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Flow cytometric analysis of C3b/iC3b fixed on antibody and complement treated human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were preincubated with either Purified NA/LE Mouse Anti-Human CD3 antibody (Cat. No. 555336; Left Plot; Anti-CD3 + Serum) or Purified NA/LE Mouse IgG2a, κ Isotype Control (Cat. No. 554645; Right Plot; Isotype Control + Serum), pelleted by centrifugation, and then resuspended in human serum (for a source of complement) for 30 min at 37°C) as indicated. After washing, the cells were stained with either PE Mouse IgG1, κ Isotype Control (Cat. No. 554680; dotted line histograms) or with PE Mouse Anti-Human C3b/iC3b antibody (Cat. No. 567548/567583; solid line histograms). The histograms showing bound levels of C3b/iC3b (or Ig Isotype control staining) were derived from gated events with the forward and side light-scatter characteristics of viable lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
BD Pharmingen™ PE Mouse Anti-Human C3b/iC3b
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Preparation And Storage
Recommended Assay Procedures
BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to BD™ CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD CompBeads to ensure that BD™ CompBeads are appropriate for your specific cellular application.
Product Notices
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Companion Products
The 3E7 monoclonal antibody specifically recognizes multifunctional C3b and iC3b fragments of the ~190 kDa plasma glycoprotein, Complement component 3 (C3). C3 is a key component of the classical, alternative and lectin complement activation pathways which participate in innate and adaptive immune responses to eliminate pathogens, dying cells, and immune complexes from the body. C3 is produced by hepatocytes, mast cells, and leucocytes including some monocytes, macrophages, dendritic cells (DC), and T cells. As a result of complement activation, eg, initiated by an antigen-antibody immune complex in the classical pathway, biologically active C3b fragments can be cleaved from C3 by enzymatic C3 convertases. C3b can function as an opsonin by binding covalently via its reactive thioester to the surfaces of target cells, including microbes. C3b can also bind to antigen-antibody immune complexes. Macrophages and neutrophils can recognize and bind to C3b by their complement receptors, such as Complement Receptor 1 (CR1, CD35), which enhances their phagocytosis of C3b-bound target cells or immune complexes. Opsonization of target surfaces, including the cell surfaces of pathogenic organisms, infected or tumor cells, through C3b deposition is central to all three pathways of complement activation during innate or adaptive immune responses. C3b can also associate with other components of the complement system to form a C5 convertase. This complex cleaves C5 into the C5a anaphylatoxin and C5b which initiates formation of the of the cytolytic membrane attack complex (MAC) that can produce holes in target cell membranes. C3b can be further cleaved into iC3b by factor I protease and its cofactors. The iC3b fragment serves as a ligand for engaging lymphoid and phagocytic cells via various receptors including CR2 (CD21), CR3 (CD11b/CD18), CR4 (CD11c/CD18), and CRIg. The 3E7 antibody van reportedly block the activation of the alternative pathway of complement.
Development References (6)
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DiLillo DJ, Pawluczkowycz AW, Peng W, et al. Selective and efficient inhibition of the alternative pathway of complement by a mAb that recognizes C3b/iC3b. Mol Immunol. 2006; 43(7):1010-1019. (Immunogen: Functional assay, Inhibition). View Reference
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Kennedy AD, Beum PV, Solga MD, et al. Rituximab infusion promotes rapid complement depletion and acute CD20 loss in chronic lymphocytic leukemia. J Immunol. 2004; 172(5):3280-3288. (Clone-specific: Cytotoxicity, Immunofluorescence). View Reference
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Kennedy AD, Solga MD, Schuman TA, et al. An anti-C3b(i) mAb enhances complement activation, C3b(i) deposition, and killing of CD20+ cells by rituximab. Blood. 2003; 101(3):1071-1079. (Clone-specific: Flow cytometry). View Reference
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Lindorfer MA, Pawluczkowycz AW, Peek EM, Hickman K, Taylor RP, Parker CJ. A novel approach to preventing the hemolysis of paroxysmal nocturnal hemoglobinuria: both complement-mediated cytolysis and C3 deposition are blocked by a monoclonal antibody specific for the alternative pathway of complement.. Blood. 2010; 115(11):2283-91. (Clone-specific: Functional assay, Inhibition). View Reference
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Lubbers R, van Essen MF, van Kooten C, Trouw LA. Production of complement components by cells of the immune system.. Clin Exp Immunol. 2017; 188(2):183-194. (Biology). View Reference
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Sokoloff MH, Nardin A, Solga MD, et al. Targeting of cancer cells with monoclonal antibodies specific for C3b(i). Cancer Immunol Immunother. 2000; 49(10):551-562. (Immunogen: Cell separation, Radioimmunoassay). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.