Abstract
Flow cytometry is the rapid analysis of populations of cells on a cell-by-cell basis by passing them individually through a beam of laser light, and the measurement of light emitted from the cells by scatter or fluorescence (1,2). Integrins have been studied by flow cytometry since the mid-1980s when immunologists started examining CD11 (integrins αM, αX, and αL) as markers of T-cell differentiation in mixed cell populations (3,4). These analyses highlight many of the advantages and utility of flow cytometry over other forms of integrin analysis. With the appropriate antibody, flow cytometry can identify individual cells in a mixed cell population. Cell-surface expression of integrins can be differentiated from intracellular pools by flow cytometry analysis, unlike Western blotting or immunoprecipitation. Multicolor analysis can simultaneously measure the levels of two or three different integrins, enabling the researcher to determine that both an α and β subunits are present on the plasma membrane. Lastly, flow cytometry can sterilely sort viable cells on the basis of their cell-surface integrin expression and retain their viability for further growth and analysis.
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References
Givan, A. L. (1992) Flow Cytometry: First Principles, Wiley-Liss, New York, NY.
Darzynkiewicz Z., Robinson, J. P., and Crissman, H. A. (1994) Methods in Cell Biology, vols. 41 and 42, Flow Cytometry, 2nd ed., Academic, San Diego, CA.
Cohen J. H., Aubry, J. P., Banchereau, J., and Revillard J. P. (1988) Identification of cell subpopulations by dual-color surface immunofluorescence using biotinylated and unlabeled monoclonal antibodies. Cytometry 9, 303–308.
Yamada, H., Martin, P. J., Bean, M. A., Braun, M. P., Beatty, P. G, Sadamoto, K., and Hansen J. A. (1985) Monoclonal antibody 9. 3 and anti-CD11 antibodies define reciprocal subsets of lymphocytes. Eur. J. Immunol. 15, 1164–1168.
Hynes, R. O. (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11–25.
Frelinger, A. L. 3d, Cohen, I., Plow, E. F., Smith, M. A., Roberts, J., Lam, S. C, and Ginsberg M. H. (1990) Selective inhibition of integrin function by antibodies specific for ligand-occupied receptor conformers. J. Biol. Chem. 265, 6346–6352.
Coopman, P. J., Thomas, D. M., Gehlsen, K. R., and Mueller, S. C. (1996) Integrin α3β1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells. Mol. Biol. Cell 7, 1789–1804.
Filardo, E. J., Deming, S. L., and Cheresh, D. A. (1996) Regulation of cell migration by the integrin beta subunit ectodomain. J. Cell. Sci. 109, 1615–1622.
Keely, P. J., Fong, A. M., Zutter, M. M., and Santoro, S. A. (1995) Alteration of collagen-dependent adhesion, motility, and morphogenesis by the expression of antisense α2 integrin mRNA in mammary cells. J. Cell Sci. 108, 595–607.
Maemura, M., Akiyama, S. K., Woods, V. L., and Dickson, R. B. (1995) Expression and ligand binding of α2β1 integrin on breast carcinoma cells. Clin. Exp. Metast. 13, 223–235.
Sonnenberg, A., Janssen, H., Hogervorst, F., Calafat, J., and Hilgers, J. (1987) A complex of platelet glycoproteins Ic and IIa identified by a rat monoclonal antibody. J. Biol. Chem. 262, 10,376–10,383.
Berlin, C, Berg, E. L., Briskin, M. J., Andrew, D. P., Kilshaw, P. J., Holzmann, B., Weissman, I. L., Hamann, A., and Butcher, E. C. (1993) α4β7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 74, 185–195.
Coller, B. S., Beer, J. H., Scudder, L. E., and Steinberg, M. H. (1989) Collagen-platelet interactions: evidence for a direct interaction of collagen with platelet GPIa/IIa and an indirect interaction with platelet GPIIb/IIIa mediated by adhesive proteins. Blood 74, 182–192.
Hangan, D., Uniyal, S., Morris, V. L., MacDonald, I. C, von Ballestrem, C, Chau, T., Schmidt, E. E., Chambers, A. F., Groom, A. C, and Chan, B. M. (1996) Integrin VLA-2 (α2β1) function in postextravasation movement of human rhabdomyosarcoma RD cells in the liver. Cancer Res. 56, 3142–3149.
Cheresh, D. A. and Spiro, R. C. (1987) Biosynthetic and functional properties of an Arg-Gly-Asp-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen, and von Willebrand factor. J. Biol. Chem. 262, 17,703–17,711.
Wayer, E. A., Orlando, R. A., and Cheresh, D. A. (1991) Integrins αvβ3 and αvβ5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface. J. Cell Biol. 113, 919–929.
Bajt, M. L., Ginsberg, M. H., Frelinger, A. L. 3d., Berndt, M. C, and Loftus, J. C. (1992) A spontaneous mutation of integrin αIIbβ3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding site. J. Biol. Chem. 267, 3789–3794.
Shattil, S. J., Hoxie, J. A., Cunningham, M., and Brass, L. F. (1985) Changes in the platelet membrane glycoprotein IIb. IIIa complex during platelet activation. J. Biol. Chem. 260, 11,107–11,114.
Gomez, M., Luque, A., del Pozo, M. A., Hogg, N., Sanchez-Madrid, F., and Cabanas, C. (1997) Functional relevance during lymphocyte migration and cellular localization of activated β1 integrins. Eur. J. Immunol. 27, 8–16.
Luque, A., Gomez, M., Puzon, W., Takada, Y., Sanchez-Madrid, F., and Cabanas, C. (1996) Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β1 chain. J. Biol. Chem. 11,067–11,075.
Freed, E., Gailit, J., van der Geer, P., Ruoslahti, E., and Hunter, T. (1989) A novel integrin β subunit is associated with the vitronectin receptor α subunit (αv) in a human osteosarcoma cell line and is a substrate for protein kinase C. EMBO J. 8, 2955–2965.
Linscott, W. D. (1998) Linscott’s Directory of Immunological and Biological Reagents, 10th ed. 1996–1997, William D. Linscott, Santa Rosa, CA.
Rouslahti, E. and Reed, J. C. (1994) Anchorage dependence, integrins and apoptosis. Cell 77, 477–478.
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Rosfjord, E.C., Dickson, R.B. (1999). Application of Flow Cytometry in the Analysis and Sterile Sorting of Cell Populations Based on Integrin Expression. In: Howlett, A. (eds) Integrin Protocols. Methods in Molecular Biology, vol 129. Humana Press. https://doi.org/10.1385/1-59259-249-X:79
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DOI: https://doi.org/10.1385/1-59259-249-X:79
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