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Alexa Fluor™ 647 Mouse Anti-Human C3b/iC3b
Alexa Fluor™ 647 Mouse Anti-Human C3b/iC3b
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 IgG2b, κ Isotype Control (Cat. No. 554645; Left Plot; Isotype Control + Serum) or Purified NA/LE Mouse Anti-Human CD3 antibody (Cat. No. 555336; Right Plot; Anti-CD3 + 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 Alexa Fluor™ Mouse IgG1, Isotype Control (Cat. No. 565571; dotted line histograms) or with Alexa Fluor™ 647 Mouse Anti-Human C3b/iC3b antibody (Cat. No. 568044/568045; solid line histograms). DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Solution (Cat. No. 564907) was added to cells right before analysis. 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 (DAPI-negative) lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
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 IgG2b, κ Isotype Control (Cat. No. 554645; Left Plot; Isotype Control + Serum) or Purified NA/LE Mouse Anti-Human CD3 antibody (Cat. No. 555336; Right Plot; Anti-CD3 + 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 Alexa Fluor™ Mouse IgG1, Isotype Control (Cat. No. 565571; dotted line histograms) or with Alexa Fluor™ 647 Mouse Anti-Human C3b/iC3b antibody (Cat. No. 568044/568045; solid line histograms). DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Solution (Cat. No. 564907) was added to cells right before analysis. 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 (DAPI-negative) lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
Product Details
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BD Pharmingen™
C3b; iC3b; C3bi; complement component C3b
Human (QC Testing)
Mouse IgG1, κ
Human C3b
Flow cytometry (Routinely Tested)
5 µl
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions and unreacted dye was removed.

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

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. This product is provided under an intellectual property license between Life Technologies Corporation and BD Businesses. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for any other use, contact Life Technologies Corporation, Cell Analysis Business Unit Business Development, 29851 Willow Creek Road, Eugene, OR 97402, USA, Tel: (541) 465-8300. Fax: (541) 335-0504.
  6. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  7. An isotype control should be used at the same concentration as the antibody of interest.
  8. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  9. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  10. Alexa Fluor™ is a trademark of Life Technologies Corporation.
  11. For U.S. patents that may apply, see bd.com/patents.
568045 Rev. 2
Antibody Details
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3E7

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.

568045 Rev. 2
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor™ 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 670-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
568045 Rev.2
Citations & References
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Development References (6)

  1. 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):1010-9. (Immunogen: Functional assay, Inhibition). View Reference
  2. 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):3280-8. (Clone-specific: Cytotoxicity, Immunofluorescence). View Reference
  3. 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):1071-9. (Clone-specific: Flow cytometry). View Reference
  4. 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
  5. 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
  6. 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):551-62. (Immunogen: Cell separation, Radioimmunoassay). View Reference
View All (6) View Less
568045 Rev. 2

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.