Guide to Red Fluorescent Proteins and Biosensors for Flow Cytometry (original) (raw)
Flow Cytometric Applications of the New Fluorochrome RED670
Annals of the New York Academy of Sciences, 1993
RED670, a newly developed energy-transfer fluorochrome,' has properties uniquely suited for use in flow cytometry. RED670 is composed of R-phycoerythrin (R-PE) covalently coupled to Cy5.18@ dye (Cy5 is a trademark of Biological Detection Systems) to form an energy-transfer fluorochrome. The absorbance and emission spectra shown in FIGURE 1 illustrate the spectral properties of the fluorochrome, including the highly efficient energy transfer. Excitation of RED670 at 488 nm results in a very intense emission with a maximum at 667 nm and a 179-nm stokes shift, properties that make the dye suitable for use with an argon ion laser in a flow cytometer. Residual fluorescent emission intensity due to the R-PE at 575 nm was only 6% of that of the Cy5.18 at 667 nm. The fluorescent emission intensities of streptavidin-and antibody-RED670 conjugates were tested as a function of time after storage at various temperatures to ascertain their stability. After four months of storage at 4 "C, room temperature, and 37 "C, the fluorescent emission intensity of both the streptavidin and the antibody conjugates at 667 nm remained constant at about 350 units. The fluorescent emission intensity at 575 nm increased slightly over four months from 21 to 23,21 to 28, and 21 to 41 units for the conjugates stored at 4 "C, room temperature, and 37 "C, respectively. The dye seems to be stable under the conditions tested. Both two-and three-color flow cytometry experiments can be readily performed with RED670 conjugates. A single argon ion laser can be used to simultaneously excite fluorescein-, R-PE-, and RED670-labeled reagents and the signals from these reagents can be differentiated by detection in three separate photomultiplier tubes (PMT) using appropriate filters. A comparison of the performance of a fluorescein/ R-PE pair to a fluorescein/RED670 pair in a two-color experiment was performed using spleen cells from Balb/c mice. The mouse cells were probed with anti-CD8afluorescein and biotinylated anti-Thyl.2; then, one set of spleen cells was incubated with streptavidin-R-PE, whereas a second set of cells was incubated with streptavidin-RED670. The fluorescein channel had to be compensated 4% due to the R-PE signal detected in the fluorescein channel, but only 1% compensation was required for the RED670 signal detected in the fluorescein channel. No compensation was required to remove fluorescein from the RED670 channel, but 26% compensation had to be employed to remove the fluorescein signal from the R-PE channel. Use of RED670 minimized the amount of color compensation required to perform a two-color flow cytometry experiment. A three-color experiment has also been performed to assess the amounts of color compensation required when fluorescein-, R-PE-, and RED670labeled probes were used. The cell surface markers, TCRa/P, CD8a, and CD4, were
Toward quantitative fluorescence measurements with multicolor flow cytometry
Cytometry Part A, 2008
A procedure is presented for calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC). The procedure involves two steps. First, each of the fluorescence channels of the flow cytometer is calibrated using Ultra Rainbow beads with assigned values of equivalent number of reference fluorophores (ERF). The objective of this step is to establish a linear relation between the fluorescence signal in a given fluorescence channel of multicolor flow cytometers and the value of ERF. The second step involves a biological standard such as a lymphocyte with a known number of antibody binding sites (e.g., CD4 binding sites). The biological standard is incubated with antibodies labeled with one type of fluorophores for a particular fluorescence channel and serves to translate the ERF scale to an ABC scale. A significant part of the two-step calibration procedure involves the assignment of ERF values to the different populations of Ultra Rainbow beads. The assignment of ERF values quantifies the relative amount of embedded fluorophore mixture in each bead population. It is crucial to insure that the fluorescence signal in a given range of fluorescence emission wavelengths is related linearly to the assigned values of ERF. The biological standard has to posses a known number of binding sites for a given antibody. In addition, this antibody has to be amenable to labeling with different types of fluorophores associated with various fluorescence channels. The present work suggests that all of the requirements for a successful calibration of a multicolor flow cytometer in terms of ABC values can be fulfilled. The calibration procedure is based on firm scientific foundations so that it is easy to envision future improvements in accuracy and ease of implementation. Published 2007 Wiley-Liss, Inc. y
Flow Cytometry: The Next Revolution
Cells
Unmasking the subtleties of the immune system requires both a comprehensive knowledge base and the ability to interrogate that system with intimate sensitivity. That task, to a considerable extent, has been handled by an iterative expansion in flow cytometry methods, both in technological capability and also in accompanying advances in informatics. As the field of fluorescence-based cytomics matured, it reached a technological barrier at around 30 parameter analyses, which stalled the field until spectral flow cytometry created a fundamental transformation that will likely lead to the potential of 100 simultaneous parameter analyses within a few years. The simultaneous advance in informatics has now become a watershed moment for the field as it competes with mature systematic approaches such as genomics and proteomics, allowing cytomics to take a seat at the multi-omics table. In addition, recent technological advances try to combine the speed of flow systems with other detection me...
