Flow cytometry allows the multiparametric physical and biochemical analysis of single cells within large cell suspensions which is most frequently for cell counting and for the measurement of antigen expression with fluorescence conjugated antibodies. Moreover, flow cytometry also represents one of the most crucial test systems for the development of therapeutic antibodies because antibodies have to bind their antigen in its natural context with relatively high affinity. The following two protocols describe the extracellular and intracellular staining of cells with recombinant antibodies and antibody fusion proteins.
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References
Bartholdi M, Meyne J, Albright K, Luedemann M, Campbell E, Chritton D, Deaven LL, Cram LS (1987) Chromosome sorting by flow cytometry. Methods Enzymol 151:252–267
Burchiel SW, Edwards BS, Kuckuck FW, Lauer FT, Prossnitz ER, Ransom JT, Sklar LA (2000) Analysis of free intracellular calcium by flow cytometry: multiparameter and pharmacologic applications. Methods 21:221–230
Givan AL, Fisher JL, Waugh MG, Bercovici N, Wallace PK (2004) Use of cell-tracking dyes to determine proliferation precursor frequencies of antigen-specific T cells. Methods Mol Biol 263:109–124
Gorczyca W, Melamed MR, Darzynkiewicz Z (1998) Analysis of apoptosis by flow cytometry. Methods Mol Biol 91:217–238
Hasbold J, Hodgkin PD (2000) Flow cytometric cell division tracking using nuclei. Cytometry 40:230–237
Johann S, Blumel G, Lipp M, Forster R (1995) A versatile flow cytometry-based assay for the determination of short- and long-term natural killer cell activity. J Immunol Methods 185:209–216
Lecoeur H, Ledru E, Gougeon ML (1998) A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes. J Immunol Methods 217:11–26
Menzel C, Schirrmann T, Konthur Z, Jostock T, Dübel S (2008) Human antibody RNase fusion protein targeting CD30+ lymphomas. Blood 111:3830–3837
Nylander S, Kalies I (1999) Brefeldin A, but not monensin, completely blocks CD69 expression on mouse lymphocytes: efficacy of inhibitors of protein secretion in protocols for intracellular cytokine staining by flow cytometry. J Immunol Methods 224:69–76
Piwnicka M, Darzynkiewicz Z, Melamed MR (1983) RNA and DNA content of isolated cell nuclei measured by multiparameter flow cytometry. Cytometry 3:269–275
Schirrmann T, Pecher G (2001) Tumor-specific targeting of a cell line with natural killer cell activity by asialoglycoprotein receptor gene transfer. Cancer Immunol Immunother 50:549–556
Seidel A, Hasmann M, Loser R, Bunge A, Schaefer B, Herzig I, Steidtmann K, Dietel M (1995) Intracellular localization, vesicular accumulation and kinetics of daunorubicin in sensitive and multidrug-resistant gastric carcinoma EPG85-257 cells. Virchows Arch 426:249–256
Shapiro HM (2000) Membrane potential estimation by flow cytometry. Methods 21:271–279
Telford WG, King LE, Fraker PJ (1992) Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry 13:137–143
Weaver JL (1998) Estimation of cell viability by flow cytometry. Methods Mol Biol 91:77–83
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Schirrmann, T. (2010). Binding Studies with Flow Cytometry. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-01144-3_50
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