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Product Information

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Contents: Human Regulatory T cell Staining Kit #3 (w/ PE-Cy5 Foxp3 PCH101, FITC CD4, PE CD25; Treg Kit) Catalog Number: 88-8995 Storage Conditions: Store at 4°C. Clone: PCH101 Isotype: Rat IgG2a

Staining of normal human peripheral blood mononuclear cells with FITC anti-human CD4 (RPA-T4) (cat. 11-0049) and PE-Cy5 Rat IgG2a Iso Cntrl (cat. 15-4321) (left) or PE-Cy5 anti-human Foxp3 (PCH101) (right). Cells in the lymphocyte gate were used for analysis.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
88-8995	Human Regulatory T cell Staining Kit #3 (w/ PE-Cy5 Foxp3 PCH101, FITC CD4, PE CD25; Treg Kit)	N/A	N/A	IC Flow

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

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Description

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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.

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:

1. Cocktail of FITC anti-human CD4 (RPA-T4) and PE anti-human CD25 (BC96): 25 tests. Store at 4°C in the dark.
2. PE-Cy5 anti-human Foxp3 (PCH101): 25 tests. Store at 4°C in the dark.
3. PE-Cy5 Rat IgG2a isotype control: 25 μg. Store at 4°C in the dark.
4. Normal Rat Serum: 100 μl
5. eBioscience Flow Cytometry Staining Buffer: 200 ml
6. 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.
7. eBioscience Fixation/Permeabilization Diluent: 100 ml. Store at 4°C. The diluent is intended to be used in combination with the Fixation/Permeabilization Concentrate.
8. 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.

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Applications Reported

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

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Applications Tested

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This PCH101 antibody has been pre-titrated and tested intracellular staining and flow cytometric analysis. This can be used at 20 μl (0.5 μg) per million cells in a 100 μl total staining volume.

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References

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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)

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)

Seddiki N, Santner-Nanan B, Martinson J, Zaunders J, Sasson S, Landay A, Solomon M, Selby W, Alexander SI, Nanan R, Kelleher A, Fazekas de St Groth B. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med. 2006 Jul 10;203(7):1693-700

Valencia X, Stephens G, Goldbach-Mansky R, Wilson M, Shevach EM, Lipsky PE.TNF down-modulates the function of human CD4+CD25hi T regulatory cells. Blood. 2006 Mar 14 [Intracellular staining for flow cytometry using PCH101]

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Related Products

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Cat. 15-4321    PE-Cy5 Rat IgG2a Isotype Control
Cat. 88-4999    Human/ Non-Human Primate (NHP) Regulatory T cell Staining Kit (w/ PE Foxp3 PCH101, FITC CD4, APC CD25; Treg Kit) (clone PCH101)
Cat. 00-5523    eBioscience Foxp3 Staining Buffer Set
Cat. 88-8111    Mouse Regulatory T cell Staining Kit (w/ PE Foxp3 FJK-16s, FITC CD4, APC CD25; Treg Kit) (clone FJK-16s)
Cat. 88-8115    Mouse Regulatory T cell Staining Kit #3 (w/ PE-Cy5 Foxp3 FJK-16s, FITC CD4, PE CD25; Treg Kit) (clone FJK-16s)
Cat. 88-8118    Mouse Regulatory T cell Staining Kit #2 (w/ APC Foxp3 FJK-16s, FITC CD4, PE CD25; Treg Kit) (clone FJK-16s)
Cat. 88-8998    Human Regulatory T cell Staining Kit #2 (w/ APC Foxp3 PCH101, FITC CD4, PE CD25; Treg Kit) (clone PCH101)

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Protocol for IC Staining

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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.

1. Add 100 µl of prepared cells (1x10⁶) to each tube.
2. Add 20 µl of CD4/25 cocktail (1 µg CD4 and 0.125 µg CD25). Incubate for 30 minutes in the dark at 4°C.
3. Wash in cold Flow Cytometry Staining Buffer (or cold PBS).
4. 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.
5. Incubate at 4°C for 30 - 60 minutes in the dark.
6. Wash once by adding 2 ml 1X Permeabilization Buffer followed by centrifugation and decanting of supernatant.
7. Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
8. [OPTIONAL] Block by adding 2 µl (2% final) normal rat serum, in approximately 100 µl volume, for 15 minutes.
9. Without washing after blocking step, add 20 μL anti-human Foxp3 (PCH101) antibody or 2.5μL 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.
10. Wash cells with 2 ml Permeabilization Buffer. Centrifuge and decant supernatant.
11. Repeat step 10.
12. 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.