Product Information

Contents: Allophycocyanin (APC) Set anti-human Foxp3 Staining Set (clone 236A/E7)
Catalog Number: 77-5774
Formulation: Phosphate buffer pH 7.2,
150 mM NaCl, 0.09% NaN₃
Storage Conditions: Store at 4°C.
DO NOT FREEZE.
LIGHT-SENSITIVE MATERIAL.
Clone: 236A/E7 Set
Isotype: Mouse IgG1

Human PBMCs were surface-stained with FITC anti-CD4 (RPA-T4) (cat. 11-0049) and PE anti-CD25 (BC96) (cat. 12-0259) and subsequently with 20 μl/million cells APC anti-human Foxp3 (236A/E7) or APC Mouse IgG1 Iso Cntrl (cat. 17-4714) using the APC anti-human Foxp3 Staining Set. The density plot on the left demonstrates co-staining of CD4 and 236A/E7 while the right plot demonstrates co-staining of CD25 and 236A/E7. Quadrant lines demarcate the isotype control. Cells in the lymphocyte gate were used for analysis.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
72-5774	Phycoerythrin (PE) anti-human Foxp3 Staining Set (clone 236A/E7)	488	575	IC Flow
73-5774	Alexa Fluor® 488 anti-human Foxp3 Staining Set (236A/E7 clone, includes isotype control, no normal rat serum)	488	519	IC Flow
77-5774	Allophycocyanin (APC) anti-human Foxp3 Staining Set (clone 236A/E7)	633	660	IC Flow

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

Description

eBioscience offers a panel of monoclonal antibodies to different epitopes of human Foxp3, providing useful tools for investigating the complete expression pattern of Foxp3 at the protein level, and discerning the precise subsets of Foxp3+ cells. Other antibodies to human Foxp3 include the recently published PCH101 (cat. 72-5776) and ebio7979 (cat. 13-7979).

The 236A/E7 antibody reacts with human foxp3 protein also known as FORKHEAD BOX P3, SCURFIN, and JM2; cross reactivity of this antibody to other proteins has not been determined. Foxp3, a 49-55 kDa protein, is a member of the forkhead/winged-helix family of transcriptional regulators, and was identified as the gene defective in ‘scurfy’ (sf) mice. Constitutive high expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg cells), and ectopic expression of foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

Intracellular staining and flow cytometric analysis of freshly isolated human peripheral blood mononuclear cells (PBMCs) with the 236A/E7 antibody using the Foxp3 Staining Buffer Set (cat. 00-5523) and protocol reveals staining of the CD4+CD25^bright population.

The epitope from 236A/E7 is different from that of PCH101 (cat. 72-5776).

Not included:
Flow Cytometry Staining Buffer (cat. 00-4222)
Surface markers such as anti-CD4 (cat. 17-0049 or 11-0049) and APC anti-CD25 (cat. 17-0259)
[For 72-5774 and 77-5774] Mouse IgG1 isotype control (cat. 12-4714 or 17-4714)
[For 73-5774] Normal Mouse Serum (24-5544)

Applications Reported

For research use only, not for diagnostic or therapeutic use. This 236A/E7 Set antibody has been reported for use in intracellular staining followed by flow cytometric analysis.

Applications Tested

This 236A/E7 antibody has been pre-titrated and tested by intracellular staining and flow cytometry of human PBMC using Foxp3 staining buffers included in this set. This can be used at 20 µl (0.125µg) per million cells in a 100 µl total staining volume.

The set components include:
1. eBioscience Fixation/Permeabilization Concentrate (cat. 00-5123, 30 mls) Store at 4ºC. 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.
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.
2. eBioscience Fixation/Permeabilization Diluent (cat. 00-5223, 100 mls). Store at 4ºC. The diluent is intended to be used in combination with the Fixation/Permeabilization Concentrate.
3. eBioscience Permeabilization Buffer (10X) (cat. 00-8333, 100 mls) Store at 4ºC. 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.
4. Normal mouse serum (cat. 24-5544, 100 μl)
5. APC anti-human Foxp3 (clone 236A/E7, 25 tests). Store at 4°C in the dark.

