Flow cytometry cell surface markers

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). 

Flow cytometry is now commonly used in immunotoxicity studies to assess changes in relative frequency and number of lymphoid and myeloid cells in the spleen, lymph nodes, bone marrow and/or peripheral blood of rodents, and in the peripheral blood of humans. A list of selected cell surface markers useful in immunotoxicity studies is shown in Table 7.3. Notably, the majority of available reagents are specific for murine antigens with human reagent availability a close second. Reagents for rat, primate, and... 

Lymphoid cell surface marker expression (Flow cytometry using fluorescent labels)... 

WG Intracellular and cell surface markers (flow cytometry)... 

Krutzik PO, Clutter MR, Nolan GP. Coordinate analysis of murine immune cell surface markers and intracellularphosphoproteins by flow cytometry. J Immunol 2005 175(4) 2357-65. 

Detection methods are critical for future automation, since detection can be a rate-limiting step in an automated process. In addition to common detection methods used in manual IA techniques, other detection methods have been applied to automation. For example, ECL technology has been used for nucleic acids in the IGEN system. Cell surface markers are detected by the Copalis system using a fluorescent conjugate and flow cytometry. New detection methods for drug IAs as well as PD measurements for efficacy and safety should prove useful in the future. 

Flow cytometry has been at the forefront of immunophenotyping (see Chapter 3.2). Immunophenotyping is the term used to describe the application of fluorescently tagged antibodies to discriminate and quantitate the various constituent cells of the immune system by flow cytometry. Immunophenotyping cells on the basis of cell surface markers can be thought of as the flrst generation application of flow cytometry in immunotoxicology. 

Hematopoietic cells can be identified by cell surface markers via flow cytometry. If testing of a purified population of either HSCs or differentiated cells is desired, hematopoietic cells can be isolated by fluorescence activated cell sorting (FACS) or magnetic separation by either positive or negative selection of known hematopoietic surface markers. Continued study of the markers which distinguish HSCs and their derivatives to clearly identify HSCs must be done in order to further examine and utilize these various cell populations. 

Flow cytometry A method of measuring the number of cells in a sample, the percentage of five cells in a sample, and certain characteristics of cells, such as size, shape, and the presence of tumor markers on the cell surface. The cells are stained with a light-sensitive dye, placed in a fluid, and passed in a stream before a laser or other type of light. The measurements are based on how the light-sensitive dye reacts to the light. 

H., Nakahata, T. (2005). Prospective characterization of neural stem cells by flow cytometry analysis using a combination of surface markers. J Neurosci Res. 80, 456-66. 

Studies of stem cell progression towards the completely differentiated mature cells have already identified several intermediary precursors, organized in a cascade (Shizuru et al., 2005). The best known and studied cellular differentiation cascade is the hematopoietic system (Figure 20.3). Within hematopoiesis, it is possible to identify many intermediary precursors between the hematopoietic stem cell and mature blood cells. This identification is based mainly on the phenotypic profile of cellular surface proteins, using flow cytometry as the main tool. This is a relatively simple technique that involves coupling a monoclonal antibody (mAb) with a fluorescent marker (fluorochrome). In this way, diverse cellular markers can be combined and thus a cellular subpopulation can be defined, as shown in Figure 20.3. 

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