Product Information

Contents: Pacific Blue® anti-human Foxp3
Catalog Number: 57-4776
Sizes: 25 tests
Formulation: Phosphate buffer pH 7.2,
150 mM NaCl, 0.09% NaN₃, gelatin and 0.2% BSA for stabilization
Storage Conditions: Store at 4°C.
DO NOT FREEZE.
LIGHT-SENSITIVE MATERIAL.
Clone: PCH101
Isotype: Rat IgG2a, κ

Human PBMCs were surface-stained with PE anti-CD4 (RPA-T4) (cat. 12-0049) and APC anti-CD25 (BC96) (cat. 17-0259) and subsequently with Pacific Blue^® anti-human Foxp3 (PCH101). The density plot on the left demonstrates co-staining of CD4 and PCH101 while the right plot demonstrates co-staining of CD25 and PCH101. Quadrant lines were drawn according to staining of the isotype control. Cells in the lymphocyte gate were used for analysis.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
11-4776	FITC anti-human Foxp3	488	518	IC Flow IH/F IHC(Paraffin)
12-4776	PE anti-human Foxp3	488	575	IC Flow
13-4776	Biotin anti-human Foxp3	N/A	N/A	IC Flow IH/F IHC(Paraffin) WB
14-4776	Affinity Purified anti-human Foxp3	N/A	N/A	IH/F IHC(Paraffin) WB
15-4776	Phycoerythrin-Cy5 (PE-Cy5) anti-human Foxp3	488	670	IC Flow
17-4776	APC anti-human Foxp3	633	660	IC Flow
45-4776	PerCP-Cy5.5 anti-human Foxp3	488	690	IC Flow
51-4776	Alexa Fluor® 647 anti-human Foxp3	633	668	IC Flow
53-4776	Alexa Fluor® 488 anti-human Foxp3	488	519	IC Flow
56-4776	Alexa Fluor® 700 anti-human Foxp3 (APC-Cy5.5 Equivalent)	633	723	IC Flow
57-4776	Pacific Blue® anti-human Foxp3	402	421	IC Flow

Questions? Please consult our answers to frequently asked questions at http://www.ebioscience.com/faq.

Description

eBioscience offers a panel of monoclonal antibodies to different epitopes of human Foxp3, providing useful tools for investigating the complete expression pattern of Foxp3 at the protein level, and discerning the precise subsets of Foxp3⁺ cells. Please contact tech@ebioscience.com or 888.810.6168 for any additional assistance.

The PCH101 antibody reacts with the amino terminus of human foxp3 protein also known as FORKHEAD BOX P3, SCURFIN, and JM2; cross reactivity of this antibody to other proteins has not been determined. Foxp3, a 49-55 kDa protein, is a member of the forkhead/winged-helix family of transcriptional regulators, and was identified as the gene defective in ‘scurfy’ (sf) mice. Constitutive high expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg cells), and ectopic expression of foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

Intracellular staining of human peripheral blood mononuclear cells (PBMCs) with PCH101 antibody using the anti-human Foxp3 Staining Set and protocol reveals approximately 0.5-4% of lymphocytes staining, with the majority of staining occurring in the CD25^bright population. This is subject to donor variability.

PCH101 crossreacts with rhesus, chimpanzee and cynomolgus. We recommend the use of CD4 (OKT4, cat. 11-0048, or RPA-T4, cat. 11-0049, depending on the species) and CD25 (BC96, cat. 17-0259). Please refer to our crossreactivity table for more information.

Applications Reported

For research use only, not for diagnostic or therapeutic use. This PCH101 antibody has been reported for use in intracellular staining followed by flow cytometric analysis.

The Pacific Blue^® emits at 456 nm and can be excited with the Violet laser. Please make sure that your instrument is capable of detecting this fluorochome.

Applications Tested

This PCH101 antibody has been pre-titrated and tested by intracellular staining and flow cytometric analysis of human peripheral blood mononuclear cells. This can be used at 20 μl (1.0 μg) per million cells in a 100 μl total staining volume.

Special Notes

Please see the following link for FAQ regarding the usage of eBioscience Foxp3 reagents:
http://www.ebioscience.com/ebioscience/Foxp3FAQs.htm

The staining protocol has been optimized with Ficoll prepped PBMCs. The use of lysed whole blood is not recommended.

It is critical that this antibody be used in conjunction with the Foxp3 Staining Buffers (cat 00-5523) for flow cytometric analysis.

References

Ahmadzadeh M, Rosenberg SA. IL-2 Administration Increases CD4+CD25hiFoxp3+ Regulatory T Cells in Cancer Patients. Blood. 2005 Nov 22; [Epub ahead of print] (PCH101, intracellular flow, PubMed)

Crellin NK, Garcia RV, Hadisfar O, Allan SE, Steiner TS, Levings MK. 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. 2005 Dec 15;175(12):8051-9. (PCH101, intracellular flow, PubMed)

Hartwig UF, Nonn M, Khan S, Meyer RG, Huber C, Herr W. Depletion of alloreactive T cells via CD69: implications on antiviral, antileukemic and immunoregulatory T lymphocytes. Bone Marrow Transplant. 2005 Dec 5; [Epub ahead of print] (Intracellular staining for flow cytometry using PCH101, PubMed)

Lim, H.W., P. Hillsamer, A.H. Banham, and C.H. Kim. 2005. Cutting Edge: Direct Suppression of B cells by CD4+CD25+ Regulatory T cells. J. Immunol. 175: 4180-4183. (PCH101, intracellular flow, PubMed)

Manigold T, Shin EC, Mizukoshi E, Mihalik K, Murthy KK, Rice CM, Piccirillo CA, Rehermann B. Foxp3+CD4+CD25+ T cells control virus-specific memory T cells in chimpanzees recovered from Hepatitis C. Blood. 2006 Feb 14; [Epub ahead of print] (PCH101 in chimpanzee, PubMed)

Hori S, Nomura T, Sakaguchi S. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science. 299(5609):1057-61.

