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Multiparameter flow cytometric analysis of CD25 expression on unstimulated Human leucocytes (Left Plots) and stimulated (Right Plot) Human lymphocytes. Left Plots- Human whole blood was stained with BD Horizon™ BV421 Mouse Anti-Human CD4 antibody (Cat. No. 562424/562425) and with either BD Horizon™ RY586 Mouse IgG1, κ Isotype Control (Cat. No. 568097; Left Plot) or BD Horizon™ RY586 Mouse Anti-Human CD25 antibody (Cat. No. 568882/568883; Right Plot). Erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). The bivariate pseudocolor density plot showing the correlated expression of CD25 (or Ig Isotype control staining) versus CD4 was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Right Plot - Peripheral blood mononuclear cells (PBMC) were stimulated for 3 days with Phytohemagglutinin (PHA) and then stained with either BD Horizon™ RY586 Mouse IgG1, κ Isotype Control (Cat. No. 568097; dashed line histogram) or BD Horizon™ RY586 Mouse Anti-Human CD25 antibody (Cat. No. 568882/568883; solid line histogram). DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Solution (Cat. No. 564907) was added to cells right before analysis. The fluorescent histogram showing CD25 expression (or Ig Isotype control staining) was derived from gated events with light-scatter characteristics of viable (DAPI-negative) lymphoblasts. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
BD Horizon™ RY586 Mouse Anti-Human CD25
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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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- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
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Companion Products
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.
Development References (6)
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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, Gel shift). 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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Li H, Zhou R, Wang C, et al. T follicular regulatory cells infiltrate the human airways during the onset of acute respiratory distress syndrome and regulate the development of B regulatory cells.. Immunol Res. 2018; 66(4):548-554. (Clone-specific: Flow cytometry). View Reference
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Nie H, Zheng Y, Li R, Zhang J. Reply to Suppressive activity of human regulatory T cells is maintained in the presence of TNF.. Nat Med. 2016; 22(1):18-9. (Clone-specific: Flow cytometry). View Reference
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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
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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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