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Contents: APC anti-human Foxp3 Staining Set
(preferentially stains CD4⁺CD25^hi)
Catalog Number: 77-5776
Formulation: Phosphate buffer pH 7.2,
150 mM NaCl, 0.09% NaN₃, 0.2% BSA
Storage Conditions: Store at 4°C.
DO NOT FREEZE.
LIGHT-SENSITIVE MATERIAL.
Clone: PCH101 Set
Isotype: Rat IgG2a, κ

Prices for This Product*
Cat. No.	Size	Price	Qty	Action
77-5776-40		$365

*International customers: Please contact your distributor for region specific pricing.

Human PBMCs were surface-stained with FITC anti-CD4 (RPA-T4) (cat. 11-0049) and PE anti-CD25 (BC96) (cat. 12-0259) and subsequently with 20 μl/million cells APC anti-human Foxp3 (clone PCH101) or APC Rat IgG2a Iso Cntrl (cat. 17-4321) using the APC anti-human Foxp3 Staining Set (cat. 77-5776). 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 demarcate 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
71-5776	FITC anti-human Foxp3 Staining Set (preferentially stains CD4+CD25hi)	488	518	IC Flow
72-5776	PE anti-human Foxp3 Staining Set (preferentially stains CD4+CD25hi)	488	575	IC Flow
77-5776	Allophycocyanin (APC) anti-human Foxp3 Staining Set (preferentially stains CD4+CD25hi)	633	660	IC Flow
73-5776	Alexa Fluor® 488 anti-human Foxp3 Staining Set (preferentially stains CD4+CD25hi)	488	519	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 Set antibody has been reported for use in intracellular staining followed by flow cytometric analysis.

Applications Tested

This PCH101 antibody has been pre-titrated and tested by intracellular staining and flow cytometry of human PBMC using Foxp3 staining buffers included in this set. This can be used at 20 μl (0.5μg) per million cells in a 100 μl total staining volume.

The set components include:
1. eBioscience Fixation/Permeabilization Concentrate (cat. 00-5123, 30 mls) Store at 4ºC. Avoid agitation.
This is a 4X stock solution that must be diluted prior to use with the Fixation/Permeabilization Diluent. Dilute 1 part Fixation/Permeabilization Concentrate with 3 parts Fixation/Permeabilization Diluent.
Caution: This solution contains Paraformaldehyde, which is toxic and a suspected carcinogen. Contact with eyes, skin and mucous membranes should be avoided. Wear proper protective clothing and gloves.
2. eBioscience Fixation/Permeabilization Diluent (cat. 00-5223, 100 mls). Store at 4ºC. The diluent is intended to be used in combination with the Fixation/Permeabilization Concentrate.
3. eBioscience Permeabilization Buffer (10X) (cat. 00-8333, 100 mls) Store at 4ºC. Dilute to 1x with deionized/distilled water and store at 4°C.
Caution: Harmful if swallowed or irritant by contact. Wear proper protective clothing and gloves.
Note: The 10x Permeabilization Buffer has a natural tendency to precipitate, however, its function is not affected by this. To clarify, the solution can be filtered after dilution to 1x working solution.
4. Normal Rat serum (cat. 24-5555, 100uL). Store at 4°C.
5. APC anti-human Foxp3 (cat 17-4776, PCH101, 25 tests) Store at 4°C

Not included:
Flow Cytometry Staining Buffer Stain Buffer (Cat. 00-4222)
Surface markers such as anti-CD4 (cat. 12-0049 or 11-0049) and PE anti-CD25 (clone BC96) (cat.12-0259)
Isotype control (Rat IgG2a)

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)

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

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

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.

Xystrakis E, Dejean AS, Bernard I, Druet P, Liblau R, Gonzalez-Dunia D, Saoudi A. 2004. Identification of a novel natural regulatory CD8 T-cell subset and analysis of its mechanism of regulation. Blood. 104(10):3294-301.

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.

Related Products

Cat. 11-0048    Fluorescein isothiocyanate (FITC) anti-human CD4 (clone OKT4 (OKT-4))
Cat. 11-0049    FITC anti-human CD4 (clone RPA-T4)
Cat. 12-0259    PE anti-human CD25 (Interleukin-2 Receptor, IL-2R, IL2R) (clone BC96)
Cat. 17-4321    APC Rat IgG2a Isotype Control
Cat. 17-4776    APC anti-human Foxp3 (clone PCH101)
Cat. 00-5521    eBioscience Foxp3 Fixation/Permeabilization Concentrate and Diluent
Cat. 00-5523    eBioscience Foxp3 Staining Buffer Set
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.