The K10.6 monoclonal antibody specifically recognizes Lyb-2.1, Lyb-2.2, and Lyb-2.4 (CD72 a, b, and d alloantigens, respectively). CD72 is a 45-kDa type-II membrane protein, containing a C-type lectin-like domain and ITIM and ITIM-like sequences in the cytoplasmic tail. CD72 is expressed at all stages of B-lymphocyte development except plasma cells, and it has been shown to negatively regulate B-cell receptor signaling. Analysis of CD72-deficient mice supports these results and shows that CD72 is involved in B-cell development. CD72-stimulated B cells show a transient association of CD19 with CD72, as well as an increase in Tyr-phosphorylation of CD19. CD72 is reported to be a ligand for CD5, although this is controversial. It is also reported to be a ligand for CD100 (Sema4D). The CD72 alloantigens Lyb-2.1 (originally identified as Ly-m19.2), Lyb-2.2 (originally identified as Ly-32.2), Lyb-2.3, and Lyb-2.4 are encoded by the Cd72[a], Cd72[b], Cd72[c], and Cd72[d] alleles, respectively. Lyb-2.1 is expressed on B lymphocytes of CBA/J, C3H/Bi, C57BR, C57L, C58, DBA/1, DBA/2, and SWR strains. Lyb-2.2 is expressed on B lymphocytes, a subset of peripheral T cells, and activated T lymphocytes in A, BALB/c, CBA/H, C3H/He, C57BL, PL, and 129 strains. Lyb-2.4 is expressed on a subset of splenocytes of the STS/A strain. K10.6 antibody does not react with Lyb-2.3 (AKR and SJL strains) nor with non-lymphoid tissues. Five serological specificities of CD72 alloantigens have been described and the nomenclature CD72.1, CD72.2, CD72.3, CD72.4, and CD72.5 proposed, which does not correspond to the names of the alleles. Some authors have referred to the specificity of mAb K10.6 as CD72.4.
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.
