Tandem Dye Research Articles

Polychromatic (eight‐color) slide‐based cytometry for the phenotyping of leukocyte, NK, and NKT subsets

Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing. Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets. In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets. With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time.

Single wavelength excitation fluorescence cross-correlation spectroscopy with spectrally similar fluorophores: Resolution for binding studies

It was shown recently that fluorescence cross-correlation spectroscopy (FCCS) can be performed using a single laser wavelength for excitation (SW-FCCS) [L. C. Hwang and T. Wohland, Chem. Phys. Chem 5, 549 (2004).]. This method simplifies the FCCS setup since it does not require the simultaneous alignment of two lasers to the same focal spot. But up to now the method was shown to work only with dyes possessing large Stokes' shifts, and thus was limited to the use of quantum dots and tandem dyes. In this work we show that standard organic dyes with overlapping emission spectra, for instance fluorescein and tetramethylrhodamine, can be used as fluorescent pairs in SW-FCCS. As a biological model system for ligand-receptor interactions we studied the binding of biotin to streptavidin. To investigate the applicability of SW-FCCS for binding studies we adapt the existing FCCS theory for SW-FCCS and calculate limits for the measurement of dissociation constants in dependence on sample concentration, sample purity, and spectral cross talk between the different detection channels.

Report of the Third Hungarian cell analysis conference: 16–18 May 2002, Budapest, Hungary

This conference was a successful continuation of previous conferences such as the Hungarian Cell Analysis Conference, Budapest, 1998 and 2000, and the ISAC-Sponsored International Conference for Flow Cytometry and Image Analysis, Epona, 1999. The Cell Analysis Section of the Hungarian Biophysical Society organized all four conferences. The popularity of the conference was hallmarked by the large number of participants (close to 300). The form of the conference proved to be very attractive: In the mornings, experts from specific fields delivered scientific lectures. In addition to Hungarian scientists, such as Margit Balazs, Gyula Hadlacky, Bela Molnar, Laszlo G. Puskas, Janos Szollosi, and Gyorgy Vereb, experts from abroad significantly raised the scientific standards of the conference. The international speakers included Peter Adorjan, Francis Mandy, Abe Schwartz, Howard M. Shapiro, and the authors of this introduction. In the afternoons, practical demonstrations were presented, ranging from basic techniques to stateof-the-art instrumentation. Practical training included cell culturing; mechanical cell separation for flow cytometry; methods for detection of cell proliferation; detection of apoptosis; fluorescence microscopy, image acquisition, and processing; confocal laser microscopy; fluorescence in situ hybridization; magnetic cell separation; magnetic mRNA isolation; real-time and traditional polymerase chain reaction; and detection of mutation. During the conference the attendees were able to choose six sessions of 11 topics. These demonstrations were headed by one of the speakers and sponsored by commercial companies that provided special kits and/or instruments to ensure a stable basis for successful practical training. An impressive selection of commercial companies provided contacts and improved awareness of advanced analytical cytology for the participants. The purpose of the conference was multifaceted. It served as a forum for presentations from Hungarian scientists, and, with the active participation of foreign experts, it opened a window to state-of-the art methods and techniques. The conference also facilitated the research cooperation between well-established scientists and Ph.D. graduate students, with the major purpose of the conference being education. The average age of the attendees was approximately 30 as more than 50% of the participants were Ph.D. students. Graduate students were able to earn credit points toward their final examination. The venue for the conference was the Medical University of Semmelweis in Budapest, the capital of Hungary. The university has great traditions, with its name reflecting the rich history of scholastic activity in Budapest. It was over 150 years ago that Ignaz Semmelweis completed his classic epidemiologic study in Budapest, studies that led to effective prophylaxis against childbed fever that still stands as the hallmark of evidence-based studies in epidemiology. At the conference both the lectures and the posters proved to be of high quality, demonstrating the high scientific standards of the community. The topics of the posters ranged from oncology (basic and applied research) to signal transduction, genetics, fertility research, and investigation in lower organisms such as Drosophila and earthworm. The methods applied were varied and highly sophisticated. Of 29 poster presentations, three prizes were awarded. Originally the poster awards committee (Margit Balazs and Attila Tarnok) was supposed to give only one award, but the decision was not easy because of the large number of excellent posters. With the financial help of commercial companies, the committee was able to present three poster awards instead of one. These awards were: First prize: Generation and Characterization by Flow Cytometry of Dendritic Cells, by Gizella Veszely, Janos Fent, Agnes Nagy, and Furesz Jozsef, Department of Pathophysiology, Institute for Health Protection of HDF, Budapest. Second prize: Hemocyte-Specific Molecular Markers in the Hematopoiesis and Innate Immunity of Drosophila melanogaster, by Istvan Nagy, Eva Kurucz, and Istvan Ando, Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Szeged. Third prize: Astrocytes Support the Neuronal Differentiation of Neuroectodermal Progenitor Cells, by Vanda Szlavik, Zsuzsanna Kornyei, and Emilia Madarasz, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest. On the following pages, the reader will find the rich program of the Third Hungarian Cell Analysis Conference through the lecture and poster abstracts.

