Foxp3 Antibodies Optimized for Flow Cytometry, IHC, WB For Mouse, Human, Rat, Non-Human Primates & Canine

Ordering Information: Foxp3
Foxp3 Antibody-Specific References
Foxp3 Antibody Data: FJK-16s, PCH101, NRRF-30, eBio7979, 236A/E7

Tregs & Foxp3: the Suppression Phenomenon

Until recently, definitive flow cytometric analysis of CD4+CD25+ regulatory T cells, expressing Foxp3, has been hindered due to lack of suitable Foxp3 antibodies. Using eBioscience Foxp3 antibodies PCH101, FJK-16s and NRRF-30, Foxp3 can now be identified at the single-cell level (see figures and table below).

Since the discovery of regulatory suppressor cells by Gershon in 1970, great controversies have ensued. These cells have broad implications ranging from immune suppression to cancer. Consequently, the interest in the transcription factor Foxp3, identified by S. Sakaguchi (2003) as a hallmark of naturally arising CD4+CD25+ regulatory T cells (Tregs), has intensified.

Recently, one splice variant of Foxp3, lacking amino acids 71-105, has been reported in human T cells. This variant is expressed in CD8+ T cells from some donors, while both forms are present in CD4+CD25+ cells. The western blot data on page two reveals two bands in human tissue; presumably from each splice variant, while only a single band is detected in mouse. The presence of this splice variant has potentially complicated the complete characterization of Foxp3 expression.

Mouse Foxp3


BALB/c splenocytes were stained with CD4-FITC (clone RM4-5; cat. 11-0042), CD25-APC (clone PC61.5; cat. 17-0251), and mouse/rat Foxp3-PE (clone FJK-16s) using the PE anti-mouse/rat Foxp3 Staining Set (cat. 72-5775). Quadrant lines demarcate the isotype control. Left: FJK-16s vs CD4 Right: FJK-16s vs CD25			The histogram demonstrates FJK-16s-FITC (cat. 71-5775) staining (blue histogram) and isotype control (yellow histogram) on CD4+CD25+ mouse spleen cells.

Human Foxp3


Human PBMCs were stained with CD4-FITC (clone RPA-T4; cat. 11-0049), CD25-APC (clone BC96; cat. 17-0259), and human Foxp3-PE (clone PCH101) using the PE anti-human Foxp3 Staining Set (cat. 72-5776). Quadrant lines demarcate the isotype control. Cells in the lymphocyte gate were used for analysis. Left: PCH101 vs CD4 Right: PCH101 vs CD25			The histogram demonstrates PCH101-FITC (cat. 71-5776) staining (blue histogram) and isotype control (yellow histogram) on CD4+CD25^bright human PBMC.

Immunological Identity, Specificity and Validation of Foxp3 Antibodies

As has been done with many cell surface and intracellular (e.g., cytokine) proteins, corroboration of staining pattern using antibodies to distinct epitopes from the same protein allows researchers to confirm staining patterns and hence specificity. To this end, eBioscience utilized protein deletion constructs to epitope-map a panel of Foxp3 antibodies. The Figure below shows the schematic representation of the Foxp3 protein with functional domains and also localizes the regions recognized by each of eBioscience's Foxp3 antibodies.

Schematic representation of the Foxp3 protein and the location of the epitopes for eBioscience antibodies. Foxp3 contains a Zn finger/Leucine zipper and a conserved Forkhead Domain. In addition, in human Exon 2 may be alternatively spliced in some cells (black box). Currently there is not evidence for alternative splicing in the mouse. The clone names for each of eBioscience antibodies are shown in the location of their individual epitopes.

In the figures below, co-staining experiments indicate that several Foxp3 antibodies stain the same cells. The anti-mouse Foxp3 antibodies, FJK-16s and NRRF-30 recognize different epitopes in the amino terminus, with the former recognizing the region corresponding to the human splice variant. These two antibodies identify the same population of lymphocytes, and co-staining experiments demonstrate 100% correlation (see figure below). These data suggest that these two antibodies, which recognize different regions of recombinant Foxp3, bind the same protein expressed in the same cells. Additionally, the same approach was taken for the human Foxp3 antibodies.


Co-staining of eBioscience foxp3 antibodies that recognize different epitopes. Left: Mouse splenocytes were co-stained with FJK-16s and eBio7979. Right: NRRF-30 co-staining with FJK-16s.			Existence of splice variant in human, not mouse: immunoblotting of PBMC (Lane A), BALB/c splenocyte (Lane B), or recombinant human Foxp3 fusion protein (Lane C); probed with eBio7979 (cat. 14-7979).


