Description
The 7E9 antibody specifically recognizes CD21/CD35, an epitope shared by mouse Complement receptor 2 (CR2) and Complement receptor 1 (CR1), which are also known as CD21 and CD35, respectively. Mouse CD21 and CD35 are ~145-kDa and ~190-kDa single pass type I transmembrane glycoproteins, respectively. Unlike human CD21 and CD35 that are encoded by separate CR2 and CR1 genes, mouse CD21 and CD35 are encoded by a single Cr2 gene and are generated by alternative mRNA splicing. CD21 and CD35 are expressed on B cells and follicular dendritic cells (FCD) but not on thymocytes, T cells, erythrocytes, or platelets. CD35 is also expressed on macrophages and activated granulocytes. CD21 binds to cleaved C3b fragments, including C3d- and C3dg-bound to antigen, whereas CD35 binds to C3b-, iC3b-, and C4b-bound complexes. These receptors play many important roles including the transport, presentation, and retention of complement fragment-tagged antigens and immune complexes crucial for generating strong B cell antibody-producing responses. Complement-tagged antigen can bind to CD21, part of the B cell coreceptor complex and to the B cell receptor for antigen (BCR) which facilitates the antigen-driven activation of B cell responses. CD35 interacts with other regulatory factors to promote C3b and C4b degradation as well as playing roles in stimulating phagocytic functions in activated leucocytes. The 7E9 antibody recognizes an epitope on CD35 distinct from the epitope recognized by the mouse CD35-specific clone, 8C12, as well as the mouse CD21/CD35-specific clone, 7G6.
Bead-based compensation or unmixing controls, such as BD® CompBeads or BD™ SpectraComp™, can be used as surrogates to assess fluorescence spillover when bound to fluorochrome-conjugated antibodies. Although these beads have spectral properties similar to cells, variations in spectral emission may occur, resulting in differing spillover values compared to biological controls. Therefore, it is considered best practice to compare the spillover obtained from cells and bead-based controls when using BD® CompBeads or BD™ SpectraComp™ for the first time, to ensure they are appropriate for the intended application.
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 that minimize unconjugated dye and antibody.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
3. For U.S. patents that may apply, see bd.com/patents.
4. 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.
5. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
6. The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity. However, verification testing has not been performed on all conjugate lots.
7. When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
9. An isotype control should be used at the same concentration as the antibody of interest.
10. Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
11. Alexa Fluor™ is a trademark of Life Technologies Corporation.