Flow Cytometry: A Blessing and a Curse
Biomedicines
Flow cytometry is a laser-based technology generating a scattered and a fluorescent light signal that enables rapid analysis of the size and granularity of a particle or single cell. In addition, it offers the opportunity to phenotypically characterize and collect the cell with the use of a variety of fluorescent reagents. These reagents include but are not limited to fluorochrome-conjugated antibodies, fluorescent expressing protein-, viability-, and DNA-binding dyes. Major developments in reagents, electronics, and software within the last 30 years have greatly expanded the ability to combine up to 50 antibodies in one single tube. However, these advances also harbor technical risks and interpretation issues in the identification of certain cell populations which will be summarized in this viewpoint article. It will further provide an overview of different potential applications of flow cytometry in research and its possibilities to be used in the clinic.
Biophysical Journal, 2010
Far-red fluorescent proteins are required for deep-tissue and whole-animal imaging and multicolor labeling in the red wavelength range, as well as probes excitable with standard red lasers in flow cytometry and fluorescence microscopy. Rapidly evolving superresolution microscopy based on the stimulated emission depletion approach also demands genetically encoded monomeric probes to tag intracellular proteins at the molecular level. Based on the monomeric mKate variant, we have developed a far-red TagRFP657 protein with excitation/emission maxima at 611/657 nm. TagRFP657 has several advantages over existing monomeric far-red proteins including higher photostability, better pH stability, lower residual green fluorescence, and greater efficiency of excitation with red lasers. The red-shifted excitation and emission spectra, as compared to other far-red proteins, allows utilizing TagRFP657 in flow cytometry and fluorescence microscopy simultaneously with orange or nearred fluorescence proteins. TagRFP657 is shown to be an efficient protein tag for the superresolution fluorescence imaging using a commercially available stimulated emission depletion microscope.
Towards in vivo flow cytometry
Journal of biophotonics, 2009
Cytometry is the characterization and measurement of cells and cellular constituents for biological, diagnostic and therapeutic purposes, embracing the fields of cell and molecular biology, biochemistry, biophysics, cell physiology, pathology, immunology, genetics, biotechnology, plant biology and microbiology. Cytometry is based on quantitative measurements of the molecular and phenotypic properties of cells using flow and image cytometry, microarrays, and proteomics. Cytomics (molecular cell systems research) aims at the understanding of the molecular architecture and functionality of cell systems (cytomes) by single-cell analysis in combination with exhaustive bioinformatic knowledge extraction. The cytomics concept has been significantly advanced by a multitude of current developments like confocal and laser scanning microscopy, multiphoton fluorescence excitation, spectral imaging, fluorescence resonance energy transfer (FRET), fast imaging in flow, optical stretching in flow, and miniaturized flow and image cytometry within laboratories on a chip or laser microdissection, as well as the use of bead arrays [1, 2]. Data sieving or data mining of the vast amounts of collected multiparameter data for exhaustive multilevel bioinformatic knowledge extraction avoids the inadvertent loss of information from unknown molecular relations being inaccessible to an a priori hypothesis. These approaches will become powerful tools for such important fields as individualized medicine, drug discovery and drug development [3-6]. This special issue is focused on state-of-the-art research in advanced cytometry and application areas with particular emphasis on novel biophotonic methods, disease diagnosis, and monitoring of disease treatment at single-cell level in stationary and flow conditions. It seeks to advance scholarly research that spans from fundamental interactions between light, cells, vascular tissue, and labeling particles, to strategies and opportunities for preclinical and clinical research. Recent advances in slide-based cytometry for in vitro application are summarized by Gerstner et al. [7]. They show that single cell based quantitation using microscope based cytometry instruments is making its way from basic research into clinical use. Calibration and quality control are essential in every cytometric analysis. Basics of standardization and
Flow cytometry: a literature review
Revista de Ciências Médicas e Biológicas, 2016
Introduction: flow cytometry is a technique that employs an optical-electronic detection apparatus to analyze the physical and chemical properties of microscopic particles suspend in a liquid medium. Objective: to review the literature in search of the main studies that used flow cytometry as the main methodology. Method: Articles were selected according to their impact factor in the Journal of Citation Reports. Literature review: a light beam is direct to a continuous flow of suspended particles marked with fluorescent substances. The light is scattered differently from the beam by the particles and is captured by sensors in line and perpendicular to the light beam. These microscopic particles are conjugated with fluorescent substances that, once excited, emit light of lower frequency than the light source. The emitted light is captured by sensors and the particles are analyzed according to fluctuations in brightness of each detector and/or fluorescence emission. The result of this process is the formation of images in real time for each cell fluorescence, scattering and transmission of light. A major problem of flow cytometry is to determine whether a subset of cells labeled with fluorochrome-conjugated monoclonal antibodies is positive or negative. Gains compensation should be determined and applied correctly, and controls should be conducted concisely with the adoption of a biological control, isotype control or Fluorescence Minus One (FMO). None of these controls are considered ideal, and must be chosen according the number of different labeling done, rarity of molecule expression on surface or intracellularly in certain cell subsets, overlap of wavelengths or unspecific binding of the fluorochrome-conjugated antibodies. Conclusion: due to its great potential, flow cytometry has been expanded to diverse fields of biological sciences, and is routinely used in clinical diagnostic, biotechnology, and basic and applied research.
