Product Information

Contents: Human/ Non-Human Primate (NHP) Regulatory T cell Staining Kit (w/ PE Foxp3 PCH101, FITC CD4, APC CD25; Treg Kit)
Catalog Number: 88-4999
Formulation: Phosphate buffer pH 7.2,
150 mM NaCl, 0.09% NaN₃, 0.2% BSA
Storage Conditions: Store at 4°C.
Clone: PCH101
Isotype: Rat IgG2a

Human PBMCs were surface-stained with cocktail of CD4-FITC (OKT-4 11-0048) and CD25-APC (BC96, cat. 17-0259) and subsequently with 20 μl/million cells PE anti-human Foxp3 (clone PCH101) using the Human/NHP Regulatory T Cell Staining Kit. The dot plot on the left demonstrates co-staining of CD4-FITC and PCH101-PE, while the right demonstrates co-staining of CD25-APC and PCH101-PE. Cells in the lymphocyte gate were used for analysis.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
88-4999	Human/ Non-Human Primate (NHP) Regulatory T cell Staining Kit (w/ PE Foxp3 PCH101, FITC CD4, APC CD25; Treg Kit)	N/A	N/A	IC Flow

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

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. 17-4714)

Components:

FITC anti-human CD4 (OKT4): 25 tests. Store at 4°C in the dark.
APC anti-human CD25 (BC96): 25 tests. Store at 4°C in the dark.
PE anti-human Foxp3 (PCH101): 25 tests. Store at 4°C in the dark.
PE 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 4 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. This PCH101 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 intracellular staining followed by flow cytometry. This can be used at 20 μl (0.25 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.

Related Products

Cat. 17-0048    Allophycocyanin (APC) anti-human CD4 (clone OKT4 (OKT-4))
Cat. 11-0259    FITC anti-human CD25 (Interleukin-2 Receptor, IL-2R, IL2R) (clone BC96)
Cat. 11-4714    Fluorescein isothiocyanate (FITC) Mouse IgG1, K Isotype Control
Cat. 17-4714    Allophycocyanin (APC) Mouse IgG1, K Isotype Control
Cat. 88-4994    Human/Non-Human Primate (NHP) Regulatory T cell Staining Kit #2 (w/ APC Foxp3 PCH101, FITC CD4, PE CD25; Treg Kit) (clone PCH101)
Cat. 00-5523    eBioscience Foxp3 Staining Buffer Set
Cat. 88-8998    Human Regulatory T cell Staining Kit #2 (w/ APC Foxp3 PCH101, FITC CD4, PE CD25; Treg Kit) (clone PCH101)
Cat. 88-8999    Human Regulatory T cell Staining Kit (w/ PE Foxp3 PCH101, FITC CD4, APC CD25; Treg Kit) (clone PCH101)

Protocol for IC Staining

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.

Add 100 µl of prepared cells (1x10⁶) to each tube.
Add 20 µl of CD4 and 20 µl of CD25 antibodies. Incubate for 30 minutes in the dark at 4°C.
Wash in cold Flow Cytometry Staining Buffer (or cold PBS).
After decanting, resuspend cell pellet in the residual buffer and add 1 ml of freshly prepared eBioscience Fixation/Permeabilization Buffer 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 followed by centrifugation and decanting of supernatant.
Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
[OPTIONAL] Block by adding 2 µl (2% final) normal rat serum, in approximately 100 µl volume, for 15 minutes.
Without washing after blocking step, add 20 µl anti-human Foxp3 (PCH101) antibody or 20 µl rat IgG2a isotype control 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 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.