The History and Future of the Fluorescence Activated Cell Sorter and Flow Cytometry: A View from Stanford

The Fluorescence Activated Cell Sorter (FACS) was invented in the late 1960s by Bonner, Sweet, Hulett, Herzenberg, and others to do flow cytometry and cell sorting of viable cells. Becton Dickinson Immunocytometry Systems introduced the commercial machines in the early 1970s, using the Stanford patent and expertise supplied by the Herzenberg Laboratory and a Becton Dickinson engineering group under Bernie Shoor. Over the years, we have increased the number of measured FACS dimensions (parameters) and the speed of sorting to where we now simultaneously measure 12 fluorescent colors plus 2 scatter parameters. In this history, I illustrate the great utility of this state-of-the-art instrument, which allows us to simultaneously stain, analyze, and then sort cells from small samples of human blood cells from AIDS patients, infants, stem cell transplant patients, and others. I also illustrate analysis and sorting of multiple subpopulations of lymphocytes by use of 8-12 colors. In addition, I review single cell sorting used to clone and analyze hybridomas and discuss other applications of FACS developed over the past 30 years, as well as give our ideas on the future of FACS. These ideas are currently being implemented in new programs using the internet for data storage and analysis as well as developing new fluorochromes, e.g., green fluorescent protein and tandem dyes, with applications in such areas as apoptosis, gene expression, cytokine expression, cell biochemistry, redox regulation, and AIDS. Finally, I describe new FACS methods for measuring activated kinases and phosphatases and redox active enzymes in individual cells simultaneously with cell surface phenotyping. Thus, key functions can be studied in various subsets of cells without the need for prior sorting.

Near‐infrared dyes for six‐color immunophenotyping by laser scanning cytometry

To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. The optical filters of the LSC were replaced-photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC.

Phycoerythrin-allophycocyanin: A resonance energy transfer fluorochrome for immunofluorescence

As immunofluorescence experiments become more complex, the demand for new dyes with different properties increases. Fluorescent dyes with large Stoke's shifts that are very bright and have low background binding to cells are especially desirable. We report on the properties of the resonance energy tandems of phycoerythrin and allophycocyanin (PE-APC). PE-APC is the original fluorescence resonance energy tandem dye described in the literature, but it has not been utilized because of the difficulty of synthesizing and preparing a consistent product. PE-APC complexes comprising different ratios of the two phycobiliproteins conjugated to streptavidin were synthesized using standard protein-protein conjugation chemistry. The PE-APC streptavidins were evaluated for flow cytometric analysis. They were compared directly to Cy5PE conjugates because Cy5PE is the fluorophore that is spectrally most like the PE-APC. PE-APC complexes showed the expected fluorescence spectral properties of a tandem: excitation was excellent at 488 nm (and best at the PE excitation maximum) and emission was greatest at the APC emission maximum at about 660 nm. The efficiency of transfer of energy from PE to APC was about 90%. PE-APC can be considered an excellent substitute for Cy5PE. Compared with Cy5PE, PE-APC has similar brightness (in staining experiments), slightly greater compensation requirements with PE but much lower compensation with Cy5.5PE or Cy5.5PerCP, and lower nonspecific background binding. PE-APC is a useful alternative to Cy5PE, especially in applications in which the use of Cy5 is impractical. Cytometry 44:24-29, 2001. Published 2001 Wiley-Liss, Inc.

