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Multicolor flow cytometric analysis of CD20 (cytoplasmic domain) expression in Human peripheral blood lymphocytes. Whole blood was treated with BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10 min at 37°C to lyse erythrocytes and fix the leucocytes in one step. The leucocytes were permeabilized with BD Phosflow™ Perm/Wash Buffer I (Cat. No. 557885) for 20 minutes. The cells were then stained with either BD Horizon™ R718 Mouse IgG2a, κ Isotype Control (Cat. No. 567089; dashed line histogram) or BD Horizon™ RY718 Mouse Anti-Human CD20 (cytoplasmic) antibody (Cat. No. 568050/568051; solid line histogram). The bivariate pseudocolor density plot showing the correlated expression of CD20 (cytoplasmatic domain) [or Ig Isotype control staining] vs side-light scatter (SSC-A) signals was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software.
BD Horizon™ R718 Mouse Anti-Human CD20
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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.
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The H1 (FB1) antibody specificially binds to a cytoplasmic domain of CD20. CD20 is a 33-37-kDa four transmembrane phosphoprotein that is expressed by B lymphocytes from the pre-B stage and most malignant B cells and is lost during plasma cell differentiation. Low level CD20 expression is observed on a subset of normal circulating T lymphocytes, and CD20-positive T-cell lymphomas have been reported. The CD20 molecule is associated with membrane lipid raft domains, acts as a channel for calcium ions, and is involved in the regulation of B cell activation and survival. The cytoplasmic domain regions are serine and threonine rich and contain multiple phosphorylation consensus sequences.
Development References (7)
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Cragg MS, Walshe CA, Ivanov AO, Glennie MJ. The biology of CD20 and its potential as a target for mAb therapy. Curr Dir Autoimmun. 2005; 8:140-174. (Biology). View Reference
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Kitamura A, Yamashita Y, Mori N. CD20-positive cytotoxic T cell lymphoma: report of two cases and review of the literature. J Clin Exp Hematop. 2005; 45(1):45-50. (Biology).
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Nozawa Y, Abe M, Ohno H, Fukuhara S, Wakasa H. Production of two monoclonal antibodies (FB1 and FB21) useful for the identification of human B lymphocytes in formalin-fixed, paraffin-embedded tissues. J Pathol. 1994; 173:347-354. (Immunogen). View Reference
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Pylayeva-Gupta Y, Das S, Handler JS, et al. IL35-Producing B Cells Promote the Development of Pancreatic Neoplasia.. Cancer Discov. 2016; 6(3):247-55. (Clone-specific: Immunofluorescence, Immunohistochemistry). View Reference
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Sachen KL, Strohman MJ, Singletary J, et al. Self-antigen recognition by follicular lymphoma B-cell receptors.. Blood. 2012; 120(20):4182-90. (Clone-specific: Flow cytometry). View Reference
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Schlossman SF. Stuart F. Schlossman .. et al., ed. Leucocyte typing V : white cell differentiation antigens : proceedings of the fifth international workshop and conference held in Boston, USA, 3-7 November, 1993. Oxford: Oxford University Press; 1995.
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Ziegler CGK, Kim J, Piersanti K, et al. Constitutive Activation of the B Cell Receptor Underlies Dysfunctional Signaling in Chronic Lymphocytic Leukemia.. Cell Rep. 2019; 28(4):923-937.e3. (Clone-specific: Flow cytometry). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.