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Contents: Alexa Fluor® 647 anti-mouse/human/rat Foxp3
Catalog Number: 51-4774
Formulation: Phosphate buffer pH 7.2,
150 mM NaCl, 0.09% NaN₃, 0.1% gelatin
Storage Conditions: Store at 4°C.
DO NOT FREEZE.
LIGHT-SENSITIVE MATERIAL.
Clone: 150D/E4
Isotype: Mouse IgG1

Prices for This Product*
Cat. No.	Size	Price	Qty	Action
51-4774-71	25 tests	$160
51-4774-73	100 tests	$315

*International customers: Please contact your distributor for region specific pricing.

Left: Mouse (BALB/c) splenocytes were surface-stained with FITC anti-mouse CD4 (RM4-5) (cat. 11-0042) and subsequently with Alexa647 anti-Foxp3 (eBio150D/E4)
Right: 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 AlexaFluor647 anti-human Foxp3 (cloneebio150D/E4) using the Foxp3 Staining Buffers (cat. 00-5523).

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
12-4774	Phycoerythrin (PE) anti-mouse/human/rat Foxp3	488	575	IC Flow
14-4774	Affinity Purified anti-mouse/human/rat Foxp3	N/A	N/A	IHC IP WB
51-4774	Alexa Fluor® 647 anti-mouse/human/rat Foxp3	633	668	IC Flow
53-4774	Alexa Fluor® 488 anti-mouse/human/rat Foxp3	488	519	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/primate and mouse/rat Foxp3 protein, 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 Foxp3 available from eBioscience, which have been used significantly in scientific literature, include the anti-human Foxp3 PCH101 (cat. 72-5776) and ebio7979 (cat. 12-7979), and the anti-mouse/rat Foxp3 FJK-16s (cat. 72-5775). The 150D/E4 has been mapped to the splice region in Exon 2 found in human cells. The splicing of this region has not been shown to occur in mouse. Characterization of this antibody to the splice variant has been limited to epitope mapping. It should be noted that other antibodies such as PCH101 and 236A/E7 will recognize both the spliced and full length forms of the Foxp3 protein and stain more intensely than 150D/E4 and FJK-16s in human cells.

The 150D/E4 antibody reacts with mouse/rat/human Foxp3 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.

Applications Reported

For research use only, not for diagnostic or therapeutic use. The 150D/E4 antibody has been reported for use in intracellular immunofluorescent staining and flow cytometric analysis.

Applications Tested

This 150D/E4 antibody has been tested by intracellular immunofluorescent staining followed by flow cytometric analysis. This can be used at less than or equal to 0.25 µg per million cells in a 100 µl total staining volume. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.

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 or mouse splenocytes. 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

Fontenot, JD., Rasmussen, JP., Williams, LM., Dooley, JL., Farr, AG., Rudensky AY. 2005. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity. 22(3): 329-41.

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

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 Jun;35(6):1681-91 (150D, IHC, PubMed).

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. 51-4714    Alexa Fluor® 647 Mouse IgG1, K Isotype Control
Cat. 17-4776    APC anti-human Foxp3 (clone PCH101)
Cat. 51-4776    Alexa Fluor® 647 anti-human Foxp3 (clone PCH101)
Cat. 00-5523    eBioscience Foxp3 Staining Buffer Set
Cat. 17-5773    Allophycocyanin (APC) anti-mouse/rat Foxp3 (clone FJK-16s)
Cat. 51-5773    Alexa Fluor® 647 anti-mouse/rat Foxp3 (clone FJK-16s)

Protocol for IC Staining

It is critical to use the Foxp3 Staining Buffer Set (cat. 00-5523).

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.
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 between 30 minutes and 18 hours in the dark for mouse tissue. (Comparable results, using the same donor, are obtained when incubated in the Fixation/Permeabilization Solution for varying times between 30 minutes and 18 hours.) If staining human cells, the incubation time is 30 - 60 minutes.
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 Fc block for mouse tissue (or normal mouse serum for human tissue) in 1X Permeabilization Buffer, in approximately 100 µl volume, at 4°C for 15 minutes.
Without washing after blocking step, add fluorochrome conjugated anti-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.