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Preparation And Storage
Recommended Assay Procedures
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.
Product Notices
- 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.
- Researchers should determine the optimal concentration of this reagent for their individual applications.
- 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.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
Companion Products
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.
Development References (12)
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Adachi T, Wakabayashi C, Nakayama T, Yakura H, Tsubata T. CD72 negatively regulates signaling through the antigen receptor of B cells.. J Immunol. 2000; 164(3):1223-9. (Biology). View Reference
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Bikah G, Lynd FM, Aruffo AA, Ledbetter JA, Bondada S. A role for CD5 in cognate interactions between T cells and B cells, and identification of a novel ligand for CD5. Int Immunol. 1998; 10(8):1185-1196. (Biology). View Reference
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Doolittle, D.P., M.T. Davisson, J.N. Guidi, and M.C. Green. M.F. Lyon, S. Rastan and S.D.M. Brown, ed. Genetic variants and strains of the laboratory mouse. Oxford : Oxford University Press; 1996:133.
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Kumanogoh A, Watanabe C, Lee I, et al. Identification of CD72 as a lymphocyte receptor for the class IV semaphorin CD100: a novel mechanism for regulating B cell signaling. Immunity. 2000; 13(5):621-631. (Biology). View Reference
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Luo W, Van de Velde H, von Hoegen I, Parnes JR, Thielemans K. Ly-1 (CD5), a membrane glycoprotein of mouse T lymphocytes and a subset of B cells, is a natural ligand of the B cell surface protein Lyb-2 (CD72). J Immunol. 1992; 148(6):1630-1634. (Biology). View Reference
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Pan C, Baumgarth N, Parnes JR. CD72-deficient mice reveal nonredundant roles of CD72 in B cell development and activation. Immunity. 1999 October; 11(4):495-506. (Biology). View Reference
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Robinson WH, Landolfi MM, Parnes JR. Allele-specific expression of the mouse B-cell surface protein CD72 on T cells. Immunogenetics. 1997; 75(3):195-200. (Biology). View Reference
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Robinson WH, Landolfi MM, Schäfer H, Parnes JR. Biochemical identity of the mouse Ly-19.2 and Ly-32.2 alloantigens with the B cell differentiation antigen Lyb-2/CD72. J Immunol. 1993 November; 151(9):4764-4772. (Biology). View Reference
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Robinson WH, Ying H, Miceli MC, Parnes JR. Extensive polymorphism in the extracellular domain of the mouse B cell differentiation antigen Lyb-2/CD72.. J Immunol. 1992; 149(3):880-6. (Biology). View Reference
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Tada N, Kimura S, Liu Y, Taylor BA, Hämmerling U. Ly-m19: the Lyb-2 region of mouse chromosome 4 controls a new surface alloantigen. Immunogenetics. 1981; 13(6):539-546. (Immunogen). View Reference
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Venkataraman C, Lu PJ, Buhl AM, Chen CS, Cambier JC, Bondada S. CD72-mediated B cell activation involves recruitment of CD19 and activation of phosphatidylinositol 3-kinase. Eur J Immunol. 1998 October; 28(10):3003-3016. (Biology). View Reference
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Ying H, Healy JI, Goodnow CC, Parnes JR. Regulation of mouse CD72 gene expression during B lymphocyte development. J Immunol. 1998; 161(9):4760-4767. (Biology). View Reference
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