Flow fluorochromes

Flame Photometry and Gas Chromatography (CyTerra) -Aerodynamic Particle Size and Shape Analysis (BIRAL) -Flow Cytometry (Luminex, LLNL) -Semiconductor-Based Ultraviolet Light (DARPA) -Polymer Fluorochrome (Echo Technology) -Laser-Induced Breakdown Spectroscopy -Raman Scattering -Infrared Absorption -Terahertz Spectroscopy -UV LIDAR... 

Anderson MT, Baumgarth N, Haugland RP et al (1998) Pairs of violet light excited fluorochromes for flow cytometry analysis. Cytometry 33 435-444... 

The digital data from the flow cytometer are collected in list mode files that contain a record of all of the fluorescent events associated with each individual cell in a sample. For any individual cell, the number of events collected is dependent on the number of fluorochromes utilized in the analysis that bind to the cell, which can be amplified by increasing the variety/number of lasers utilized to excite the fluorochrome. These raw data can then be analyzed on-line or off-line by software provided by the manufacturer of... 

Representative Fluorochromes/Dyes and Applications for Functional Flow Cytometry3... 

J. A. Steinkamp and H. A. Crissman, Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry, Cytometry 14, 210-216 (1993). 

A key feature of apoptosis is that, similar to the cell cycle, the duration of apoptosis is variable and the process is asynchronous in most cell populations. Consequently, there are variable proportions of cells in distinct phases of apoptosis and a sample, collected from a culture in which apoptosis has been induced, will contain cells in all the phases of apoptosis. This could represent a major technical problem in the investigation of apoptosis and requires measurement of metabolic apoptotic changes concomitantly with established markers of apoptosis progression. Using the same cellular model described above, we investigated metabolic alterations induced by X-rays using flow cytometry and a panel of fluorochromes... 

Fluorochromes are stains that interact directly with cellular components, or are used to form conjugates with antibodies or ligands, yielding fluorescent reporter molecules. O Table 13-1 lists a selection of fluoro- D Table 13-1 Fluorochromes for flow cytometry ... 

Fixation procedures are necessary when biohazardous samples are analyzed, and are sometimes used to allow access of membrane impermeant fluorochromes, or to stabilize samples for short-term storage. Optimal fixatives are those that have low autofluorescence and do not significantly affect staining. Paraformaldehyde, at concentrations of 0.5-2%, and ethanol (70%, 4 C) are widely used fixatives for flow cytometry. Combinations of paraformaldehyde with Triton X-100 or saponin have been employed in procedures that fix and permeabilize cells. 

The analysis of cell-cycle progression was one of the earliest applications of flow cytometry (for review, see Darzynkiewicz et al., 2004). In this assay, fluorescence signals from cells stained with DNA-binding fluorochromes are plotted as DNA content histograms that may be analyzed by using histogram deconvolution software to quantify cell-cycle phase distributions (Rabinovitch 1994). Fluorochromes that are useful for this purpose are the plasma membrane-impermeant DNA stains, propidium iodide (PI),... 

Telford WG, Hawley TS, Hawley RG. 2003. Analysis of violet-excited fluorochromes by flow cytometry using a violet laser diode. Cytometry 54A 48-55. 

A major limitation of flow cytometric analysis is that it provides data from individual cells at a single point in time and the same cells are not available for further analysis once they have passed through the flow cell of the instrument. Therefore, it is not possible to monitor a given cell over time for changes in fluorescence intensity or distribution of fluorescence signal. Such studies require microinjection of the fluorochrome into individual cells and fluorescence microscopy analysis. 

If iV-formyl peptide chemoattractants conjugated with fluorochrome are purchased from a commercial source, the reagents listed in Subheading 2. referencing this note are not needed. Although this chapter has focused on iV-formyl peptides, other chemoattractants, such as complement-derived C5a, have also been fluorescently labeled and evaluated in flow cytometric assays (9-11). 

A limitation of the flow cytometric binding assay has been the precise determination of the receptor affinity and calculation of the receptors per cell. This limitation appears to have been overcome by the development of fluorescein and phycoerythrin compensation-calibration standards (Flow Cytometry Standards Corp., Research Triangle Park, NC). These standards have made it possible to quantify the fluorescence intensity of samples labeled with fluorescein or phycoerythrin, and relate the intensity to molecules of equivalent soluble fluorochrome. These standards have been utilized in quantitative studies of neutrophil chemoattractant-ligand interaction (4). 

Calibrator beads with known fluorescence intensity defined in terms of equivalent molecules of soluble fluorochrome (e.g., Quantum Beads, Flow Cytometry Standards Corporation). 

Having considered conditions for the binding of fluorochrome-labeled antibody to reflect antigen density, the most appropriate flow cytometric analysis method can then be selected. Some of these considerations will be specific to particular manufacturers flow cytometers and cannot be addressed in detail here there are, however, common approaches, that are generally applicable, and appreciation of the principles involved will at least allow some of the more obvious pitfalls to be avoided. 

A similar method is used for beads with defined fluorochrome content (e g, Quantum beads, Flow Cytometry Standards Corporation), except that the beads are not subjected to the antibody-staining procedure The number of fluorochrome molecules per cell is deduced from the standard curve, and is converted to bound antibody molecules from knowledge of the fluorochrome protein ratio of the conjugate. 

The potential for multi-fluorochrome analysis has increased further with the availability of dyes excited by shorter wavelengths (such as Cascade blue) and longer wavelengths (such as Allophycocyamn and Texas red). With multiple laser flow cytometers, five cell-surface antigens (1) or more may be analyzed simultaneously. In principle, these methods are a natural extension of the methods outlined here, but require more sophisticated flow cytometers, and much more powerful computer facilities to explore the exponentially more complex data sets arising from such multiparameter analyses. 

Because multiple photodetectors are available, a flow cytometer has the ability to measure two or more fluorescence signals simultaneously from the same cell. To use several fluorochromes at the same time, cytometrists with only one laser required a group of stains, all of which absorb 488 nm light but which have different Stokes shifts so that they emit fluorescent light at different wavelengths and thereby can be distinguished from each other by the color of their fluorescence. Propidium iodide and fluorescein are a pair of fluorochromes that fulfill these criteria (having different Stokes shifts) and can be... 

To be of use in microscopy or flow cytometry, this bond needs to be visualized (to the eye or to the photodetector) by the addition of a fluorescent tag. Visualization can be accomplished by one of two different methods. With direct staining, cells are incubated with a monoclonal antibody that has been previously conjugated to a fluorochrome (for example, fluorescein or phycoerythrin or any fluorochrome with appropriate absorption and emission spectra). This procedure is quick and direct it merely involves a half-hour incubation of cells with antibody (at 4°C), followed by several washes to remove weakly or nonspecifically bound antibodies. Cells thus treated are ready for flow analysis (although final fixation with 1% electron microscopic-grade formaldehyde will provide a measure of biological safety and long-term stability). 

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