Human Regulatory T Cell Whole Blood Staining Kit

Also known as: Forkhead Box P3, Scurfin, JM2, Treg, Foxp3 PCH101 PE-Cy5; CD4 FITC; CD25 PE

RUO: For Research Use Only

Human Regulatory T Cell Whole Blood Staining Kit

Staining of whole blood with Anti-Human CD4 FITC (cat. 11-0048) (left) or Anti-Human CD25 APC (cat. 17-0259) (right) followed by RBC lysis and intracellular staining using the Whole Blood Foxp3 Staining kit. Cells in the lymphocyte gate were used for analysis.

Contents: Human Regulatory T Cell Whole Blood Staining Kit
Catalog Number: 88-8996
Storage Conditions: Store at 2-8°C. Light sensitive material. Use within 6 months of receipt or by date indicated on the bottle.
Host/Isotype: Rat IgG2a

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Research Related Products
Cat. No.	Format
12-4321	Rat IgG2a K Isotype Control PE

Available Formats
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications	Regulatory Status
88-8996	Human Regulatory T Cell Whole Blood Staining Kit	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.

This kit (25 tests) has been optimized for staining normal human blood cells from lysed whole blood.

Not included:
Antibody or Isotype control for CD4 and CD25 (cocktails cat. 22-0425 and cat. 22-1714)

Components

eBioscience 1X RBC Lysis Buffer: 200 ml
eBioscience Flow Cytometry Staining Buffer: 600 ml
eBioscience Fixation/Permeabilization Concentrate: 30 ml
Foxp3 LWB Diluent: 100 ml
PE anti-human Foxp3 (PCH101): 25 tests

Applications Reported

For research use only, not for diagnostic or therapeutic use. Foxp3 Whole Blood Staining Kit has been reported for use in intracellular staining followed by flow cytometric analysis.

Applications Tested

Foxp3 Whole Blood Staining Kit has been pre-titrated and tested by flow cytometric analysis of human blood cells following protocol below. Please refer to the antibody vial for the recommended test volume to use per test in a 100 μl total staining volume.

Foxp3 Staining Protocol for Human Lysed Whole Blood Cells:

The following protocol is for intracellular staining of Foxp3 in human lysed whole blood using Phycoerythrin (PE) conjugated Foxp3 antibody (clone PCH101).

Prior to staining, dilute the Fixation/Permeabilization Concentrate (1 part) into the Foxp3 LWB Fixation/Permeabilization Diluent (3 parts) to the desired volume of working solution. This Foxp3 LWB Fixation/Permeabilization working solution should not be stored for more than 1 day. For example: For 12 samples, use 3 ml Fixation/Permeabilization Concentrate and 9 ml Foxp3 LWB Fixation/Permeabilization Diluent.

1. Add 100 µl of human blood to each 12x75mm tube.
2. Stain surface molecules such as CD4, CD8, CD25, etc. following the Surface Staining Protocol.
3. After incubation with antibodies DO NOT WASH THE CELLS, instead add 2-3 ml of room temperature 1X RBC Lysis Buffer (cat. 00-4333) to each tube. Mix well.
4. Incubate for 10 minutes at room temperature.
5. Centrifuge at 300-500 x g for 5 minutes and decant supernatant.
6. Wash by adding 2-3 ml FSB (Flow Staining Buffer Cat. 00-4222).
7. Centrifuge and decant. Repeat.
8. Resuspend cell pellet with pulse vortex and add 1 ml of freshly prepared Foxp3 LWB Fixation/Permeabilization working solution to each sample. Pulse vortex again.
9. Incubate at 4°C for 30 - 60 minutes in the dark.
10. Wash with 2-3 ml FSB, followed by centrifugation and decanting. Repeat.
11. Add one test (volume indicated on antibody vial) of PE PCH101 (anti-human Foxp3) antibody or 0.25μg PE rat IgG2a isotype control in a final volume of approximately 100 μl. Incubate at 4°C for at 30-60 minutes in the dark.
12. Wash cells with 2 ml FSB. Centrifuge and decant .
13. Resuspend in appropriate volume FSB and analyze on a 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.

It is critical to use the optimized Foxp3 LWB Staining Buffer Set. Although the current Foxp3 buffers (cat. 00-5523) can be used, we find results are brighter and more consistent using the adapted Foxp3 LWB Staining Buffers included in this kit.

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)

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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