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PE-CF594 Mouse Anti-Human CD25
PE-CF594 Mouse Anti-Human CD25
Multicolor flow cytometric analysis of CD25 expression on unstimulated peripheral blood lymphocytes. Human whole blood was stained with BD Horizon™ BUV395 Mouse Anti-Human CD4 antibody (Cat. No. 564724) and either BD Horizon™ PE-CF594 Mouse IgG1, κ Isotype Control (Cat. No. 562292; Left Plot) or BD Horizon™ PE-CF594 Mouse Anti-Human CD25 antibody (Cat. No. 567489/567490; Right Plot). Erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). The bivariate pseudocolor density plots showing the correlated expression of CD25 (or Ig Isotype control staining) versus CD4 were 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™ X-20 Cell Analyzer System and FlowJo™ software.
PE-CF594 Mouse Anti-Human CD25

CD25 expression on activated lymphocytes. Human PBMC were stimulated with PHA (3 days). Cells were stained with BD Horizon™ PE-CF594 Mouse IgG1, κ Isotype Control (dashed line histogram) or BD Horizon™ PE-CF594 Mouse Anti-Human CD25 (solid line histogram). Fluorescent histograms showing CD25 expression (or Ig Isotype control staining) histogram was derived from gated events with light-scatter characteristics of viable (DAPI-negative) lymphoblasts. BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software were used.

Multicolor flow cytometric analysis of CD25 expression on unstimulated peripheral blood lymphocytes. Human whole blood was stained with BD Horizon™ BUV395 Mouse Anti-Human CD4 antibody (Cat. No. 564724) and either BD Horizon™ PE-CF594 Mouse IgG1, κ Isotype Control (Cat. No. 562292; Left Plot) or BD Horizon™ PE-CF594 Mouse Anti-Human CD25 antibody (Cat. No. 567489/567490; Right Plot). Erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). The bivariate pseudocolor density plots showing the correlated expression of CD25 (or Ig Isotype control staining) versus CD4 were 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™ X-20 Cell Analyzer System and FlowJo™ software.

CD25 expression on activated lymphocytes. Human PBMC were stimulated with PHA (3 days). Cells were stained with BD Horizon™ PE-CF594 Mouse IgG1, κ Isotype Control (dashed line histogram) or BD Horizon™ PE-CF594 Mouse Anti-Human CD25 (solid line histogram). Fluorescent histograms showing CD25 expression (or Ig Isotype control staining) histogram was derived from gated events with light-scatter characteristics of viable (DAPI-negative) lymphoblasts. BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software were used.

Product Details
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BD Horizon™
IL-2Rα; IL2RA; TAC antigen; TCGFR; p55
Human (QC Testing)
Mouse BALB/c IgG1, κ
Recombinant Human CD25
Flow cytometry (Routinely Tested)
5 µl
C012
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 to the dye under optimum conditions and unconjugated antibody and free dye were removed.

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

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. 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.
  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. 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).
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. An isotype control should be used at the same concentration as the antibody of interest.
  8. Texas Red is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  9. CF™ is a trademark of Biotium, Inc.
  10. When excited by the yellow-green (561-nm) laser, the fluorescence may be brighter than when excited by the blue (488-nm) laser.
  11. This product is provided under an Agreement between BIOTIUM and BD Biosciences. The manufacture, use, sale, offer for sale, or import of this product is subject to one or more patents or pending applications owned or licensed by Biotium, Inc. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Biotium, Inc. For information on purchasing a license to this product including for purposes other than research, contact Biotium, Inc., 3159 Corporate Place, Hayward, CA 94545, Tel: (510) 265-1027. Fax: (510) 265-1352. Email: btinfo@biotium.com.
  12. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using multi-laser cytometers, which may directly excite both PE and CF™594.
  13. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
Antibody Details
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BC96

The BC96 monoclonal antibody specifically binds to human CD25, the low-affinity alpha subunit of the Interleukin-2 Receptor (IL-2Rα), which is also known as TAC antigen. IL2RA encodes CD25 which is a 55 kDa type I transmembrane glycoprotein comprised of an extracellular region with two Complement Control Protein domains (CCP) followed by a transmembrane region and a short cytoplasmic tail. CD25 is constitutively expressed at high levels on natural T regulatory cells and variably expressed on conventional T cells and B cells and their precursors, NK cells, monocytes, and macrophages. CD25 expression can be highly upregulated upon antigenic or mitogenic stimulation of T cells or B cells. A soluble form of CD25 is found in biological fluids due to proteolytic cleavage of the extracellular region of transmembrane CD25. CD25 noncovalently associates with CD122 (IL-2Rβ chain) and CD132 (IL-2Rγ, also known as the common γ chain or γc) to form the high-affinity signal-transducing IL-2R complex (IL-2Rαβγ).  This heterotrimeric receptor mediates biological activities of IL-2 which can act as a cellular activation, growth, and differentiation factor and regulator of cell viability. Analysis of CD25 expression can be used to characterize the nature of normal leucocytes in their resting states or activated during inflammatory or immune responses as well as those present in certain disease states.

This antibody is conjugated to BD Horizon™ PE-CF594, which has been developed exclusively by BD Biosciences as a better alternative to PE-Texas Red®. PE-CF594 excites and emits at similar wavelengths to PE-Texas Red® yet exhibits improved brightness and spectral characteristics. Due to PE having maximal absorption peaks at 496 nm and 564 nm, PE-CF594 can be excited by the blue (488-nm), green (532-nm) and yellow-green (561-nm) lasers and can be detected with the same filter set as PE-Texas Red® (eg, 610/20-nm filter).

Format Details
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PE-CF594
BD Horizon™ PE-CF594 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 with an emission maximum (Em Max) at 615-nm. PE-CF594, driven by BD innovation, is designed to be excited by the blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and detected using an optical filter centered near 615 nm (e.g., a 610/20-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 green (532-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-CF594
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
615 nm
Citations & References
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Development References (4)

  1. Chapel A, Bensussan A, Vilmer E, Dormont D. Differential human immunodeficiency virus expression in CD4+ cloned lymphocytes: from viral latency to replication.. J Virol. 1992; 66(6):3966-70. (Immunogen: Flow cytometry). View Reference
  2. Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:1-1182.
  3. Poszepczynska E, Bagot M, Echchakir H, et al. Functional characterization of an IL-7-dependent CD4(+)CD8alphaalpha(+) Th3-type malignant cell line derived from a patient with a cutaneous T-cell lymphoma.. 2000; 96(3):1056-63. (Clone-specific). View Reference
  4. 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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Please refer to Support Documents for Quality Certificates


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.