Flow cytometry biology

Darzynkiewicz, Z., Crissman, H.A. (eds.), Methods in Cell Biology Flow Cytometry, Vol. 33 Academic Press, New York (1990). 

Although the concept of flow cytometry originated in studies of blood cells and tumor cells (for review, see Darzynkiewicz et ak, 2004), flow cytometric procedures are now used routinely in disciplines as diverse as immunology, the neurosciences, nutritional sciences, pharmacology, parasitology, and marine biology. This chapter offers an introduction to the theory and practice of analytical flow cytometry, with emphasis on applications in the neurosciences. 

It was shown by Creech and Jones (1) in 1940 that proteins, including antibodies, could be labeled with a fluorescent dye (phenylisocyanate) without biological or immunological effects to the intended target. In theory, fluorescent reporters (tracers, probes, antibodies, stains, and so on) can be used to detect or measure any cell constituent, provided that the tag reacts specifically and stoichiometrically with the cellular constituent in question (2). Today, the repertoire of fluorescent probes is expanding almost daily see Chapter 14). One area that has benefited from the ever-increasing number of fluorescent probes is flow cytometry. 

Keren, D. F. (1989) Clinical molecular cytometry merging flow cytometry with molecular biology in laboratory medicine, in Flow Cytometry in Clinical Diagnosis (Keren, D. F., Hanson, C. A., and Hurtubise, P. E., eds.), Chicago, ASCP, pp. 614-634. 

The combination of the specificity of the antigen-antibody interaction with the exquisite sensitivity of fluorescence detection and quantitation yields one of the most widely applicable analytical tools in cell biology (1). Within the last decade, flow cytometry (FCM) has become an integral part of basic immunological research. Elaboration of this technology has been intensively stimulated by a rapidly growing sophistication in monoclonal antibody technology and vice versa (2). 

Chapter 1 in Melamed et al., Chapter 3 in Shapiro, and Chapter 1 in Darzynkiewicz are good historical reviews of flow cytometry. Alberto Cambrosio and Peter Keating (2000) have used flow cytometry as a model for looking at historical changes in the way scientists use instrumentation to view the world Of lymphocytes and pixels The techno-visual production of cell populations. Studies in History and Philosophy of Biological and Biomedical Sciences 31 233-270. 

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). 

Flow cytometry has been applied in many creative ways to the science of molecular biology. Flow sorting, based on Floechst 33258 and chromomycin A3 fluorescence, turns out to be one of the best ways available for obtaining relatively pure preparations of each type of chromosome. Even those chromosomes that are not distinguishable by their fluorescence (e.g., 9-12) can usually be sorted from hamster-human hybrid cell lines. These preparations of reasonably... 

Fixation Fixation is the process by which the protein of cells is denatured, or cross-linked, and preserved. Fixation in flow cytometry is used to inactivate hazardous biological material and also to preserve stained cells when there is not immediate access to a flow cytometer. Fixation is also important in preserving proteins before detergent permeabilization for intracellular staining. Formaldehyde is often the fixative of choice for flow cytometry because it preserves the forward and side scatter characteristics of cells (but does cause some increase in their autofluorescence). 

Sometimes, cellular analysis was performed with cells in a flow stream, which is also termed flow cytometry or FACS in the field of cell biology. For instance, human blood cell (WBC, RBC) rheology was studied in channels fabricated on the Si-Pyrex substrates. The channels were either uncoated or coated with albumin [825]. 

Fig. 6.3. Cutoff fluorescence selection for screening. Instrumentation, labeling, and biological noise introduce spreading into a fluorescence measurement, such that the fluorescence probability distributions for wild-type and mutant cells overlap. The logarithm of single-cell fluorescence as measured by flow cytometry is generally well-approximated by a symmetrical Gaussian curve. A cutoff fluorescence value is selected for screening, with all cells above that value sorted out. The enrichment factor forthe mutants is the ratio of (dotted + striped areas)/(striped area), and the probability of retention of a given mutant clone at a single pass is the (striped + dotted area)/(all area under mutant curve).

The difficulty of separation is highly dependent on peak spreading, as shown in Fig. 6.5. It is therefore critical to minimize the peak width as far as possible. This would be difficult for cell display methods if only single color fluorescent labeling were used, because the primary source of variability is biological. Flow cytometry instrumentation point spread functions generally contribute below 2 % to the overall coefficient of variance (CV = standard deviation/mean), but typical overall CVs for yeast display are approximately 50 - 100 % for the logarithmic fluorescence intensity. 

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