Guide to Red Fluorescent Proteins and Biosensors for Flow Cytometry (original) (raw)
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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.
Near infrared in vivo flow cytometry for tracking fluorescent circulating cells
Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2015
The in vivo flow cytometry (IVFC) is now a powerful technique in biomedical research, especially for tracking specific cells in circulatory system. The current fluorescence-based IVFC is limited to visible spectrum, while near infrared (NIR) dyes have their advantages, such as deeper penetration, less absorption and less scattering for NIR fluorescence. Here, using an NIR in vivo flow cytometer with a 785 nm laser excitation, the measurement of fluorescent dye IR-780 labeled circulating cells is demonstrated. Representative peaks corresponding to NIR fluorescent circulating cells are detected and quantified. In addition, blood flow information, including the blood flow velocity and flow volume per unit time, is obtained. By simultaneous detection of IR-780 and enhanced green fluorescent protein (EGFP) signals from dual labeled cells, the IR-780 is shown to be a suitable fluorescent dye for multicolor detection by IVFC, including NIR. Thus, the IVFC is extended to the NIR range and s...
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.