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PE-Cy™7 Mouse Anti-Human CD20
PE-Cy™7 Mouse Anti-Human CD20

Flow cytometric analysis of CD20 (cytoplasmic domain) expression by human peripheral blood lymphocytes.  Human 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 leukocytes in one step. The leukocytes were permeabilized with BD Phosflow™ Perm Buffer I (Cat. No. 557885) for 20 minutes. The cells were then stained with either PE-Cy™7 Mouse IgG2a, κ Isotype Control (Cat. No. 560906; dashed line histogram) or PE-Cy™7 Mouse anti-human CD20 antibody (Cat. No. 561175; solid line histogram).  The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of viable lymphocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.

Flow cytometric analysis of CD20 (cytoplasmic domain) expression by human peripheral blood lymphocytes.  Human 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 leukocytes in one step. The leukocytes were permeabilized with BD Phosflow™ Perm Buffer I (Cat. No. 557885) for 20 minutes. The cells were then stained with either PE-Cy™7 Mouse IgG2a, κ Isotype Control (Cat. No. 560906; dashed line histogram) or PE-Cy™7 Mouse anti-human CD20 antibody (Cat. No. 561175; solid line histogram).  The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of viable lymphocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.

Product Details
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BD Phosflow™
MS4A1; membrane-spanning 4-domains subfamily A member 1; B1; Bp35; LEU-16
Human (QC Testing)
Mouse BALB/c IgG2a, κ
Human B lymphoma cell line
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
V cB010
AB_10562032
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

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 with PE-Cy7 under optimum conditions, and unconjugated antibody and free PE-Cy7 were removed.

Product Notices

  1. 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).
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. PE-Cy7 is a tandem fluorochrome composed of R-phycoerythrin (PE), which is excited by 488-nm light and serves as an energy donor, coupled to the cyanine dye Cy7, which acts as an energy acceptor and fluoresces maximally at 780 nm. PE-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher. 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 PE may be observed. Therefore, we recommend that individual compensation controls be performed for every PE-Cy7 conjugate. PE-Cy7 is optimized for use with a single argon ion laser emitting 488-nm light, and there is no significant overlap between PE-Cy7 and FITC emission spectra. When using dual-laser cytometers, which may directly excite both PE and Cy7, we recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
  7. 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.
  8. Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
  9. Cy is a trademark of GE Healthcare.
  10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
561175 Rev. 3
Antibody Details
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H1

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.

561175 Rev. 3
Format Details
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PE-Cy7
PE-Cy7 dye is a part of the BD PE family of dyes. This tandem fluorochrome is comprised of a R-Phycoerythrin (PE) donor that has excitation maxima (Ex Max) of 496-nm and 566-nm and an acceptor dye, Cy™7, with an emission maximum (Em Max) at 781-nm. PE can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and detected using an optical filter centered near 781 nm (e.g., a 760/60-nm bandpass filter). The donor dye can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and the acceptor dye can be excited by the Red (627–640-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE-Cy7
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
781 nm
561175 Rev.3
Citations & References
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Development References (5)

  1. 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
  2. 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).
  3. 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
  4. Nozawa Y, Abe M, Wakasa H. Three mAb, FUN-1, FB1, and FB21, that recognize B-cell antigens in frozen or paraffin-embedded tissue sections. In: 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; 1995:705-706.
  5. 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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561175 Rev. 3

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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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.