Description
The Ab93 monoclonal antibody (aka, Antibody93) specifically recognizes a common epitope on the extracellular domains of mouse CD16 (Fc gamma RIII/FcγRIII encoded by Fcgr3) and CD32 (Fc gamma RIIB/FcγRIIB encoded by Fcgr2b). Therefore, Ab93 is referred to as an Anti-CD16/32 or Anti-FcgRII/III antibody. CD16 is variably expressed on neutrophils, macrophages, and natural killer (NK) cells whereas CD32 is expressed on B cells, monocytes, granulocytes, platelets and endothelial cells. CD16 and CD32 serve as low affinity receptors for IgG Fc constant regions and are involved in regulating various cellular functions including antibody-dependent cellular toxicity (ADCC), phagocytosis, effector cell degranulation, and B cell proliferation. In addition to identifying CD16- or CD32-positive cells, the Ab93 antibody is useful in phenotyping studies for blocking nonspecific staining due to Fc receptor-mediated binding of other antibodies. Ab93 is also useful in functional studies due to its Fc receptor blocking capability or by its capacity to crosslink Fc receptors leading to signal transduction that triggers cellular responses. Ab93 (Rat IgG2a, λ) and clone 2.4G2 (Rat IgG2b, κ), another mouse CD16/32-specific antibody, reportedly have similar specificities. The differences in the Ig heavy chain or Ig light chain isotypes of these CD16/32-specific antibodies afford flexibility in the design of experimental model systems involving other antibodies.
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.
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. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
11. 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.
12. Tandem fluorochromes contain both an energy donor and an energy acceptor. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from the donor may be observed. Additionally, multi-laser cytometers may directly excite both the donor and acceptor fluorochromes. Therefore, we recommend for every tandem conjugate, a matched individual single-stain control be acquired for generating a compensation or spectral unmixing matrix.
