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Multicolor flow cytometric analysis of FoxP3 expressed in human lymphocytes. Human peripheral blood mononuclear cells were stained with BD Horizon™ BUV395 Mouse Anti-Human CD4 (Cat. No. 564724; Upper Plots) and FITC Anti-Human CD25 (Cat. No. 555431/560990; Lower Plots) antibodies. The cells were washed, fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574/562725). The cells were then stained with either BD Horizon™ R718 Mouse IgG1, κ Isotype Control (Cat. No. 566928; Left Plots) or BD Horizon™ R718 Mouse Anti-Human FoxP3 antibody (Cat No. 567243/567067; Right Plots). Bivariate pseudocolor density plots showing the correlated expression of FoxP3 versus either CD4 or CD25 [or Ig Isotype control staining] were derived from gated events with the light scattering characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
BD Horizon™ R718 Mouse Anti-Human FoxP3
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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 CompBead to ensure that BD CompBeads are appropriate for your specific cellular application.
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- 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).
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The 236A/E7 antibody reacts with the human FoxP3 (Forkhead box protein P3) transcription factor, a member of the forkhead or winged helix family of transcription factors. The expression of FoxP3, also known as Scurfin, IPEX and JM2, has been found to be associated with CD4+ CD25+ regulatory T cells and represents a specific marker for these cells. Flow cytometric analysis has shown that FoxP3 is expressed by the majority of CD4+ CD25+ (high) T cells in peripheral blood while less than half of CD4+ CD25+ (intermediate) cell population are FoxP3 positive. Approximately 5-10% of peripheral CD4+ cells are CD4+ CD25+ T regulatory cells. T regulatory cells are thought to play a critical role in the control of T cell mediated autoimmunity by suppressing the proliferation and cytokine production of other T cells. To support this hypothesis, it has been found that Foxp3 is mutated in scurfy (sf) mice. Cumulative evidence suggests that the 236A/E7 antibody recognizes epitopes from both isoforms of Exon 2 alternatively-spliced variants and is located in the region between Exon 2 and the Zn/LZ domains (aa 105-235).
The antibody was conjugated to BD Horizon Red 718, which has been developed exclusively by for BD Biosciences as a better alternative to Alexa Fluor™ 700. BD Horizon Red 718 can be excited by the red laser (628 – 640 nm) and, with an Em Max around 718 nm, it can be detected using a 730/45 nm filter. Due to similar excitation and emission properties, we do not recommend using R718 in combination with APC-R700 or Alexa Fluor™ 700.
Development References (8)
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Alvaro T, Lejeune M, Salvado MT, et al. Outcome in Hodgkin's lymphoma can be predicted from the presence of accompanying cytotoxic and regulatory T cells. Clin Cancer Res. 2005; 11(4):1467-1473. (Clone-specific: Immunohistochemistry). View Reference
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Brunkow ME, Jeffery EW, Hjerrild KA, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. 2001; 27(1):68-73. (Biology). View Reference
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Fox BC, Bignone PA, Brown PJ, Banham AH. Defense of the clone: antibody 259D effectively labels human FOXP3 in a variety of applications. Blood. 2008; 111(7):3897-3899. (Clone-specific: Western blot). View Reference
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Lennon G, Auffray C, Polymeropoulos M, Soares MB. Consortium: an integrated molecular analysis of genomes and their expression. Genomics. 1996; 33(1):151-152. (Biology). View Reference
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Roncador G, Brown PJ, Maestre L, et al. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur J Immunol. 2005; 35(6):1681-1691. (Immunogen: Flow cytometry). View Reference
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Roncador G, Garcia JF, Maestre L, et al. FOXP3, a selective marker for a subset of adult T-cell leukaemia/lymphoma. Leukemia. 2005; 19(12):2247-2253. (Immunogen: Immunohistochemistry). View Reference
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Wildin RS, Ramsdell F, Peake J, et al. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet. 2001; 27(1):18-20. (Biology). View Reference
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Wolf D, Wolf AM, Rumpold H, et al. The expression of the regulatory T cell-specific forkhead box transcription factor FoxP3 is associated with poor prognosis in ovarian cancer. Clin Cancer Res. 2005; 11(23):8326-8331. (Biology). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.