The 6H6 monoclonal antibody specifically recognizes the Interleukin-3 receptor alpha chain (IL-3Ra) which is also known as CD123. IL-3Ra (CD123) is a ~70 kDa type I transmembrane glycoprotein that is encoded by IL3RA (interleukin 3 receptor subunit alpha) which belongs to the type I cytokine receptor family within the immunoglobulin gene superfamily. This receptor chain consists of an extracellular region that contains an immunoglobulin-like N-terminal domain (NTD) with a fibronectin type III (FnIII) fold followed by two more FnIII domains that form the cytokine receptor module (CRM), a transmembrane region, and an intracellular tail. IL-3Ra (CD123) binds IL-3 specifically and with low affinity. IL-3Ra (CD123) forms a high-affinity signaling receptor for IL-3 (IL-3R) with the ß common chain (ßc; also known as, CD131) that is shared with the heterodimeric IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors. IL-3Ra (CD123) is variably expressed on certain hematopoietic progenitor cells, basophils, eosinophils, mast cells, monocytes, macrophages, dendritic cells, megakaryocytes, and on some B cells, endothelial cells, and leukemia cells. Bound IL-3 can signal through IL-3R to promote the activation, proliferation, differentiation, and viability of these cells. Amongst monoclonal antibodies specific for human IL-3Ra (CD123), the 6H6 and 9F5 antibodies do not block IL-3 binding to the IL-3R whereas the 7G3 antibody does block IL-3 binding to its receptor in a dose-dependent manner.
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. 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.
13. 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.
