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Human Regulatory T Cell Staining Kit #2

Also known as: Forkhead Box P3, Scurfin, JM2, Treg, Foxp3 PCH101 APC; CD4 FITC; CD25 PE

RUO: For Research Use Only

human CD4/Foxp3 antibody, Cytokine Kits conjugate

Normal human peripheral blood cells were surface stained with a cocktail of Anti-Human CD4 FITC and CD25 PE (cat. 22-8425) then fixed and permeabilized using the Foxp3 Staining Buffers and subsequently stained with Anti-Human Foxp3 APC or Rat IgG2a κ Isotype Control APC (cat. 17-4321). Cells in the lymphocyte gate were used for analysis.

Contents: Human Regulatory T Cell Staining Kit #2
Catalog Number: 88-8998
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

Product Options
Cat. No.	Size	Price	Add Qty to Cart
88-8998-40		To view relevant pricing, please select your country and resume browsing.

Note: Several countries will continue to be supplied via distributors. Country specific prices may apply.

Research Related Products
Cat. No.	Format
00-5521	Foxp3 Fixation/Permeabilization Concentrate and Diluent
00-5523	Foxp3 Staining Buffer Set
11-0049	Anti-Human CD4 FITC (RPA-T4)
11-4714	Mouse IgG1 K Isotype Control FITC
12-0259	Anti-Human CD25 PE (BC96)
12-4714	Mouse IgG1 K Isotype Control PE
17-4321	Rat IgG2a K Isotype Control APC
17-4776	Anti-Human Foxp3 APC (PCH101)
22-8425	eZFluor® Anti-Human CD4 FITC and CD25 PE Cocktail (RPA-T4, BC96)
88-8999	Human Regulatory T Cell Staining Kit (PCH101)

Available Formats
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications	Regulatory Status
88-8998	Human 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 Regulatory T Cell Staining Kit contains all the necessary reagents to stain natural Tregs including monoclonal antibodies against CD4, CD25 and Foxp3.

This kit has been optimized for staining normal hPBMCs.

Not included:
Isotype control for anti-CD4 and anti-CD25 (cocktail of FITC & PE mouse IgG1, cat. 22-2714)

Components

Cocktail of FITC anti-human CD4 (RPA-T4) and PE anti-human CD25 (BC96): 25 tests. Store at 2-8°C in the dark.
APC anti-human Foxp3 (PCH101): 25 tests. Store at 2-8°C in the dark.
APC Rat IgG2a isotype control: 25 tests. Store at 2-8°C in the dark.
Normal Rat Serum: 100 μl. Store at 2-8°C.
eBioscience Flow Cytometry Staining Buffer: 200 ml. Store at 2-8°C.
eBioscience Fixation/Permeabilization Concentrate: 30 ml. Store at 2-8°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 2-8°C. The diluent is intended to be used in combination with the Fixation/Permeabilization Concentrate.
eBioscience Permeabilization Buffer (10X): 100 ml. Store at 2-8°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 Tested

This Human Regulatory T cell Staining kit is tested by intracellular staining and flow cytometric analysis using included Foxp3 Buffer Set and protocol. Please click here for Staining Protocol (refer to Protocol B: One-step protocol for intracellular (nuclear) proteins). This PCH101 antibody has been pre-titrated and tested by intracellular staining and flow cytometric analysis of normal human peripheral blood mononuclear cells. 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 freshly Ficoll prepped PBMCs. The use of lysed whole blood is not recommended.

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] (PCH101, intracellular flow, 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)

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