Journal of Histochemistry & Cytochemistry, 2012
Imaging flow cytometry (IFC) platforms combine features of flow cytometry and fluorescent microscopy with advances in data-processing algorithms. IFC allows multiparametric fluorescent and morphological analysis of thousands of cellular events and has the unique capability of identifying collected events by their real images. IFC allows the analysis of heterogeneous cell populations, where one of the cellular components has low expression (<0.03%) and can be described by Poisson distribution. With the help of IFC, one can address a critical question of statistical analysis of subcellular distribution of proteins in a cell. Here the authors review advantages of IFC in comparison with more traditional technologies, such as Western blotting and flow cytometry (FC), as well as new high-throughput fluorescent microscopy (HTFM), and discuss further developments of this novel analytical technique.
Flow Cytometry and Its Applications to Molecular Biology and Diagnosis 2.0
International Journal of Molecular Sciences
Flow cytometry is a single-cell based technology aimed to quantify the scattering of light and the emission of multiple fluorescence signals by individual cells, biological vesicles, or synthetic microscopical particles when examined one by one at high speed using lasers or other suitable illumination sources [...]
Flow cytometry for high-throughput, high-content screening
Current Opinion in Chemical Biology, 2004
Flow cytometry is a mature platform for quantitative multiparameter measurement of cell fluorescence. Recent innovations allow up to 30-fold faster serial processing of bulk cell samples. Homogeneous discrimination of free and cellbound fluorescent probe eliminates wash steps to streamline sample processing. Compound screening throughput may be further enhanced by multiplexing of assays on color-coded bead or cell suspension arrays and by integrating computational techniques to create smaller, focused compound libraries. Novel bead-based assay systems allow studies of real-time interactions between solubilized receptors, ligands and molecular signaling components that recapitulate and extend measurements in intact cells. These new developments, and its broad usage, position flow cytometry as an attractive analysis platform for high-throughput, high-content biological testing and drug discovery.
mScarlet: a bright monomeric red fluorescent protein for cellular imaging
Nature Methods, 2016
We report the engineering of mscarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mscarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mscarlet's crystal structure reveals a planar and rigidified chromophore. mscarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a förster resonance energy transfer (fret) acceptor in ratiometric imaging. Fluorescent proteins (FPs) have become indispensable in biological research 1. After the cloning of GFP from the jellyfish Aequorea victoria 2 , several GFP spectral variants were developed, including blue, cyan and yellow FPs 3. The palette of FPs was greatly expanded after the cloning of red FP (RFP) homologs from corals and other Anthozoa species 4,5. However, all Anthozoa RFPs form obligate tetramers, which can seriously interfere with localization and functioning of RFP-fusion proteins. Monomerization of tetrameric RFPs was accompanied by a serious deterioration of the brightness and incomplete and/or partial green maturation of the resulting monomer 6. After the development of the first monomeric RFP (mRFP), mRFP1 (ref. 6), several improved mRFPs have been reported: mCherry 7 , mApple 8 , TagRFP(-T) 8,9 , mKate2 (ref. 10), mRuby2 (ref. 11), mRuby3 (ref. 12) and FusionRed 13 (reviewed in ref. 14). But all these mRFPs are dimmer than their tetrameric ancestors; they have quantum yields below 50%, and several still harbor additional problems due to incomplete or partial green maturation and a residual tendency to dimerize 15. Spectral variants of FPs can be applied in FRET-based biosensors to probe molecular interactions, conformational changes and metabolite concentrations within living cells 16. While good FP-based FRET pairs are available with cyan FPs (CFPs) as donors
Fluorogen activating proteins in flow cytometry for the study of surface molecules and receptors
Methods, 2012
The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g. EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extracellular milieu. FAPs are GFPsized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes ('activate the fluorogen'). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000-fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (b2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well.
