH 7.5, were excited with light emitted by the 100-Wmercury arc lamp of the Nikon Eclipse E400 fluorescence mi-croscope. Using the
 40 objective, integrated fluorescenceemission intensity under continuous illumination was mea-sured initially and then every 5 sec for 95 sec, and the observedfluorescence intensities were normalized to the initial values.
Labeling Reactions
 Protein conjugates containing the Alexa Fluor dyes wereprepared, purified, and characterized as described previously(Panchuk–Voloshina et al. 1999; Haugland 2000; Hahn etal. 2001). Cy, Atto, and Dyomics dye conjugates wereprepared according to manufacturers’ instructions. TheDOL of each conjugate was determined spectrophotometri-cally as previously described (Panchuk–Voloshina et al. 1999;Haugland 2000). To evaluate dye R-PE SA tandem conju-gates by FRET, samples were matched for equal absorptionby equalizing their optical density at the excitation wave-length of 488 nm.
Flow Cytometry
 Flow cytometry was used to compare the fluorescence inten-sity of cells labeled with dye–protein conjugates prepared forthis study as described above or labeled with commerciallyavailable conjugates. For comparison of dye–GAM conju-gates, human peripheral blood was collected in a Vacutainertube containing sodium heparin (BD Biosciences). An ali-quot (100
 l) of the whole blood was added to a 3-ml plas-tic tube and blocked with 10% normal goat serum (NGS) inPBS on ice for 10 min. After addition of mouse anti-humanCD3 antibody (1
 g) (Caltag Laboratories) to label T-cells,or PBS (5
 l) as a control, the tubes were incubated on icefor 30 min. Cells were washed, resuspended to 100
 l withPBS, and incubated with secondary reagent (dye–GAM con-jugates; 0.5
 g) on ice for another 30 min. Erythrocytes inthe sample were lysed by the addition of 2.5 ml ammoniumchloride lysis buffer (0.15 M ammonium chloride, 0.01 Mpotassium bicarbonate, and 0.1 mM EDTA) (Stewart andStewart 2001). The labeled cells were analyzed by flow cy-tometry with gating for lymphocytes.The fluorescence intensity of cells labeled with AlexaFluor 647 R-PE SA or Cy5 R-PE SA tandem conjugates wascompared by flow cytometry as described previously (Tel-ford et al. 2001a,b). Briefly, washed EL4 lymphoma cells(ATCC; Manassas, VA) were incubated first with a biotiny-lated anti-CD44 mouse monoclonal antibody, then labeledwith either the Alexa Fluor 647 R-PE SA tandem conjugateor the Cy5 R-PE SA tandem conjugate and analyzed.Flow cytometry was also used to compare a Cy7 APC SAtandem conjugate to APC SA conjugates of Alexa Fluor 680,Alexa Fluor 700, or Alexa Fluor 750 dye. For tandem conju-gates, human peripheral blood was collected in a Vacutainertube as above and centrifuged at 3300
 g for 30 min atroom temperature (RT). Mononuclear cells were harvestedfrom the white-colored layer directly below the plasmalayer. 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
 cells/ml. An aliquot (100
 l) of cell suspension was trans-ferred to a reaction tube and blocked for 10 min at RT withnonspecific mouse IgG antibody (0.1
 g). Cells werewashed, resuspended to 100
 l with PBS, and then incu-bated with 5
 l of either biotinylated mouse anti-humanCD3 antibody (Caltag Laboratories) to label T-cells or withPBS as a control, at RT for 15 min. Cells were washed andsecondary reagents containing streptavidin (tandem con-structs) (0.1–1
 g per reaction) were added. Samples wereprotected from light, incubated at RT for 15 min, washed,and analyzed by flow cytometry with gating for lympho-cytes.
Immunofluorescence Microscopy
 Immunocytochemistry experiments with the bioconjugateswere conducted on prepared slides with fixed HEp-2 human

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