Not included:
Flow Cytometry Staining Buffer Stain Buffer (Cat. 00-4222)
Surface markers such as anti-CD4 (cat. 12-0049 or 11-0049) and anti-CD25 (clone BC96) (cat. 12-0259)
Mouse IgG1 isotype control (cat. 12-4714)

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.

It is critical that this antibody be used in conjunction with the Foxp3 Staining Buffers (cat 00-5523) for flow cytometric analysis.

References

Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science. 2003 299(5609):1057-61.

Lim, H.W., P. Hillsamer, A.H. Banham, and C.H. Kim. Cutting Edge: Direct Suppression of B cells by CD4+CD25+ Regulatory T cells. J. Immunol. 2005 175: 4180-4183. (PCH101 FC, and 236A/E7 IHC PubMed)

Manavalan JS, Kim-Schulze S, Scotto L, Naiyer AJ, Vlad G, Colombo PC, Marboe C, Mancini D, Cortesini R, Suciu-Foca N. Alloantigen specific CD8+CD28- FOXP3+ T suppressor cells induce ILT3+ ILT4+ tolerogenic endothelial cells, inhibiting alloreactivity. Int Immunol. 2004 16(8):1055-68.

Roncador G, Brown PJ, Maestre L, Hue S, Martinez-Torrecuadrada JL, Ling KL, Pratap S, Toms C, Fox BC, Cerundolo V, Powrie F, Banham AH. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur J Immunol. 2005 35(6):1681-91.

Sakaguchi S. The origin of FOXP3-expressing CD4+ regulatory T cells: thymus or periphery. J Clin Invest. 2003 112(9):1310-2.

Takahata Y, Nomura A, Takada H, Ohga S, Furuno K, Hikino S, Nakayama H, Sakaguchi S, Hara T. 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. 2004 32(7):622-9.

Xystrakis E, Dejean AS, Bernard I, Druet P, Liblau R, Gonzalez-Dunia D, Saoudi A. Identification of a novel natural regulatory CD8 T-cell subset and analysis of its mechanism of regulation. Blood. 2004 104(10):3294-301.

Related Products

Cat. 11-0048    Fluorescein isothiocyanate (FITC) anti-human CD4 (clone OKT4 (OKT-4))
Cat. 11-0049    FITC anti-human CD4 (clone RPA-T4)
Cat. 12-0259    PE anti-human CD25 (Interleukin-2 Receptor, IL-2R, IL2R) (clone BC96)
Cat. 17-4714    Allophycocyanin (APC) Mouse IgG1, K Isotype Control
Cat. 17-4777    Allophycocyanin (APC) anti-human Foxp3 (clone 236A/E7)
Cat. 00-5521    eBioscience Foxp3 Fixation/Permeabilization Concentrate and Diluent
Cat. 00-5523    eBioscience Foxp3 Staining Buffer Set
Cat. 00-8333    eBioscience Permeabilization Buffer(10X)

Protocol for IC Staining

It is critical to use the Foxp3 Staining Buffer Set (cat. 00-5523). The buffer set is included with all Foxp3 Staining Sets.

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. The Permeabilization Buffer is supplied as a 10X concentrate. The 10X stock should be diluted in distilled water to a 1X solution prior to use. The 1X permeabilization buffer should be made fresh before each experiment.

Add 100 µl of prepared cells (1x10⁶) to each tube.
Stain surface molecules such as CD4, CD8, CD25, etc. following the Surface Staining Protocol (http://www.ebioscience.com/ebioscience/appls/FCS.htm).
Wash in cold Flow Cytometry Staining Buffer (or cold PBS).
Resuspend cell pellet with pulse vortex and add 1 ml of freshly prepared Fixation/Permeabilization working solution 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 (made from 10X Permeabilization Buffer) followed by centrifugation and decanting of supernatant.
Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
[OPTIONAL] Block with 2% (2 µl) normal rat (or mouse) serum in 1X Permeabilization Buffer, in approximately 100 µl volume, at 4°C for 15 minutes.
Without washing after blocking step, add 20 µl fluorochrome conjugated anti-human Foxp3 antibody or isotype control in 1X Permeabilization Buffer 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 1X 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.