Flow cytometric analysis of CD25 expression on unstimulated and PHA-stimulated human peripheral blood lymphocytes Left and Middle Plots: Human whole blood was stained with APC Mouse Anti-Human CD4 antibody (Cat. No. 561840/555349/561841) and with either BD Horizon™ RY586 Mouse IgG1, κ Isotype Control (Cat. No. 568097) or BD Horizon™ RY586 Mouse Anti-Human CD25 antibody (Cat. No. 568459/568460). Erythrocytes were lysed with BD FACS Lysing™ Solution (Cat. No. 349202). The bivariate 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: Human peripheral blood mononuclear cells were stimulated for 3 days with Phytohemagglutinin (PHA). The cells were 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. 568459/568460; solid line histogram). BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. The fluorescence histogram showing CD25 expression (or Ig Isotype control staining) was derived from events with the forward and side light-scatter characteristics of viable (7-AAD-negative) lymphoblasts. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
Flow cytometric analysis of CD25 expression on unstimulated and PHA-stimulated human peripheral blood lymphocytes Left and Middle Plots: Human whole blood was stained with APC Mouse Anti-Human CD4 antibody (Cat. No. 561840/555349/561841) and with either BD Horizon™ RY586 Mouse IgG1, κ Isotype Control (Cat. No. 568097) or BD Horizon™ RY586 Mouse Anti-Human CD25 antibody (Cat. No. 568459/568460). Erythrocytes were lysed with BD FACS Lysing™ Solution (Cat. No. 349202). The bivariate 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: Human peripheral blood mononuclear cells were stimulated for 3 days with Phytohemagglutinin (PHA). The cells were 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. 568459/568460; solid line histogram). BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. The fluorescence histogram showing CD25 expression (or Ig Isotype control staining) was derived from events with the forward and side light-scatter characteristics of viable (7-AAD-negative) lymphoblasts. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
Product Details
BD Horizon™
IL-2R; IL2RA; IL-2Rα; TCGFR; TAC antigen; p55
Human (QC Testing)
Mouse BALB/c IgG1, κ
Phytohemagglutinin stimulated human lymphocytes
Flow cytometry (Routinely Tested)
5 µl
IV A053
3559
Aqueous buffered solution containing ≤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 unreacted dye was 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
- 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.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- CF™ is a trademark of Biotium, Inc.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
RY586 Mouse IgG1, κ Isotype Control RUO
Size
50 µg
Cat No.
568097
Lysing Solution 10X Concentrate IVD
Size
100 mL
Cat No.
349202
APC Mouse Anti-Human CD4 RUO
Size
500 Tests
Cat No.
561841
Lysing Buffer RUO
Size
100 mL
Cat No.
555899
568460 Rev. 2
Antibody Details
M-A251
The M-A251 monoclonal antibody specifically binds to the 55 kDa type I transmembrane glycoprotein known as low-affinity interleukin-2 receptor alpha chain subunit (IL-2Rα). CD25 is expressed on regulatory T cells, activated lymphocytes (T and B), and monocytes. It associates with the IL-2Rβ/CD122 and IL-2Rγ/CD132 receptor chains to form the high-affinity IL-2R complex. CD25 expression on T and B lymphocytes is upregulated by antigenic or mitogenic stimulation. Soluble CD25/IL-2Rα is produced as a consequence of lymphocyte stimulation and is found in biological fluids following inflammatory responses.
568460 Rev. 2
Format Details
RY586
The BD Horizon RealYellow™ 586 (RY586) Dye is part of the BD family of yellow-green dyes. It is a small organic fluorochrome with an excitation maximum (Ex Max) at 565-nm and an emission maximum (Em Max) at 586-nm. Driven by BD innovation, RY586 can be used on both spectral and conventional cytometers and is designed to be excited by the Yellow-Green laser (561-nm) with minimal excitation by the 488-nm Blue laser. For conventional instruments equipped with a Yellow-Green laser (561-nm), RY586 can be used as an alternative to PE and we recommend using an optical filter centered near 586-nm (eg, a 586/15-nm bandpass filter). For spectral instruments equipped with a Yellow-Green laser (561-nm), it can be used in conjunction with PE. Compared to PE, RY586 is similar in brightness, minimal spillover into Blue detectors, and increased spillover into the 610/20-nm (PE-CF594) detector. Please ensure that your instrument configuration (lasers and optical filters) is appropriate for this dye.
RY586
Yellow-Green 561 nm
564 nm
586 nm
568460 Rev.2
Citations & References
View product citations for antibody "568460" on CiteAb
Development References (7)
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Janszen M, Buck D, Maino VC. Functional and molecular properties of CD25 monoclonal antibodies. In: Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:403-406.
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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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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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Nakamura K, Kitani A, Fuss I, et al. TGF-β1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice. J Immunol. 2004; 172(2):834-842. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting). View Reference
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Ravoet AM, Latinne D, Couvreur B, et al. CD25 mab: Epitopes recognized, effect on lymphocyte activation, mediation of ADCC. In: Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:408-411.
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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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Schwarting R, Stein H. Cluster report: CD25. In: Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:399-403.
568460 Rev. 2
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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.