Manavalan JS, Kim-Schulze S, Scotto L, Naiyer AJ, Vlad G, Colombo PC, Marboe C, Mancini D, Cortesini R, Suciu-Foca N.2004. Alloantigen specific CD8+CD28- FOXP3+ T suppressor cells induce ILT3+ ILT4+ tolerogenic endothelial cells, inhibiting alloreactivity. Int Immunol. 16(8):1055-68.

Takahata Y, Nomura A, Takada H, Ohga S, Furuno K, Hikino S, Nakayama H, Sakaguchi S, Hara T. 2004. CD25+CD4+ T cells in human cord blood: an immunoregulatory subset with naive phenotype and specific expression of forkhead box p3 (Foxp3) gene. Exp Hematol. 32(7):622-9.

Sakaguchi S. 2003. The origin of FOXP3-expressing CD4+ regulatory T cells: thymus or periphery. J Clin Invest. 112(9):1310-2.

Clark, Rachael, Kupper, Thomas S. (2007) “IL-15 and dermal fibroblasts induce proliferation of natural regulatory cells isolated from human skin” Blood 109:194-202

Epple, Hans-Jorg et al (2006) “Mucosal but not peripheral FOXP3 regulatory T cells are highly increased in untreated HIV infection and normalize after suppressive HAART” Blood 108: 3072-3078

Related Products

Cat. 57-4321    Pacific Blue® Rat IgG2a Isotype Control
Cat. 14-4774    Affinity Purified anti-mouse/human/rat Foxp3 (clone 150D/E4)
Cat. 14-4777    Affinity Purified anti-human Foxp3 (clone 236A/E7)
Cat. 00-5523    eBioscience Foxp3 Staining Buffer Set
Cat. 14-7979    Affinity Purified anti-mouse/human Foxp3 (clone eBio7979)
Cat. 00-8333    eBioscience Permeabilization Buffer(10X)

Protocol for IC Staining

It is critical to use the Foxp3 Staining Buffer Set (cat. 00-5523). The buffer set is included with all Foxp3 Staining Sets.

Prior to staining, dilute the Fixation/Permeabilization Concentrate (1 part) into the Fixation/Permeabilization Diluent (3 parts) to the desired volume of Fixation/Permeabilization working solution. This buffer should not be stored for more than 1 day. For example: For 12 samples, use 3 ml Fixation/Permeabilization Concentrate and 9 ml Fixation/Permeabilization Diluent. The Permeabilization Buffer is supplied as a 10X concentrate. The 10X stock should be diluted in distilled water to a 1X solution prior to use. The 1X permeabilization buffer should be made fresh before each experiment.

Add 100 µl of prepared cells (1x10⁶) to each tube.
Stain surface molecules such as CD4, CD8, CD25, etc. following the Surface Staining Protocol (http://www.ebioscience.com/ebioscience/appls/FCS.htm).
Wash in cold Flow Cytometry Staining Buffer (or cold PBS).
Resuspend cell pellet with pulse vortex and add 1 ml of freshly prepared Fixation/Permeabilization working solution to each sample. Pulse vortex again.
Incubate at 4°C for 30 - 60 minutes in the dark.
Wash once by adding 2 ml 1X Permeabilization Buffer (made from 10X Permeabilization Buffer) followed by centrifugation and decanting of supernatant.
Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
[OPTIONAL] Block with 2% (2 µl) normal rat (or mouse) serum in 1X Permeabilization Buffer, in approximately 100 µl volume, at 4°C for 15 minutes.
Without washing after blocking step, add 20 µl fluorochrome conjugated anti-human Foxp3 antibody or isotype control in 1X Permeabilization Buffer and incubate at 4°C for at least 30 minutes in the dark. Please perform further titration for optimal staining in your own assay system.
Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
Repeat step 10.
Resuspend in appropriate volume Flow Cytometry Staining Buffer and analyze on cytometer. Please note that due to the fixation and permeabilization procedure, the FSC/SSC distribution of the cell population will be different than live cells. Therefore the gate and voltages will need to be modified.

Alexa Fluor® and Pacific Blue® are registered trademarks of and licensed under patents assigned to Molecular Probes, Inc. for research use only. This product is subject to an agreement between Molecular Probes, Inc. and eBioscience, and the manufacture, use, sale or import of this product may be subject to one or more U.S. patents, pending applications and corresponding foreign equivalents, owned by Molecular Probes, Inc. (a wholly owned subsidiary of Invitrogen Corp). The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product for life science research or as an ASR. The buyer cannot use this product for manufacturing or for any other screening (specifically including use in combination with microarrays or High Content Screening) or testing purpose, other than as an ASR. For information on purchasing a license to this product for purposes other than life science research or use as an ASR, contact Molecular Probes, Inc.