Four color compensation

Four-color immunophenotyping can now be routinely performed using either a single laser or dual laser flow cytometer. When a single laser instrument is used, the fluorochromes evaluated are usually FITC, PE, PE-TR and PE-CY5 (or PerCP). For two-laser excitation APC is generally used in place of PE-TR. Since each tandem dye construct contains PE, three of the four detectors are affected and compensation can be problematic. In this report we show that each tandem conjugated antibody, whether different batches from the same supplier or conjugates from different suppliers all require unique compensation. This inconsistency results in erroneous data, negates the use of single labeled particles as a method for providing adequate compensation and requires dual and triple labeled cells of known pattern to verify compensation. It is also shown that improper compensation can reduce or eliminate completely the detection of fluorescence emission from PECY5 conjugated antibodies. These problems are caused by a variation in energy transfer between PE and either TR or CY5 because the chemistry involved in preparation and conjugation to antibodies is not sufficiently controlled to produce reagents with uniform compensation requirements. The variation in tandem dye compensation can be addressed by either using the same tandem conjugated antibody, by using the same second step tandem reagent to an appropriate first step antibody or by using software compensation. The latter provides an easy solution because a unique compensation matrix can be produced for each antibody tandem conjugate. Cytometry (Comm. Clin. Cytometry) 38:161–175, 1999. © 1999 Wiley-Liss, Inc.

Four color compensation.

Four-color immunophenotyping can now be routinely performed using either a single laser or dual laser flow cytometer. When a single laser instrument is used, the fluorochromes evaluated are usually FITC, PE, PE-TR and PE-CY5 (or PerCP). For two-laser excitation APC is generally used in place of PE-TR. Since each tandem dye construct contains PE, three of the four detectors are affected and compensation can be problematic. In this report we show that each tandem conjugated antibody, whether different batches from the same supplier or conjugates from different suppliers all require unique compensation. This inconsistency results in erroneous data, negates the use of single labeled particles as a method for providing adequate compensation and requires dual and triple labeled cells of known pattern to verify compensation. It is also shown that improper compensation can reduce or eliminate completely the detection of fluorescence emission from PECY5 conjugated antibodies. These problems are caused by a variation in energy transfer between PE and either TR or CY5 because the chemistry involved in preparation and conjugation to antibodies is not sufficiently controlled to produce reagents with uniform compensation requirements. The variation in tandem dye compensation can be addressed by either using the same tandem conjugated antibody, by using the same second step tandem reagent to an appropriate first step antibody or by using software compensation. The latter provides an easy solution because a unique compensation matrix can be produced for each antibody tandem conjugate.

Validation of a flow cytometric assay detecting in vitro basophil activation for the diagnosis of muscle relaxant allergy

Background: Anaphylactic reactions during anesthesia are mainly the result of muscle-relaxant (MR) drugs. Skin tests, serologic detection of specific IgE, and in vitro leukocyte histamine release are used to investigate MR allergy. Objective: We describe a new assay that is based on the detection by flow cytometry of the altered expression of plasma membrane molecules of MR-activated basophils. Methods: For this assay, which we have named the BASIC assay, basophils are incubated in vitro with MR, after which they are fixed and then triple labeled with fluorescein-conjugated anti-CD63, tandem dye R-phycoerythrin-cyanin 5.1 conjugated anti-CD45, and R-phycoerythrin conjugated anti-IgE. The resulting B asophils’ A ltered S urface I mmunofluorescence is detected by flow C ytometry (BASIC). Results: Forty-one patients who had an allergic reaction during general anesthesia and 23 control subjects without such a history were studied. All included subjects’ basophils were tested in the BASIC assay with at least 4 MR: suxamethonium, gallamine, vecuronium, and pancuronium. After reaction of the basophils of the MR-allergic patients with MRs, increased surface expression of CD63 and CD45 and decreased expression of IgE were detected. Increased expression of CD63 was observed most frequently and it was stronger than the alteration of the 2 other markers. Cross-reactivity between MRs commonly occurred. MRs diluted 10 –1 activate the basophils of the control subjects, suggesting that at relatively high concentrations MRs are also nonspecific basophil activators. Conclusion: In the diagnosis of MR allergy, the BASIC assay has a good specificity but a low sensitivity, and it correlates strongly with skin test results. It is currently appraised for the diagnosis of anaphylactic reaction induced by other classes of drugs. (J Allergy Clin Immunol 1999;104:411-8.)

