Human/Non-Human Primate Regulatory T Cell Staining Kit #2

Also known as: Treg Kit; Foxp3 PCH101 APC; CD4, PE CD25 PE

RUO: For Research Use Only

human CD4/Foxp3 antibody, Cytokine Kits conjugate

Normal human peripheral blood cells were surface stained with Anti-Human CD4 FITC (cat. 11-0049) and Anti-Human CD25 PE (cat. 12-0259) then fixed and permeabilized using the Foxp3 Staining Buffers and subsequently stained with Rat IgG2a κ Isotype Control APC (cat. \lt{17-4321) or Anti-Human Foxp3 APC. Cells in the lymphocyte gate were used for analysis.

Contents: Human/Non-Human Primate Regulatory T Cell Staining Kit #2
Catalog Number: 88-4994
Concentration: 20 µl (0.5 µg)/test
Formulation: aqueous buffer, 0.09% sodium azide, contains stabilizer if necessary
Storage Conditions: Store at 2-8°C. Light sensitive material. Use within 6 months of receipt or by date indicated on the bottle.
Clone: PCH101
Host/Isotype: Rat IgG2a

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Research Related Products
Cat. No.	Format
00-5523	Foxp3 Staining Buffer Set
11-0048	Anti-Human CD4 FITC (OKT4 (OKT-4))
11-4714	Mouse IgG1 K Isotype Control FITC
12-0259	Anti-Human CD25 PE (BC96)
14-4714	Mouse IgG1 K Isotype Control Purified
17-4321	Rat IgG2a K Isotype Control APC
88-4999	Human/Non-Human Primate Regulatory T Cell Staining Kit #1 (PCH101)
88-8998	Human Regulatory T Cell Staining Kit #2 (PCH101)
88-8999	Human Regulatory T Cell Staining Kit (PCH101)

Available Formats
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications	Regulatory Status
88-4994	Human/Non-Human Primate Regulatory T Cell Staining Kit #2	N/A	N/A	IC Flow	RUO
Flow Cytometry Product Notes: Test Sizes: To accommodate multicolor flow cytometry, eBioscience is in the process of reducing test size volumes from 20 µl to 5 µl. Please check your antibody vial for the recommended test size. Fluorochrome Replacements: eBioscience is in the process of replacing all Alexa Fluor® 647 conjugated products with eFluor® 660 conjugated products.

Description

Regulatory T cells/Tregs (also known as suppressor T cells) are a specialized subpopulation of T cells that act to suppress immune responses thereby maintaining homeostasis and self tolerance. Intense investigation has tried to determine exclusive protein markers for Tregs. This has been complicated by the identification of four regulatory T cells subsets; natural, anergic, Tr1 and CD8+CD28- suppressor T cells. There are still no cell surface molecules that uniquely distinguish functional regulatory cells from conventional T cells. It has been proposed that the “natural” regulatory T cells originally recognized by their constitutive expression of CD4 and CD25 are further defined by expression of the transcription factor Foxp3. Additionally Foxp3 has also been characterized in CD8+CD28- suppressor T cells.

The Complete Regulatory T Cell Staining Kit contains everything needed to stain the natural Tregs including CD4, CD25 and Foxp3 from human, rhesus, chimpanzee or cynomolgus PBMCs. In baboon, CD25 and Foxp3 can be stained, while CD4 cannot be stained using this kit. Other primate species have not been tested. Please refer to Non-Human Primate Reagent page.

This kit has been optimized for staining normal hPBMCs. It is critical to use freshly isolated PBMCs to assure optimal Foxp3 staining.

Not included:
Isotype controls for anti-CD4 (mouse IgG1, cat. 11-4714) and anti-CD25 (mouse IgG1, cat. 12-4714)

Components

FITC anti-human CD4 (OKT4): 25 tests. Store at 4°C in the dark.
PE anti-human CD25 (BC96): 25 tests. Store at 4°C in the dark.
APC anti-human Foxp3 (PCH101): 25 tests. Store at 4°C in the dark.
APC Rat IgG2a isotype control: 25 tests. Store at 4°C in the dark.
Normal Rat Serum: 100 μl. Store at 4°C.
eBioscience Flow Cytometry Staining Buffer: 200 ml. Store at 4°C.
eBioscience Fixation/Permeabilization Concentrate: 30 ml. Store at 4°C. 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. Use within 6 months of receipt. 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.
eBioscience Fixation/Permeabilization Diluent: 100 ml. Store at 4°C. The diluent is intended to be used in combination with the Fixation/Permeabilization Concentrate.
eBioscience Permeabilization Buffer (10X): 100 ml. Store at 4°C. 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.

Applications Reported

For research use only, not for diagnostic or therapeutic use.

Applications Tested

This PCH101 antibody has been pre-titrated and tested intracellular staining by flow cytometric analysis. Please refer to the antibody vial for the recommended test volume to use for one 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; (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; (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; (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 naïve 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.

Protocol

To view protocol, please download the TDS by clicking the PDF button above.

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Unless indicated otherwise, all products are For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for further distribution without written consent. Not all products available in all regions.