Co-staining of eBioscience Foxp3 antibodies that recognize different epitopes. Left: Human PBMCs were co-stained with PCH101 and 236A/E7. Right: Co-staining of PCH101 and eBio7979.

The expanding panel of antibodies reactive with distinct epitopes of Foxp3 is useful for investigating the complete Foxp3 protein expression profile at the single-cell level in both human and mouse, particularly with regards to its relationship to functionally-defined Regulatory T cells. In addition, the presence of an alternatively spliced transcript in human adds another layer of complexity to the Foxp3 expression pattern and functional significance. Currently this phenomenon appears to be unique to human since both co-staining at the single-cell level using several different combinations of antibodies to different domains and immunoblotting data suggests that only one isoform exists in the mouse. (See flow analysis and immunoblot data above.) In the human, two isoforms can be present; the anti-mouse/rat Foxp3 antibody, FJK-16s, crossreacts to human Foxp3 but recognizes the region that can be alternatively spliced in some transcripts. Therefore, the FJK-16s antibody labels the Foxp3 protein isoform that has not been spliced, while PCH101 and 236A/E7 antibodies recognize both isoforms of the protein. Using these reagents together will allow researchers to investigate the function of these two isoforms of human Foxp3 protein in the same cell population.

The Foxp3 antibodies have been validated for intracellular staining with flow cytometric analysis. The protocols and staining buffers have been developed for optimal and consistent staining Foxp3 staining while preserving surface antigen staining. Furthermore, the protocol was designed for robustness, reproducibility, ease-of-use, as well as flexibility, to allow researchers to stain at their convenience. In addition, the antibodies have been reported useful for immunohistological staining (both frozen and paraffin sections) and western blotting of endogenous protein from total human PBMCs or mouse splenocytes, without the need for enrichment of Tregs.


C57BL6 spleen cryosections were stained with anti-mouse/rat Foxp3 antibody, FJK-16s (cat. 14-5773) and revealed with colorimetric methods. Image courtesy of Cintia De Paiva.		Staining of human tonsillar tissue section with PCH101 (cat. 14-4776). Image courtesy of Roger Sutmuller.

New Information

The specificity of all Foxp3 monoclonal antibodies has been confirmed by numerous publications. (see reference list below) A recent manuscript by Tran et al (Blood 2007) suggests that monoclonal antibiody PCH101 is not specific on activated/cultured Tregs. Leading scientists in the Regulatory T Cell field have shown PCH101 is an excellent antibody for examining Foxp3 expression in resting and cultured cells. The data in the Tran paper suggested that PCH101 staining is found on all cells when cultured with TGF-β or with neutralizing antibodies to TGF-β. The cells cultured with TGF-β clearly show 2 distinct populations albeit shifted higher off the axis. It is the opinion of eBioscience that the 2 populations consist of a positive Foxp3 population and a negative population. This interpretation would then be consistent with all the subsequent findings (RT-PCR and siRNA). The siRNA can only knock down the brightest staining population but not the dim population, since the dim population is in fact negative. The shift/increase in background staining is not seen in other publications nor in any eBioscience data comparing PCH101 to any other Foxp3 antibody. The cause for the shift seen by Tran et al is not known.

Several articles recently published utilizing PCH101 in cultured/activated Tregs and confirming specificity:
Ahmadzadeh et al J Immunother 2007;30:294–302
Pillai et al Clin Immunol. 2007 Apr;123(1):18-29
Wang et al Eur. J. Immunol. 2007. 37: 129–138

In particular the Wang paper shows costaining of 236A and PCH101 suggesting the antibodies, that are known to recognize different epitopes, see the same cell population. This is additional evidence confirming specificity of PCH101 for Foxp3. Additionally please see the following link for a letter from the authors with additional supporting data.

The transient expression pattern of Foxp3 in in vitro cultured cells shown by Pillai et al further supports the specificity of PCH101 for Foxp3. The figure below illustrates a distinct population of Foxp3 negative and positive population an be identified in activated Treg cultures.

