The 3D3 monoclonal antibody specifically recognizes FcγRII (CD32), a 40 kDa, polymorphic type I transmembrane glycoprotein that serves as a low affinity receptor for aggregated IgG. This highly glycosylated molecule (encoded by at least two different genes) is expressed on monocytes, granulocytes, platelets and B cells. Unlike the FLI28.26 mAb, the 3D3 mAb detected a polymorphic CD32 antigen expressed on B cells of all donors, but only on platelets, monocytes and granulocytes of some donors. The platelets from 3D3+ donors respond to certain stimulatory mAb such as CD165 (clone SN2) which results in aggregation. On the other hand, the platelets from 3D3 negative donors do not form aggregates after stimulation. Individuals can be divided into two groups as responder and non-responder depending on expression, or non-expression, of 3D3. In comparison to the 3D3 mAb, the FLI8.26 mAb detects a monomorphic CD32 antigen expressed on all human donors.
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. This product is sold under license. Purchase of this product does not include rights to (i) incorporate this product into the purchaser's own products for resale to end-users, or (ii) use this product to conduct for-profit research for or on behalf of another party. For information on obtaining a license to this product for such prohibited uses, contact INSERM, 7 rue Watt, 75013 Paris. Telephone: +33 1 55 03 01 60. Facsimile: +33 1 55 03 01 18. Email:
[email protected]8. 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.
9. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
10. An isotype control should be used at the same concentration as the antibody of interest.
11. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
12. 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.
13. 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.
14. Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
