Flow cytometry fluorescent antibodies

EIA was originally developed as a histological technique to localize specific ceUular sites using the specificity of an immunological reaction (23). The resulting fluorescent antibodies can be detected in animal tissues at levels as low as 1 /tg/mL of body fluid. Eluorophore-labeled antibodies have also been used widely for flow cytometry appHcations using fluorescein antibodies to cell surface markers to detect and quantify specific cells (24). 

Covalent attachment of antibody molecules to liposomes can provide a targeting capacity to the vesicle that can modulate its binding to specific antigenic determinants on cells or to molecules in solution. Antibody-bearing liposomes may possess encapsulated components that can be used for detection or therapy (Figure 22.17). For instance, fluorescent molecules encapsulated within antibody-targeted vesicles can be used as imaging tools or in flow cytometry... 

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 ... 

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. 

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). 

BrdU/DNA flow cytometry offers flexibility and diversity in the study of cell kinetics from cells in culture to human tumors in vivo. The essence of the procedure is to pulse label with BrdU by a short-term incubation in vitro or by a single injection in vivo samples are then taken at time intervals thereafter and stained after fixation in ethanol. The cells are then stained with a monoclonal antibody against BrdU that can be either directly conjugated to a fluoro-chrome (usually fluorescein isothiocyanate [FITC]) or, alternatively, bound to a second antibody conjugated with FITC. The cells are then counterstained with propidium iodide (PI) to measure the DNA content and analyzed on the flow cytometer. The results are displayed as linear-red fluorescence on the x-axis vs linear or log-green fluorescence on they-axis. 

This method illustrates well the precision with which antibody binding can be measured by flow cytometry, and the use of linear amplification of the fluorescence signal. 

Successful cell surface display of the protein can be verified by inducing gene expression via addition of anhydrotetracycline to the culture medium and immunofluorescence staining of the cells using an antibody directed against the protein to be displayed (Protocol 1). Analysis can be performed by fluorescence microscopy or flow cytometry. 

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). 

Although many applications of flow cytometry involve the staining of cells for proteins expressed on the outer membrane, cells also have many proteins that are not displayed on their surface. With appropriate procedures, flow cytometry can provide a means to analyze these intracellular proteins. The outer cell membrane is impermeable to large molcules like antibodies however, if we intentionally fix cells to stabilize proteins and then disrupt the outer membrane, the cells can be stained with fluorochrome-conjugated monoclonal antibodies against intracellular proteins. After time to allow the antibodies to pass through the now-permeabilized membrane, the cells are washed to remove loosely bound antibodies and then are run through the flow cytometer to measure their fluorescence intensity. 

Lymphocyte A lymphocyte is a particular type of white blood cell that is involved in many of an organism s immune responses. Subpopulations of lymphocytes with microscopically identical anatomy can be distinguished because their surface membranes contain different arrays of proteins. The staining of these proteins with fluorescently tagged monoclonal antibodies allows the subpopulations to be enumerated by flow cytometry. 

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