CD4+CD25- T-cells upregulate FOXP3 transiently following activation. Bead sorted ‘untouched’ CD3+CD25- T-cells or CD4+CD25- T-cells were stained with CFSE and activated with either allogeneic T-cell-depleted PBMC. CD25 and FOXP3 expression and CFSE dilution were monitored over time. Color-coded contour plots of gated CD4+ T-cells from allostimulated cultures are shown, with CFSE on the x-axis and CD25 (top row) or FOXP3 (bottom row) on the y-axis. The vertical lines separate dividing (CFSE^low) from non-dividing (CFSE^high) cells. Courtesy of Pillai V, Ortega SB, Wang CK, Karandikar NJ. University of Texas Southwestern Medical Center

New! For Non-Human Primate Studies and Beyond

The anti-human Foxp3 antibodies, PCH101 and 236A/E7, crossreact to several non-human primate species in intracellular staining studies. Additionally, antibodies to CD4 (OKT4) and CD25 (BC96) also stain rhesus and cynomolgus lymphocytes, making characterization of natural Tregs in non-human primates more comprehensive.

It has been confirmed that FJK-16s crossreacts with canine Foxp3 protein (Biller BJ, Elmslie RE, Burnett RC, Avery AC, Dow SW. Use of FoxP3 expression to identify regulatory T cells in healthy dogs and dogs with cancer. Vet Immunol Immunopathol. 2007 Mar 15;116(1-2):69-78, pubmed).

As Researchers Learn, the Partnership with eBioscience Grows

As researchers learn more about the importance of Foxp3 as a master control of immune regulation, the commitment of eBioscience to providing critically-important, well-validated reagents becomes clear. The Foxp3 antibodies are available in a variety of fluorochromes (including tandem dyes and Alexa Fluors^®*) that is constantly expanding to meet your needs. In combination with our vast offering of fluorochrome-conjugated antibodies reactive with cell surface antigens, Foxp3 staining can be analyzed against more than 6 colors allowing for the intricate analysis of cell populations and subsets of populations.

Publications using eBioscience Foxp3 Clones for IC Staining & Flow Cytometric Analysis

Mouse Foxp3

Biller, BJ., et al. 2007. Use of FoxP3 expression to identify regulatory T cells in healthy dogs and dogs with cancer. Vet Immunol Immunopathol. 116(1-2):69-78. [Intracellular staining for flow cytometry using FJK-16s in canine]
Sakaguchi, S. et al. 2005. Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+CD25+CD4+ regulatory T cells. J. Exp. Med. 202: 885-891. [Intracellular staining for flow cytometry using FJK-16s]
McGeachy, MJ., et al. 2005. Natural Recovery and Protection from Autoimmune Encephalomyelitis: Contribution of CD4+CD25+ Regulatory Cells within the Central Nervous System. J. Immunol. 175: 3025 - 3032. [Intracellular staining for flow cytometry using FJK-16s]
Fields, ML., et al. 2005. CD4+CD25+Regulatory T Cells Inhibit the Maturation but Not the Initiation of an Autoantibody Response. J. Immunol. 175: 4255 - 4264. [Intracellular staining for flow cytometry using FJK-16s]
Lu, LF., et al. 2005. NFB-Inducing Kinase Deficiency Results in the Development of a Subset of Regulatory T Cells, which Shows a Hyperproliferative Activity upon Glucocorticoid-Induced TNF Receptor Family-Related Gene Stimulation. J. Immunol. 175: 1651 - 1657. [Intracellular staining for flow cytometry using FJK-16s]
Beyersdorf, S., et al. 2005. Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis. J Exp Med. 202: 445-55. [Intracellular staining for flow cytometry using FJK-16s (correction; not mFoxy)]
Anderson, M.S., et al. 2005. The cellular mechanism of Aire control of T cell tolerance. Immunity. 28: 227-39. [Intracellular staining for flow cytometry using FJK-16s]
Chang, X., et al. 2005. The Scurfy mutation of FoxP3 in the thymus stroma leads to defective thymopoiesis. 202:1141-51. [Intracellular staining for flow cytometry using FJK-16s]
Aswad, F., et al. 2005. High sensitivity of CD4+CD25+ regulatory T cells to extracellular metabolites nicotinamide adenine dinucleotide and ATP: a role for P2X7 receptors. J. Immunol. 175:3075-83. [Intracellular staining for flow cytometry using FJK-16s]
Siegmund, R., et al. 2005. Migration matters: regulatory T-cell compartmentalization determines suppressive activity in vivo. Blood. 106: 3097-104. [Intracellular staining for flow cytometry using FJK-16s]
Romagnoli, S., et al. 2005. Genetic control of thymic development of CD4+CD25+FoxP3+ regulatory T lymphocytes. Eur J Immunol. 35: 1-8. [Intracellular staining for flow cytometry using FJK-16s]
Wilson, M.S., et al. 2005. Suppression of allergic airway inflammation by helminth-induced regulatory T cells. J Exp Med. 202: 1199-1212. [Intracellular staining for flow cytometry using FJK-16s]
Knoechel, B., et al. 2005. Sequential development of interleukin 2-dependent effector and regulatory T cells in response to endogenous systemic antigen J. Exp. Med. Nov (Epub ahead of print). [Intracellular staining for flow cytometry using FJK-16s]
Kretschmer, K., et al. 2005. Inducing and expanding regulatory T cells population by foreign antigen. Nat. Immunol. 6(12): 1219-1227. [Intracellular staining for flow cytometry using FJK-16s]
Krupnick, AS., et al. 2005. Cutting Edge: Murine Vascular Endothelium Activates and Induces the Generation of Allogeneic CD4+25+Foxp3+ Regulatory T Cells. J. Immunol. 175: 6265-6270. [Intracellular staining for flow cytometry using FJK-16s]
Liu, R., et al. 2005. Cooperation of Invariant NKT Cells and CD4+CD25+ T Regulatory Cells in the Prevention of Autoimmune Myasthenia. J. Immunol. 175(12):7898-7904. [Intracellular staining for flow cytometry using FJK-16s]
Moore, AC., et al. 2005. Anti-CD25 Antibody Enhancement of Vaccine-Induced Immunogenicity: Increased Durable Cellular Immunity with Reduced Immunodominance. J. Immunol. 175: 7264-7273.
Stephens, LA., et al. 2005. CD4+CD25+ regulatory T cells limit the risk of autoimmune disease arising from T cell receptor crossreactivity. PNAS 102:17418-17423.