Journal of Biomedical Optics, 2007
The recent introduction of the in vivo flow cytometer for real-time, noninvasive detection and quantification of cells circulating in the vasculature of small animals has provided a powerful tool for tracking the roles of different types of cells in disease progression. We describe a portable version of the device, which provides the capability to: a͒ excite and detect fluorescence at two distinct colors simultaneously, and b͒ perform data analysis in real time. These advances improve significantly the utility of the instrument and provide a means of increasing detection specificity. As examples, we present the depletion kinetics of circulating green fluorescent protein ͑GFP͒labeled breast cancer cells in the vasculature of mice, and the specific detection of circulating hematopoietic stem cells labeled in vivo with two antibodies.
The Relevance of Flow Cytometry for Biochemical Analysis
IUBMB Life, 2001
Flow cytometry (FCM) allows the simultaneous measurement of multiple fluorescences and light scatter induced by illumination of single cells or microscopic particles in suspension, as they flow rapidly through a sensing area. In some systems, individual cells or particles may be sorted according to the properties exhibited. By using appropriate fluorescent markers, FCM is unique in that multiple structural and functional parameters can be quantified simultaneously on a single‐particle basis, whereas up to thousands of biological particles per second may be examined. FCM is increasingly used for basic, clinical, biotechnological, and environmental studies of biochemical relevance. In this critical review, we summarize the main advantages and limitations of FCM for biochemical studies and discuss briefly the most relevant parameters and analytical strategies. Graphical examples of the biological information provided by multiparametric FCM are presented. Also, this review contains spec...
Flow cytometry in biotechnology
Applied Microbiology and Biotechnology, 2001
Flow cytometry is a general method for rapidly analyzing large numbers of cells individually using light-scattering, fluorescence, and absorbence measurements. The power of this method lies both in the wide range of cellular parameters that can be determined and in the ability to obtain information on how these parameters are distributed in the cell population. Flow cytometric assays have been developed to determine both cellular characteristics such as size, membrane potential, and intracellular pH, and the levels of cellular components such as DNA, protein, surface receptors, and calcium. Measurements that reveal the distribution of these parameters in cell populations are important for biotechnology, because they better describe the population than the average values obtained from traditional techniques. This Mini-Review provides an overview of the principles of flow cytometry, with descriptions of methods used to measure various cellular parameters and examples of the application of flow cytometry in biotechnology. Finally, a discussion of the challenges and limitations of the method is presented along with a future outlook. A flow cytometry system consists of five main operating units (Fig. ): a light source (mercury lamp or laser), flow cell, optical filter units for specific wavelength detection over a broad spectral range, photodiodes or pho-
High-throughput flow cytometry compatible biosensor based on fluorogen activating protein technology
Cytometry Part A, 2013
Monitoring the trafficking of multiple proteins simultaneously in live cells is of great interest because many receptor proteins are found to function together with others in the same cell. However, existing fluorescent labeling techniques have restricted the mechanistic study of functional receptor pairs. We have expanded a hybrid system combining fluorogen-activating protein (FAP) technology and high-throughput flow cytometry to a new type of biosensor that is robust, sensitive, and versatile. This provides the opportunity to study multiple trafficking proteins in the same cell. Human beta2 adrenergic receptor (b2AR) fused with FAP AM2.2 and murine CC chemokines receptor type 5 fused with FAP MG13 was chosen for our model system. The function of the receptor and the binding between MG13 and fluorogen MG-2p have been characterized by flow cytometry and confocal microscopy assays. The binding of fluorogen and the FAP pair is highly specific, while both FAP-tagged fusion proteins function similarly to their wild-type counterparts. The system has successfully served as a counter screen assay to eliminate false positive compounds identified in a screen against NIH Molecular Libraries Small Molecule Repository targeting regulators of the human b2AR.
Modern flow cytometry: a practical approach
Clinics in laboratory …, 2007
In this presentation, we outline ways in which current users of Fluorescence Activated Cell Sorting (FACS) can get more from their FACS work without undue effort. FACS technology development, and the emergence of new software support for various aspects of this technology are now cooperating in this effort.
Microfluidic Flow Cytometer for Quantifying Photobleaching of Fluorescent Proteins in Cells
2012
Here we present additional photophysical information on mOrange2, mCherry, TagRFP, and TagRFP-T including in vitro studies to determine the percent fluorescence recovery after photobleaching, photostabilities, and lifetimes. Furthermore, additional experimental details regarding in vitro analysis of the proteins, theoretical calculations and experimental setup are provided. Lastly, cytometry intensity calibration plots using commercially available fluorescent polystyrene beads are presented.