Multicolor flow cytometric analysis of CD25 expression on unstimulated Human peripheral blood lymphocytes. Human whole blood was stained with APC Mouse Anti-Human CD4 antibody (Cat. No. 561841) and with either BD Horizon™ RB780 Mouse IgG1 κ Isotype Control (Cat. No. 568532; Left Plot) or BD Horizon™ RB780 Mouse Anti-Human CD25 antibody (Cat. No. 568688/568689; Right Plot). The erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). The two-parameter 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. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Flow Cytometer System and FlowJo™ software.
Flow cytometric analysis of CD25 expression on stimulated Human peripheral blood lymphocytes. Human PBMC were stimulated for 3 days with Phytohemagglutinin (PHA). Cells were stained with either BD Horizon™ RB780 Mouse IgG1, κ Isotype Control (Cat No. 568532, dashed line histogram) or BD Horizon™ RB780 Mouse Anti-Human CD25 antibody (Cat No. 568688/568689, solid line histogram). The fluorescence histogram showing CD25 expression (or Ig Isotype control staining) was derived from gated events with the forward and side light-scatter characteristics of viable lymphoblasts. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Flow Cytometer System and FlowJo™ software.
.png){kind=spacer}
Multicolor flow cytometric analysis of CD25 expression on unstimulated Human peripheral blood lymphocytes. Human whole blood was stained with APC Mouse Anti-Human CD4 antibody (Cat. No. 561841) and with either BD Horizon™ RB780 Mouse IgG1 κ Isotype Control (Cat. No. 568532; Left Plot) or BD Horizon™ RB780 Mouse Anti-Human CD25 antibody (Cat. No. 568688/568689; Right Plot). The erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). The two-parameter 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. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Flow Cytometer System and FlowJo™ software.
Flow cytometric analysis of CD25 expression on stimulated Human peripheral blood lymphocytes. Human PBMC were stimulated for 3 days with Phytohemagglutinin (PHA). Cells were stained with either BD Horizon™ RB780 Mouse IgG1, κ Isotype Control (Cat No. 568532, dashed line histogram) or BD Horizon™ RB780 Mouse Anti-Human CD25 antibody (Cat No. 568688/568689, solid line histogram). The fluorescence histogram showing CD25 expression (or Ig Isotype control staining) was derived from gated events with the forward and side light-scatter characteristics of viable lymphoblasts. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Flow Cytometer 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
Human Phytohemagglutinin-activated T Cells
Flow cytometry (Routinely Tested)
5 µl
III A769,T153; IV A8
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
- When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- An isotype control should be used at the same concentration as the antibody of interest.
- 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).
- 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.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
- 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.
568689 Rev. 2
Antibody Details
2A3
The 2A3 monoclonal antibody specifically binds to human CD25, the low-affinity alpha subunit of the Interleukin-2 Receptor (IL- 2Rα). CD25 associates with CD122 (IL-2Rβ chain) and CD132 (common γ chain or γc) to form the high-affinity signal-transducing IL-2R complex. CD25 is expressed by subsets of thymocytes and peripheral blood lymphocytes including CD4+CD25+ regulatory T cells and memory T cells. CD25 antigen density increases on activated T cells including phytohemagglutinin (PHA)-, concanavalin A (Con A)-, and CD3-activated T lymphocytes. High levels of CD25 can be expressed by T lymphocytes from mixed lymphocyte cultures and by human T-lymphocyte leukemia virus (HTLV)-infected T-lymphocyte leukemia lines, for example, HUT-102. CD25 can also be expressed by activated B cells and macrophages. Recombinant IL-2 blocks the binding of the 2A3 antibody to PHA-activated T lymphocytes.
568689 Rev. 2
Format Details
RB780
The BD Horizon RealBlue™ 780 (RB780) Dye is part of the BD family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 498-nm and an emission maximum (Em Max) at 781-nm. Driven by BD innovation, RB780 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with minimal excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), RB780 can be used as an alternative to PE-Cy7 and we recommend using an optical filter centered near 780-nm (eg, a 780/60-nm bandpass filter). For spectral instruments equipped with a Blue laser (488-nm), it can be used in conjunction with PE-Cy7. RB780 is on average brighter than PE-Cy7 and has minimal spillover into Yellow-Green detectors.
RB780
Blue 488 nm
498 nm
781 nm
568689 Rev.2
Citations & References
View product citations for antibody "568689" on CiteAb
Development References (13)
-
Dower SK, Hefeneider SH, Alpert AR, Urdal DL. Quantitative measurement of human interleukin 2 receptor levels with intact and detergent-solubilized human T-cells. Mol Immunol. 1985; 22(8):937-947. (Clone-specific). View Reference
-
Greene WC, Leonard WJ. The human interleukin-2 receptor. Annu Rev Immunol. 1986; 4:69-95. (Clone-specific). View Reference
-
Jackson AL, Matsumoto H, Janszen M, Maino V, Blidy A, Shye S. Restricted expression of p55 interleukin 2 receptor (CD25) on normal T cells. Clin Immunol Immunopathol. 1990; 54(1):126-133. (Clone-specific: Flow cytometry). View Reference
-
Lando Z, Sarin P, Megson M, et al. Association of human T-cell leukaemia/lymphoma virus with the Tac antigen marker for the human T-cell growth factor receptor. Nature. 1983; 305(5936):733-736. (Biology). View Reference
-
Leonard WJ, Depper JM, Uchiyama T, Smith KA, Waldmann TA, Greene WC. A monoclonal antibody that appears to recognize the receptor for human T-cell growth factor; partial characterization of the receptor. Nature. 1982; 300(5889):267-269. (Biology). View Reference
-
Ng WF, Duggan PJ, Ponchel F, et al. Human CD4(+)CD25(+) cells: a naturally occurring population of regulatory T cells. Blood. 2001; 98(9):2736-2744. (Biology). View Reference
-
Rambaldi A, Young DC, Herrmann F, Cannistra SA, Griffin JD. Interferon-gamma induces expression of the interleukin 2 receptor gene in human monocytes. Eur J Immunol. 1987; 17(1):153-156. (Clone-specific). View Reference
-
Robb RJ, Greene WC, Rusk CM. Low and high affinity cellular receptors for interleukin 2. Implications for the level of Tac antigen. J Exp Med. 1984; 160(4):1126-1146. (Biology). View Reference
-
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.
-
Sereti I, Martinez-Wilson H, Metcalf JA, et al. Long-term effects of intermittent interleukin 2 therapy in patients with HIV infection: characterization of a novel subset of CD4(+)/CD25(+) T cells. Blood. 2002; 100(6):2159-2167. (Clone-specific: Flow cytometry). View Reference
-
Siegel JP, Sharon M, Smith PL, Leonard WJ. The IL-2 receptor beta chain (p70): role in mediating signals for LAK, NK, and proliferative activities. Science. 1987; 238(4823):75-78. (Biology). View Reference
-
Teshigawara K, Wang HM, Kato K, Smith KA. Interleukin 2 high-affinity receptor expression requires two distinct binding proteins. J Exp Med. 1987; 165(1):223-238. (Biology). View Reference
-
Urdal DL, March CJ, Gillis S, Larsen A, Dower SK. Purification and chemical characterization of the receptor for interleukin 2 from activated human T lymphocytes and from a human T-cell lymphoma cell line. Proc Natl Acad Sci U S A. 1984; 81(20):6481-6485. (Immunogen: Blocking, Dot Blot, Immunoaffinity chromatography, Inhibition, Radioimmunoassay). View Reference
568689 Rev. 2
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.