Human Foxp3 (PCH101):

Ahmadzadeh, M., et al. 2005. IL-2 Administration Increases CD4+CD25hiFoxp3+ Regulatory T Cells in Cancer Patients. Blood (Nov Epub). [Intracellular staining for flow cytometry using PCH101]
Ahmadzadeh et al 2007. IL-2 and IL-15 each mediate de novo induction of FOXP3 expression in human tumor antigen-specific CD8 T cells. J Immunother 30:294–302
Crellin, NK., et al. 2005. Human CD4+ T Cells Express TLR5 and Its Ligand Flagellin Enhances the Suppressive Capacity and Expression of FOXP3 in CD4+CD25+ T Regulatory Cells. J. Immunol. 175(12): 8051-9. [Intracellular staining for flow cytometry using PCH101]
Hartwig, UF., et al. 2005. Depletion of alloreactive T cells via CD69: implications on antiviral, antileukemic and immunoregulatory T lymphocytes. Bone Marrow Transplant (Dec 5 Epub ahead of print). [Intracellular staining for flow cytometry using PCH101]
Lim, HW., et al. 2005. Cutting Edge: Direct Suppression of B Cells by CD4+CD25+ Regulatory T Cells. J. Immunol. 175: 4180 - 4183. [IHC of frozen sections using 236A/E; Intracellular staining for flow cytometry using PCH101]
Pillai et al. 2007 Transient regulatory T-cells: a state attained by all activated human T-cells. Clin Immunol Apr;123(1):18-29
Wang et al 2007. Transient expression of FOXP3 in human activated nonregulatory CD4+ T cells. Eur J Immunol. Jan;37(1):129-38. Eur. J. Immunol. 2007. 37: 129–138

Human Foxp3 (236A/E7):

Alvaro, T., et al. 2005. Outcome in Hodgkin's lymphoma can be predicted from the presence of accompanying cytotoxic and regulatory T cells. Clin. Cancer Res. 11(4):1467-73. [IHC paraffin using 236A/E7]
Roncador, G., et al. 2005. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur. J. Immunol. 35: 1681-91. [IHC of paraffin sections using 236A/E; Intracellular staining for flow cytometry using 236A/E]
Roncador, G., et al. 2005. FOXP3, a selective marker for a subset of adult T-cell leukaemia/lymphoma. Leukemia (Epub ahead of print). [IHC paraffin sections using 236A/E7]
Wolf, D., et al. 2005. The expression of the regulatory T cell-specific forkhead box transcription factor FoxP3 is associated with poor prognosis in ovarian cancer. Clin. Cancer Res. 11(23): 8326-31. [IHC paraffin using 236A/E7]

