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RB670 Mouse Anti-Human FoxP3
RB670 Mouse Anti-Human FoxP3
Two-color flow cytometric analysis of FoxP3 expression in resting Human peripheral blood lymphocytes.  Human peripheral blood mononuclear cells (PBMC) were stained with BD Horizon™ BUV395 Mouse Anti-Human CD4 antibody (Cat. No. 563550). The cells were fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574) followed by staining with either BD Horizon™ RB670 Mouse IgG1, κ Isotype Control (Cat. No. 571784; Left Plot) or BD Horizon™ RB670 Mouse Anti-Human FoxP3 antibody (Cat. No. 571930/571932; Right Plot). The bivariate pseudocolor density plot showing the correlated expression of FoxP3 versus CD4 [or Ig Isotype control staining] was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Samples were acquired using the BD FACSymphony™ A5 SE Cell Analyzer System and were spectrally unmixed with autofluorescence using FlowJo™ v10.10 Software.
Two-color flow cytometric analysis of FoxP3 expression in resting Human peripheral blood lymphocytes.  Human peripheral blood mononuclear cells (PBMC) were stained with BD Horizon™ BUV395 Mouse Anti-Human CD4 antibody (Cat. No. 563550). The cells were fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574) followed by staining with either BD Horizon™ RB670 Mouse IgG1, κ Isotype Control (Cat. No. 571784; Left Plot) or BD Horizon™ RB670 Mouse Anti-Human FoxP3 antibody (Cat. No. 571930/571932; Right Plot). The bivariate pseudocolor density plot showing the correlated expression of FoxP3 versus CD4 [or Ig Isotype control staining] was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Samples were acquired using the BD FACSymphony™ A5 SE Cell Analyzer System and were spectrally unmixed with autofluorescence using FlowJo™ v10.10 Software.
Product Details
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BD Horizon™
scurfin; IPEX; JM2; AIID; XPID; DIETER; PIDX
Human (QC Testing), Rhesus,Cynomolgus,Baboon (Tested in Development)
Mouse BALB/c IgG1
FoxP3
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl/test
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

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. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

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

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. 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.
  3. 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).
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. An isotype control should be used at the same concentration as the antibody of interest.
  6. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  7. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  8. 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.
  9. 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.
  10. Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
  11. Tandem fluorochromes contain both an energy donor and an energy acceptor. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from the donor may be observed. Additionally, multi-laser cytometers may directly excite both the donor and acceptor fluorochromes. Therefore, we recommend for every tandem conjugate, a matched individual single-stain control be acquired for generating a compensation or spectral unmixing matrix.
  12. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
  13. For U.S. patents that may apply, see bd.com/patents.
571932 Rev. 1
Antibody Details
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259D/C7

The 259D/C7 monoclonal antibody specifically recognizes human Forkhead box protein P3 (FoxP3) that is also known as Scurfin. FoxP3 is encoded by FOXP3 (Forkhead box P3), likewise known as IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-Linked) and JM2, that belongs to the forkhead/winged-helix family of transcriptional regulators. FoxP3 is expressed in CD4+ regulatory T cells (Treg) and represents a specific marker for these cells. Flow cytometric analyses have shown that FoxP3 is expressed by the majority of CD4+CD25+high T cells in peripheral blood while less than half of the 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 regulation of T cell-mediated immunity and to protect against autoimmunity by suppressing the proliferation and cytokine production of other T cells. In support of this hypothesis, it has been found that Foxp3 is mutated in Scurfy (sf) mice that have defective T cell tolerance leading to an X-linked lymphoproliferative disease. The 259D/C7 antibody recognizes all currently-identified isoforms of human FoxP3 and is crossreactive with FoxP3 from Cynomolgus, Rhesus and Baboon primates.

571932 Rev. 1
Format Details
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RB670
The BD Horizon RealBlue™ 670 (RB670) Dye is part of the BD® family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 492 nm and an emission maximum (Em Max) at 670 nm as measured using an antibody-dye conjugate. Driven by BD® innovation, RB670 can be used on both spectral and conventional cytometers and is designed to be primarily excited by the Blue laser (488-nm). For conventional instruments equipped with only a Blue laser (488-nm), RB670 can be used as an alternative to PE-Cy5 and we recommend using an optical filter centered near 670-nm (eg, a 670/30-nm bandpass filter). For conventional and spectral instruments equipped with both a Blue (488-nm) and Yellow-Green (561-nm) laser and appropriate detectors, it can be used in conjunction with PE-Cy5.
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RB670
Blue 488 nm
492 nm
670 nm
571932 Rev.1
Citations & References
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View product citations for antibody "571932" on CiteAb

Development References (7)

  1. Lauer FT, Denson JL, Beswick E, Burchiel SW. Intracellular Cytokine Detection by Flow Cytometry in Surface Marker-Defined Human Peripheral Blood Mononuclear T Cells.. Curr Protoc Toxicol. 2017; 73:18.19.1-18.19.14. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
  2. Law JP, Hirschkorn DF, Owen RE, Biswas HH, Norris PJ, Lanteri MC. The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cells.. Cytometry A. 2009; 75(12):1040-50. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
  3. Mailer RKW. Alternative Splicing of FOXP3-Virtue and Vice.. Front Immunol. 2018; 9:530. (Biology). View Reference
  4. 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: Immunohistochemistry, Intracellular Staining/Flow Cytometry, Western blot). View Reference
  5. Ruitenberg JJ, Boyce C, Hingorani R, Putnam A, Ghanekar SA. Rapid assessment of in vitro expanded human regulatory T cell function.. J Immunol Methods. 2011; 372(1-2):95-106. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
  6. Sadlon TJ, Wilkinson BG, Pederson S, et al. Genome-wide identification of human FOXP3 target genes in natural regulatory T cells. J Immunol. 2010; 185(2):1071-1081. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
  7. Santegoets SJ, Dijkgraaf EM, Battaglia A, et al. Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry.. Cancer Immunol Immunother. 2015; 64(10):1271-86. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
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571932 Rev. 1

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.