Application of fluorescence resonance energy transfer in the clinical laboratory: Routine and research

Fluorescence resonance energy transfer (FRET) phenomenon has been applied to a variety of scientific challenges in the past. The potential utility of this biophysical tool will be revisited in the 21st century. The rapid digital signal processing in conjunction with personal computers and the wide use of multicolor laser technology in clinical flow cytometry opened an opportunity for multiplexed assay systems. The concept is very simple. Color-coded microspheres are used as solid-phase matrix for the detection of fluorescent labeled molecules. It is the homogeneous assay methodology in which solid-phase particles behave similarly to the dynamics of a liquid environment. This approach offers a rapid cost-effective technology that harnesses a wide variety of fluorochromes and lasers. With this microsphere technology, the potential applications for clinical flow cytometry in the future are enormous. This new approach of well-established clinically proven methods sets the stage to briefly review the theoretical and practical aspects of FRET technology. The review shows various applications of FRET in research and clinical laboratories. Combination of FRET with monoclonal antibodies resulted in a boom of structural analysis of proteins in solutions and also in biological membranes. Cell surface mapping of cluster of differentiation molecules on immunocompetent cells has gained more and more interest in the last decade. Several examples for biological applications are discussed in detail. FRET can also be used to improve the spectral characteristics of fluorescent dyes and dye combinations, such as the tandem dyes in flow and image cytometry and the FRET primers in DNA sequencing and polymerase chain reactions. The advantages and disadvantages of donor-acceptor dye combinations are evaluated. In addition, the sensitivity of FRET provides the basis for establishing fast, robust, and accurate enzyme assays and immunoassays. Benefits and limitations of FRET-based assays are thoroughly scrutinized. At the end of the paper we review the future of FRET methodology.

Conjugation of Fluorochromes to Monoclonal Antibodies

Detection of cell surface molecules labeled by monoclonal or polyclonal antibodies conjugated to a fluorochrome is probably the most widely used application of flow cytometry. This unit contains protocols for tagging monoclonal antibodies with fluorescein, biotin, Texas Red, and phycobiliproteins. In addition, it provides a procedure for preparing a PE-Texas Red tandem conjugate dye that can then be used for antibody conjugation. These protocols enable investigators to label antibodies of their choice with multiple fluorochromes and permit more combinations of antibodies for multicolor flow applications. flow cytometry; monoclonal antibodies; fluorochromes; antibody labeling.

Allo-7: a new fluorescent tandem dye for use in flow cytometry.

This report describes the development of a novel tandem dye by combining allophycocyanine (APC) and cyanine dye indotricarbocyanine (CY7) to create ALLO-7 for use in flow cytometry. The APC donor fluorophore was excited at 647 nm and, through resonance energy transfer to the CY7 acceptor, produced fluorescence at > 780 nm. To test the applicability of this tandem in single and multicolor immunofluorescence, a streptavidin conjugate of the tandem (SA-ALLO-7) was used for the detection of cell surface antigens on human peripheral blood leukocytes (PBL) by indirect immunofluorescence. Human PBL were stained with CD4/ GaM-APC, CD3-fluorescein isothiocyanate (FITC), CD14-phycoerythrin (PE), CD19-energy-coupled dye (phycoerythrin-Texas Red) (ECD), and CD8-biotin with SA-ALLO-7 and analyzed for fluorescence on a FACS Vantage using dual-laser excitation (488 and 647 nm). The results indicated that the percentage of cells positive for each of the surface antigens was comparable for single-color controls and multicolor samples. The ALLO-7 fluorescence, which was collected with a 730-shortpass dichroic mirror and a 790/50-bandpass filter, was clearly resolved from the APC fluorescence and that from FITC, PE, and ECD. The SA-ALLO-7 exhibited minimal nonspecific binding to PBL monocytes. However, the specific binding of the tandem to high-density antigens was clearly identified by positive fluorescence. This unique tandem reagent, ALLO-7, provided the capability for dual-color immunofluorescence with a 647-nm laser line (or a helium neon laser at 633 nm) and provides the potential to perform three-color analysis with a dye-head laser (Texas Red, APC, ALLO-7).