Ordering Information

Foxp3 Reagents
Description	Clone	Application	Format	Catalog No.
Human Foxp3	PCH101	Flow (IC), WB, IH (paraffin & frozen)	Purified	14-4776
(reacts w/ Monkey)		Flow (IC), WB, IH (paraffin & frozen)	Biotin	13-4776
		Flow (IC), IH (paraffin & frozen)	FITC	11-4776
		Flow (IC)	FITC Staining Set	71-5776
		Flow (IC)	PE	12-4776
		Flow (IC)	PE Staining Set	72-5776
		Flow (IC)	PE-Cy5	15-4776
		Flow (IC), IH (paraffin & frozen)	APC	17-4776
		Flow (IC)	APC Staining Set	77-5776
		Flow (IC), IH (paraffin & frozen)	Alexa Fluor^® 488	53-4776
		Flow (IC)	Alexa Fluor^® 488 Staining Set	73-5776
		Flow (IC)	Alexa Fluor^® 647	51-4776
		Flow (IC)	Alexa Fluor^® 700	56-4776
		Flow (IC)	eFluor® 450	48-4776
		Flow (IC)	Staining Buffer Set	00-5523
		Flow (IC)	Treg Staining Kit (w/ PE Foxp3 PCH101)	88-8999
		Flow (IC)	Treg Staining Kit #2 (w/ APC Foxp3 PCH101)	88-8998
		Flow (IC)	Treg Staining Kit #3 (w/ PE-Cy5 Foxp3 PCH101)	88-8995
		Flow (IC)	Human/NHP Treg Staining Kit (w/ PE Foxp3 PCH101)	88-4999
		Flow (IC)	Human/NHP Treg Staining Kit #2 (w/ APC Foxp3 PCH101)	88-4994
NEW!		Flow (IC)	Human Foxp3 Whole Blood Staining Kit	88-8996
Human Foxp3	236A/E7	Flow (IC), IHC (paraffin & frozen), WB	Purified	14-4777
(reacts w/ Monkey)		Flow (IC), IH (paraffin & frozen)	Biotin	13-4777
		Flow (IC)	FITC	11-4777
		Flow (IC)	PE	12-4777
		Flow (IC)	PE Staining Set	72-5774
		Flow (IC)	APC	17-4777
		Flow (IC)	APC Staining Set	77-5774
		Flow (IC), IH (paraffin & frozen)	Alexa Fluor^® 488	53-4777
		Flow (IC)	Alexa Fluor^® 488 Staining Set	73-5774
		Flow (IC)	Alexa Fluor^® 647	51-4777
		Flow (IC)	eFluor® 450	57-4777
		Flow (IC)	Staining Buffer Set	00-5523
Human/Mouse Foxp3	eBio7979	WB, IH (paraffin)	Purified	14-7979
		Flow (IC)	PE	12-7979
		Flow (IC)	Alexa Fluor^® 488	53-7979
		Flow (IC)	Alexa Fluor^® 647	51-7979
		Flow (IC)	Staining Buffer Set	00-5523
Mouse/Rat Foxp3	FJK-16s	Flow (IC), WB, IH (frozen)	Purified	14-5773
		Flow (IC), WB, IH (frozen)	Biotin	13-5773
		Flow (IC)	FITC	11-5773
		Flow (IC)	FITC Staining Set	71-5775
		Flow (IC)	PE	12-5773
		Flow (IC)	PE Staining Set	72-5775
		Flow (IC)	PE-Cy5	15-5773
		Flow (IC), IH (frozen)	APC	17-5773
		Flow (IC)	APC Staining Set	77-5775
		Flow (IC), IH (frozen)	Alexa Fluor^® 488	53-5773
		Flow (IC)	Alexa Fluor^® 647	51-5773
		Flow (IC)	Alexa Fluor^® 700	56-5773
		Flow (IC)	eFluor® 450	57-5773
		Flow (IC)	Staining Buffer Set	00-5523
		Flow (IC)	Treg Staining Kit (w/ PE Foxp3 FJK-16s)	88-8111
		Flow (IC)	Treg Staining Kit #2 (w/ APC Foxp3 FJK-16s)	88-8118
		Flow (IC)	Treg Staining Kit #3 (w/ PE-Cy5 Foxp3 FJK-16s)	88-8115
Mouse Foxp3	NRRF-30	WB, IH (frozen)	Purified	14-4771
		Flow (IC)	PE	12-4771
		Flow (IC)	Staining Buffer Set	00-5523
Mouse/Human Rat Foxp3	150D/E4	Flow (IC), WB, IH	Purified	14-4774
		Flow (IC)	PE	12-4774
		Flow (IC)	Alexa Fluor^® 488	53-4774
		Flow (IC)	Alexa Fluor^® 647	51-4774