Contents: Pacific Blue® anti-human Foxp3
Catalog Number: 57-4777
Formulation: Phosphate buffered saline, pH 7.2, less than or equal to 0.09% NaN₃, BSA and/or gelatin for protein stabilization
Storage Conditions: Store at 4°C.
DO NOT FREEZE.
LIGHT-SENSITIVE MATERIAL.
Clone: 236A/E7
Isotype: Mouse IgG1

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Human PBMCs were surface-stained with FITC anti-CD4 (clone RPA-T4) (cat. 11-0049) and PE anti-CD25 (clone BC96) (cat. 12-0259) and subsequently with 20 µl/million cells Pacific Blue anti-human Foxp3 (clone 236A/e7) using the Foxp3 Buffer Set (cat. 00-5523). The density plot on the left demonstrates co-staining of CD25 and 236A/E7 while the right plot demonstrates co-staining of CD4 and 236A/E7. Cells in the lymphocyte gate were used for analysis.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
14-4777	Affinity Purified anti-human Foxp3	N/A	N/A	IC Flow IH/F IHC(Paraffin) WB
13-4777	Biotin anti-human Foxp3	N/A	N/A	IC Flow IH/F IHC(Paraffin)
11-4777	Fluorescein isothiocyanate (FITC) anti-human Foxp3	488	518	IC Flow
12-4777	Phycoerythrin (PE) anti-human Foxp3	488	575	IC Flow
17-4777	Allophycocyanin (APC) anti-human Foxp3	633	660	IC Flow
51-4777	Alexa Fluor® 647 anti-human Foxp3	633	668	IC Flow
53-4777	Alexa Fluor® 488 anti-human Foxp3	488	519	IC Flow
48-4777	eFluor^TM 450 anti-human Foxp3 (Pacific Blue® Replacement)	405	450	FC
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 Pacific Blue® and APC-Alexa Fluor® 750 conjugated products with eFluor™ 450 and APC-eFluor™ 780 conjugated products, respectively.

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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. 14-7979).

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).

Applications Reported

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

The Pacific Blue® emits at 456 nm and can be excited with the Violet laser. Please make sure that your instrument is capable of detecting this fluorochome.

Applications Tested

This 236A/E7 antibody has been pre-titrated and tested by intracellular staining and flow cytometric analysis of normal human peripheral blood mononuclear cells. This can be used at 20 μl (0.5 μg) per million cells in a 100 μl total staining volume.

References

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

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

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.

Alvaro T, Lejeune M, Salvado MT, Bosch R, Garcia JF, Jaen J, Banham AH, Roncador G, Montalban C, Piris MA. 2005. Outcome in Hodgkin's lymphoma can be predicted from the presence of accompanying cytotoxic and regulatory T cells. Clin Cancer Res. 11(4):1467-73. [IHC paraffin using 236A/E7, 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. (236A/E7, IH(frozen), PCH101, IC (flow), PubMed)

Roncador, G., P.J. Brown, L. Maestre, S. Hue, J.L. Martinez-Torrecuadrada, K.L. Ling, S. Pratap, C. Toms, B.C. Fox, V. Cerundolo, F. Powrie, and A.H. Banham. 2005. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur J Immunol. 35: 1681-91. [236A/E7, IC(flow), IH(paraffin), PubMed]

Roncador G, Garcia JF, Garcia JF, Maestre L, Lucas E, Menarguez J, Ohshima K, Nakamura S, Banham AH, Piris MA. 2005. FOXP3, a selective marker for a subset of adult T-cell leukaemia/lymphoma. Leukemia [Epub ahead of print] [IHC paraffin sections using 236A/E7, PubMed]

Wolf D, Wolf AM, Rumpold H, Fiegl H, Zeimet AG, Muller-Holzner E, Deibl M, Gastl G, Gunsilius E, Marth C.The expression of the regulatory T cell-specific forkhead box transcription factor FoxP3 is associated with poor prognosis in ovarian cancer. Clin Cancer Res. 2005 Dec 1;11(23):8326-31. [IHC paraffin using 236A/E7, PubMed]

Related Products

Cat. 57-4714  Pacific Blue® Mouse IgG1, K Isotype Control
Cat. 57-4776  Pacific Blue® anti-human Foxp3 (clone PCH101)
Cat. 57-5773  Pacific Blue® anti-mouse/rat Foxp3 (clone FJK-16s)

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 recommended volume (indicated on antibody vial) 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.

Alexa Fluor® and Pacific Blue® are registered trademarks of and licensed under patents assigned to Molecular Probes, Inc. for research use only. This product is subject to an agreement between Molecular Probes, Inc. and eBioscience, and the manufacture, use, sale or import of this product may be subject to one or more U.S. patents, pending applications and corresponding foreign equivalents, owned by Molecular Probes, Inc. (a wholly owned subsidiary of Invitrogen Corp). The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product for life science research or as an ASR. The buyer cannot use this product for manufacturing or for any other screening (specifically including use in combination with microarrays or High Content Screening) or testing purpose, other than as an ASR. For information on purchasing a license to this product for purposes other than life science research or use as an ASR, contact Molecular Probes, Inc.