Flow cytometry antigens

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... 

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... 

R. B. Alexander, E. S. Bolton, S. Koenig, G. M. Jones, S. L. Topalian, C. H. June, and S. A. Rosenberg, Detection of antigen specific T lymphocytes by determination of intracellular calcium concentration using flow cytometry, J. Immunol. Methods 148, 131-141 (1992). 

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). 

In another experimental setting in vitro data on the cell surface antigen expression were obtained for PBMCs isolated from chronic lymphocytic leukemia (CLL) patient blood samples and cultured in the presence of DIMS (according to standard procedures). Cultured cells were collected and stained with the corresponding antibodies for detection of the expression of cell surface antigens. Subsequendy they were analyzed by flow cytometry. 

Positive selection is not recommended as these procedures may induce signaling pathways in CLL cells due to antibody/antigen interactions. Purity of CLL cells is assessed by flow cytometry using anti-CD19, anti-CD5, and matching isotype control... 

Roe, R., Robins, R. A., Laxton, R R., and Baldwin, R W (1985) Kinetics of divalent monoclonal antibody binding to tumor cell surface antigens using flow cytometry—standardization and mathematical analysis Mol Immunol. 22, 11—21... 

Traill, K. N., Bock, G, Winter, U, Hilchenbach, M., Jurgens, G., and Wick, G. (1986) Simple method for comparing large numbers of flow-cytometry histograms exemplified by analysis of the CD4 (T4) antigen and LDL receptor on human peripheral-blood lymphocytes. J Histochem. Cytochem. 34, 1217-1221... 

Many proliferation-associated antigens have been reported as clinically useful indicators of proliferative activity (1). Of these, the so-called proliferating cell nuclear antigen (PCNA) and Ki-67 have been identified as the most useful in both immunohistochemistry (see Chapter 27) and flow cytometry (FCM). PCNA is an auxiliary protein to DNA polymerase 8 (2,3) and is intimately associated with DNA replication, but also DNA repair (4,5). Ki-67 is a large protein associated with nuclear nonhistone proteins (6,7), and is expressed in all actively proliferating cells (8,9). Expression of these two proteins, in a cell population should equate to the growth fraction, i.e., the proportion of cells involved in an active cell cycle. However, there are apparent inconsistencies when these two proteins have been compared with one another (10) and with other methods of assessing cell proliferation (11). 

Fig. 1. Scheme showing the basic strategies available for the detection of nuclear antigens by flow cytometry. PF, paraformaldehyde EtOH, ethanol MeOH, methanol. 

Prosperi, E, Stivala, N. A., Sala, E., Scovassi, A. I., and Bianchi, L (1993) Proliferating cell nuclear antigen complex formation induced by ultraviolet irradiation in human quiescent fibroblasts as detected by lmmunostaming and flow cytometry Exp Cell Res 205, 320-325. 

This will depend on the affinity of the MAb and the density of the antigen on the cells, but a final concentration of 5 pg/mL will be adequate for most commercially available MAbs If necessary, the concentration can be determined empirically by using serial dilutions of MAb and analyzing uptake by flow cytometry (see Chapter 33)... 

The limitations of the application of conventional detergents mentioned above can be circumvented by replacing this approach with cell membrane permeabilization by microwave heating. Improved detection of intracellular antigens can be obtained with microwave heating used in combination with flow cytometry. This approach yields histogram patterns that show clear discrimination between intact cells and cell debris (Fig. 9.5). 

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