Flow cytometric analysis of CD25 expression on unstimulated and stimulated human peripheral blood lymphocytes. Left and Middle Panels: Human whole blood was stained with either BD Horizon™ BV421 Mouse IgG1, κ Isotype Control (Cat. No. 562438; Left Panel) or BD Horizon™ BV421 Mouse Anti-Human CD25 antibody (Cat. No. 564033; Middle Panel). Erythrocytes were lysed with BD FACS Lysing Solution (Cat. No. 349202). Two-color flow cytometric dot plots show the correlated expression patterns for Ig Isotype control staining (Left Panel) or CD25 expression (Middle Panel) versus autofluorescence for events with the forward and side light-scatter characteristics of intact lymphocytes. Right Panel: Human peripheral blood mononuclear cells were stimulated for 3 days with Phytohemagglutinin. The cells were stained with either BD Horizon™ BV421 Mouse IgG1, κ Isotype Control (dashed line histogram) or BD Horizon™ BV421 Mouse Anti-Human CD25 antibody (solid line histogram). The fluorescence histograms were derived from events with the forward and side light-scatter characteristics of viable lymphoblasts. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Flow cytometric analysis of CD25 expression on unstimulated and stimulated human peripheral blood lymphocytes. Left and Middle Panels: Human whole blood was stained with either BD Horizon™ BV421 Mouse IgG1, κ Isotype Control (Cat. No. 562438; Left Panel) or BD Horizon™ BV421 Mouse Anti-Human CD25 antibody (Cat. No. 564033; Middle Panel). Erythrocytes were lysed with BD FACS Lysing Solution (Cat. No. 349202). Two-color flow cytometric dot plots show the correlated expression patterns for Ig Isotype control staining (Left Panel) or CD25 expression (Middle Panel) versus autofluorescence for events with the forward and side light-scatter characteristics of intact lymphocytes. Right Panel: Human peripheral blood mononuclear cells were stimulated for 3 days with Phytohemagglutinin. The cells were stained with either BD Horizon™ BV421 Mouse IgG1, κ Isotype Control (dashed line histogram) or BD Horizon™ BV421 Mouse Anti-Human CD25 antibody (solid line histogram). The fluorescence histograms were derived from events with the forward and side light-scatter characteristics of viable lymphoblasts. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
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
AB_2738555
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 BD Horizon BV421 under optimum conditions, and unconjugated antibody and free BD Horizon BV421 were removed.
Product Notices
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
- Brilliant Violet™ 421 is a trademark of Sirigen.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Lysing Solution 10X Concentrate IVD
Size
100 mL
Cat No.
349202
Lysing Buffer RUO
Size
100 mL
Cat No.
555899
BV421 Mouse IgG1, k Isotype Control RUO
Size
50 µg
Cat No.
562438
564033 Rev. 1
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-2R β chain) and CD132 (common γ chain or γc) to form the high-affinity signal-transducing IL-2R complex. CD25 is expressed by a subset of peripheral blood lymphocytes including CD4+CD25+ natural regulatory 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 also 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. Recombinant IL-2 blocks the binding of the 2A3 antibody to PHA-activated T lymphocytes.
The antibody was conjugated to BD Horizon™ BV421 which is part of the BD Horizon™ Brilliant Violet™ family of dyes. With an Ex Max of 407-nm and Em Max at 421-nm, BD Horizon™ BV421 can be excited by the violet laser and detected in the standard Pacific Blue™ filter set (eg, 450/50-nm filter). BD Horizon™ BV421 conjugates are very bright, often exhibiting a 10 fold improvement in brightness compared to Pacific Blue™ conjugates.
564033 Rev. 1
Format Details
BV421
The BD Horizon Brilliant Violet™ 421 (BV421) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This polymer-technology based dye has an excitation maximum (Ex Max) of 407-nm and an emission maximum (Em Max) at 423-nm. Driven by BD innovation, BV421 is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 420-nm (e.g., a 431/28-nm or 450/50-nm bandpass filter). BV421 is an ideal alternative for V450 as it is approximately ten times brighter with less spillover into the BV510/V500 detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
BV421
Violet 405 nm
407 nm
423 nm
564033 Rev.1
Citations & References
Development References (12)
-
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
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Greene WC, Leonard WJ. The human interleukin-2 receptor. Annu Rev Immunol. 1986; 4:69-95. (Clone-specific). 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
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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
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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
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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
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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.
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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
564033 Rev. 1
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.
