Abstract
Interaction of the complement system, directly or indirectly (e.g., via antibodies), with cells activates the early and late complement components and culminates in the deposition of a membrane-spanning C5b-9 complex on the cell surface. At a high copy number, this C5b-9 will activate cell death, whereas at a low copy number, it will transmit various signals into cells. Quantification of C5b-9 deposition is useful for assessments of the capacity of cells and antibodies to activate complement. By using an antibody that identifies a novel antigen of the C5b-9 complex, the amount of C5b-9 complexes on cells can be quantified by flow cytometry. The detailed protocol is described in this chapter.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Walport MJ (2001) Complement. Second of two parts. N Engl J Med 344:1140–1144
Muller-Eberhard HJ (1986) The membrane attack complex of complement. Annu Rev Immunol 4:503–528
Mollnes TE, Harboe M (1987) Immunohistochemical detection of the membrane and fluid-phase terminal complement complexes C5b-9(m) and SC5b-9. Consequences for interpretation and terminology. Scand J Immunol 26:381–386
DiScipio RG, Berlin C (1999) The architectural transition of human complement component C9 to poly(C9). Mol Immunol 36:575–585
Podack ER, Tschopp J (1982) Polymerization of the ninth component of complement (C9): formation of poly(C9) with a tubular ultrastructure resembling the membrane attack complex of complement. Proc Natl Acad Sci U S A 79:574–578
Tschopp J, Podack ER, Muller-Eberhard HJ (1985) The membrane attack complex of complement: C5b-8 complex as accelerator of C9 polymerization. J Immunol 134:495–499
Whitlow MB, Ramm LE, Mayer MM (1985) Penetration of C8 and C9 in the C5b-9 complex across the erythrocyte membrane into the cytoplasmic space. J Biol Chem 260:998–1005
Ramm LE, Whitlow MB, Mayer MM (1982) Size of the transmembrane channels produced by complement proteins C5b-8. J Immunol 129:1143–1146
Ramm LE, Whitlow MB, Mayer MM (1982) Transmembrane channel formation by complement: functional analysis of the number of C5b6, C7, C8, and C9 molecules required for a single channel. Proc Natl Acad Sci U S A 79:4751–4755
Preissner KP, Podack ER, Muller-Eberhard HJ (1989) SC5b-7, SC5b-8 and SC5b-9 complexes of complement: ultrastructure and localization of the S-protein (vitronectin) within the macromolecules. Eur J Immunol 19:69–75
Bhakdi S, Tranum-Jensen J, Klump O (1980) The terminal membrane C5b-9 complex of human complement. Evidence for the existence of multiple protease-resistant polypeptides that form the trans-membrane complement channel. J Immunol 124:2451–2457
Kim SH, Carney DF, Hammer CH, Shin ML (1987) Nucleated cell killing by complement: effects of C5b-9 channel size and extracellular Ca2+ on the lytic process. J Immunol 138:1530–1536
Papadimitriou JC, Ramm LE, Drachenberg CB, Trump BF, Shin ML (1991) Quantitative analysis of adenine nucleotides during the prelytic phase of cell death mediated by C5b-9. J Immunol 147:212–217
Morgan BP, Luzio JP, Campbell AK (1986) Intracellular Ca2+ and cell injury: a paradoxical role of Ca2+ in complement membrane attack. Cell Calcium 7:399–411
Cragg MS, Howatt WJ, Bloodworth L, Anderson VA, Morgan BP, Glennie MJ (2000) Complement mediated cell death is associated with DNA fragmentation. Cell Death Differ 7:48–58
Fishelson Z, Attali G, Mevorach D (2001) Complement and apoptosis. Mol Immunol 38:207–219
Nauta, A. J., M. R. Daha, O. Tijsma, B. van de Water, F. Tedesco, and A. Roos (2002) The membrane attack complex of complement induces caspase activation and apoptosis. Eur J Immunol 32:783–792
Gancz D, Donin N, Fishelson Z (2009) Involvement of the c-jun N-terminal kinases JNK1 and JNK2 in complement-mediated cell death. Mol Immunol 47:310–317
Ziporen L, Donin N, Shmushkovich T, Gross A, Fishelson Z (2009) Programmed necrotic cell death induced by complement involves a Bid-dependent pathway. J Immunol 182:515–521
Bohana-Kashtan O, Ziporen L, Donin N, Kraus S, Fishelson Z (2004) Cell signals transduced by complement. Mol Immunol 41:583–597
Gancz D, Fishelson Z (2009) Cancer resistance to complement-dependent cytotoxicity (CDC): problem-oriented research and development. Mol Immunol 46:2794–2800
Morgan BP, Dankert JR, Esser AF (1987) Recovery of human neutrophils from complement attack: removal of the membrane attack complex by endocytosis and exocytosis. J Immunol 138:246–253
Moskovich O, Fishelson Z (2007) Live cell imaging of outward and inward vesiculation induced by the complement c5b-9 complex. J Biol Chem 282:29977–29986
Pilzer D, Gasser O, Moskovich O, Schifferli JA, Fishelson Z (2005) Emission of membrane vesicles: roles in complement resistance, immunity and cancer. Springer Semin Immunopathol 27:375–387
Dalmasso AP, Falk RJ, Raij L (1989) The pathobiology of the terminal complement complexes. Complement Inflamm 6:36–48
Vakeva A, Meri S (2000) Complement deposition in tissues. Methods Mol Biol 150:113–121
Accardo-Palumbo A, Triolo G, Casiglia D, Salli L, Giardina E, Triolo G (1993) Two-site ELISA for quantification of the terminal C5b-9 complement complex in plasma. Use of monoclonal and polyclonal antibodies against a neoantigen of the complex. J Immunol Methods 163:169–172
Hugo F, Kramer S, Bhakdi S (1987) Sensitive ELISA for quantitating the terminal membrane C5b-9 and fluid-phase SC5b-9 complex of human complement. J Immunol Methods 99:243–251
Matsell DG, Roy S III, Tamerius JD, Morrow PR, Kolb WP, Wyatt RJ (1991) Plasma terminal complement complexes in acute poststreptococcal glomerulonephritis. Am J Kidney Dis 17:311–316
Wurzner R (2000) Immunochemical measurement of complement components and activation products. Methods Mol Biol 150:103–112
Moskovich O, Herzog LO, Ehrlich M, Fishelson Z (2012) Caveolin-1 and dynamin-2 are essential for removal of the complement C5b-9 complex via endocytosis. J Biol Chem 287:19904–19915
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Moskovich, O., Fishelson, Z. (2014). Quantification of Complement C5b-9 Binding to Cells by Flow Cytometry. In: Gadjeva, M. (eds) The Complement System. Methods in Molecular Biology, vol 1100. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-724-2_8
Download citation
DOI: https://doi.org/10.1007/978-1-62703-724-2_8
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-723-5
Online ISBN: 978-1-62703-724-2
eBook Packages: Springer Protocols