The 8C12 antibody recognizes an epitope present on the 190-kDa complement receptor protein, originally designated CR1 (CD35), but not the 145-150-kDa CR2 (CD21) molecule. Unlike the human system, in which these proteins are products of independent genes, both of these mouse receptors are membrane proteins resulting from the alternative splicing of mRNA transcribed from the Cr2 gene. Therefore, an alternative nomenclature has been proposed, designating the proteins Cr2-190 (CD21b) and Cr2-145 (CD21a), respectively. The epitope recognized by 8C12 mAb is only present on CD35/CD21b. Moreover, it has also been proposed that Crry is the true mouse genetic homologue of human CR1 (CD35). In the mouse, CD35 is expressed on the majority of peripheral B cells, on the majority of resident peritoneal macrophages, on peripheral blood granulocytes after treatment with N-formyl-Met-Leu-Phe, and on follicular dendritic cells, but not on thymocytes, T cells, erythrocytes, or platelets. In addition, it has not been detected, at the protein or mRNA level, in the macrophage cell line J774, bone marrow-derived macrophages, or thioglycollate-elicited peritoneal macrophages. The 8C12 mAb has been reported to inhibit rosette formation by C3bbearing sheep erythrocytes, to block the complement-dependent trapping of immune complexes by follicular dendritic cells, and to down-regulate mouse CD35 expression upon in vivo application, inhibiting only some primary antibody responses to immunization. B lymphocytes of Cr2[null] mice display impaired humoral immune responses in vivo. The 8C12 mAb recognizes an epitope on mouse CD35 distinct from the epitope recognized by anti-mouse CD21/CD35 mAb 7G6, and it does not block binding by 7G6 mAb to CD35. *Please note that the isotype of 8C12 mAb was originally reported to be Rat IgG2c. Further investigations have demonstrated that the isotype of 8C12 mAb is Rat IgG2a.
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.