ALLO‐7: A new fluorescent tandem dye for use in flow cytometry

ALLO‐7: A new fluorescent tandem dye for use in flow cytometry

Cy7PE and Cy7APC: bright new probes for immunofluorescence.

We demonstrate the utility of indotricarbocyanine (Cy7) conjugates of the phycobiliproteins phycoerythrin (PE) and allophycocyanin (APC) in flow cytometry. This is the first demonstration of the use of an APC tandem dye for fluorescence measurements. These resonance energy transfer tandem dyes can be excited by the phycobiliprotein-specific excitation wavelengths and fluoresce at wavelengths above 780 nm. The tandem dyes, when conjugated to antibodies, are suitable for flow cytometry and other immunofluorescence applications. These conjugates are easily detectable above the very low autofluorescence in this part of the spectrum. Indeed, the Cy7-conjugated PE tandem (Cy7PE) has a "brightness" (fluorescence signal over cellular autofluorescence) comparable to that of fluorescein, and the Cy7APC tandem has a "brightness" comparable to that of APC. These tandems are also easily distinguished from other commonly used fluorophores, making them suitable for high-order multiparametric analysis. We show an example of six-color immunofluorescence analysis by flow cytometry, simultaneously measuring fluorescences from fluorescein, PE, Cy5PE, Texas red, APC, and Cy7APC.

Cy7PE and Cy7APC: Bright new probes for immunofluorescence

We demonstrate the utility of indotricarbocyanine (Cy7) conjugates of the phycobiliproteins phycoerythrin (PE) and allophycocyanin (APC) in flow cytometry. This is the first demonstration of the use of an APC tandem dye for fluorescence measurements. These resonance energy transfer tandem dyes can be excited by the phycobiliprotein-specific excitation wavelengths and fluoresce at wavelengths above 780 nm. The tandem dyes, when conjugated to antibodies, are suitable for flow cytometry and other immunofluorescence applications. These conjugates are easily detectable above the very low autofluorescence in this part of the spectrum. Indeed, the Cy7-conjugated PE tandem (Cy7PE) has a “brightness” (fluorescence signal over cellular autofluorescence) comparable to that of fluorescein, and the Cy7APC tandem has a “brightness” comparable to that of APC. These tandems are also easily distinguished from other commonly used fluorophores, making them suitable for high-order multiparametric analysis. We show an example of six-color immunofluorescence analysis by flow cytometry, simultaneously measuring fluorescences from fluorescein, PE, Cy5PE, Texas red, APC, and Cy7APC. © 1996 Wiley-Liss, Inc.

Application of a flow cytometric method using autofluorescence and a tandem fluorescent dye to analyze human alveolar macrophage surface markers

Human resident alveolar macrophages (AM) exhibit autofluorescence when excited by light from 488 nm lasers used by most flow cytometers. Because this autofluorescence occurs at peak 540 nm, it obscures fluorescence generated by commonly used immunofluorescent reagent (e.g., antibodies conjugated to fluorescein isothiocyanate (FITC) or R-phycoerythrin (R-PE)) applied for cell surface marker analysis. Therefore, a two color flow cytometric method has been developed that permits the quantitative phenotypic analysis of AM without influence by their natural autofluorescence. In this method, a commercially available preparation of secondary polyclonal antibodies (recognizing primary specific mouse IgG monoclonal antibodies) that are conjugated to a tandem fluorochrome dye (containing R-PE and Cy5) is used. Using this method, the expression of 12 different surface markers on AM obtained from bronchoalveolar lavage (BAL) of 13 subjects was analyzed and compared with their expression on the surface of peripheral blood monocytes. This method will facilitate analysis of surface markers on AM in a variety of disorders.