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Two-color flow cytometric analysis of CD20 expression on human peripheral blood lymphocytes. Whole blood was stained with PE Mouse Anti-Human CD19 antibody (Cat. No. 555413/561741) and either BD Horizon™ BUV737 Mouse IgG2b, κ Isotype Control (Left Plot) or BD Horizon BUV737 Mouse Anti-Human CD20 antibody (Cat. No. 612848/612849; Right Plot). Erythrocytes were lysed with BD FACS Lysing Solution (Cat. No. 349202). Two-color flow cytometric contour plots showing the correlated expression of CD20 (or Ig Isotype Control staining) versus CD19 were derived from gated events with the forward light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD FACSCelesta™ Flow Cytometer System and FlowJo™ software.
BD Horizon™ BUV737 Mouse Anti-Human CD20
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
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. This will ensure that BD CompBeads are appropriate for your specific cellular application.
For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).
Note: When using high concentrations of antibody, background binding of this dye to erythroid cell subsets (mature erythrocytes and precursors) has been observed. For researchers studying these cell populations, or in cases where light scatter gating does not adequately exclude these cells from the analysis, this background may be an important factor to consider when selecting reagents for panel(s).
Product Notices
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- BD Horizon Brilliant Ultraviolet 737 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,575,303; 8,354,239.
- BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
The 2H7 monoclonal antibody specifically binds to CD20, encoded by the MS4A1 (Membrane-spanning 4-domains, subfamily A, member 1) gene. CD20 is a 33-37 kDa, unglycosylated four-transmembrane phosphoprotein. CD20 is expressed on pre-B-cells, resting and activated B cells, and follicular dendritic cells, but not plasma cells. Low level CD20 expression is observed on a small subset of normal circulating T lymphocytes. The CD20 molecule is involved in the regulation of B-cell activation.
This clone also cross-reacts with a subset of peripheral blood lymphocytes, but not monocytes nor granulocytes, of baboon and both rhesus and cynomolgus macaque monkeys. The distribution on lymphocytes is similar to that seen with normal human donor lymphocytes, namely bright staining on B lymphocytes and weak reactivity on a small subset of CD3-positive T lymphocytes.
The antibody was conjugated to BD Horizon BUV737 which is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This dye is a tandem fluorochrome with an Ex Max near 350 nm and an Em Max near 737 nm. BD Horizon Brilliant BUV737 can be excited by the ultraviolet laser (355 nm) and detected with a 740/35 nm filter. Due to the excitation of the acceptor dye by the red laser line, there may be significant spillover into red laser detectors with filters in the 700-720 nm range.
Development References (8)
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Clark EA, Yokochi T. Human B cell and B cell blast-associated surface molecules defined with monoclonal antibodies. In: Bernard A, Boumsell L, Dausset J, Milstein C, Schlossman SF, ed. Leukocyte Typing. Berlin: Springer-Verlag; 1984:339-346.
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Hultin LE, Hausner MA, Hultin PM, Giorgi JV. CD20 (pan-B cell) antigen is expressed at a low level on a subpopulation of human T lymphocytes. Cytometry. 1993; 14(2):193-204. (Clone-specific: Flow cytometry). View Reference
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Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:1-1182.
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Ledbetter JA, Clark EA. Surface phenotype and function of tonsillar germinal center and mantle zone B cell subsets. Hum Immunol. 1986; 15:30-43. (Immunogen: Blocking, Flow cytometry). View Reference
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Lin G-X, Yang X, Hollemweguer E, et al. Cross-reactivity of CD antibodies in eight animal species. In: Mason D. David Mason .. et al., ed. Leucocyte typing VII : white cell differentiation antigens : proceedings of the Seventh International Workshop and Conference held in Harrogate, United Kingdom. Oxford: Oxford University Press; 2002:519-523.
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Loken MR, Shah VO, Dattilio KL, Civin CI. Flow cytometric analysis of human bone marrow. II. Normal B lymphocyte development. Blood. 1987; 70(5):1316-1324. (Biology). View Reference
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Reinherz EL. Ellis L. Reinherz .. et al., ed. Leukocyte typing II. New York: Springer-Verlag; 1986:1-560.
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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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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.