Product Information

Contents: Alexa Fluor® 647 anti-mouse/human Foxp3
Catalog Number: 51-7979
Sizes: 25 ug
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: eBio7979
Isotype: Mouse IgG1

Mouse (BALB/c) splenocytes were surface-stained with FITC anti-mouse CD4 (RM4-5) (cat. 11-0042) and subsequently with Alexa647 anti-Foxp3 (eBio7979) using the Foxp3 Buffer Set (cat. 00-5523).

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
12-7979	Phycoerythrin (PE) anti-mouse/human Foxp3	488	575	IC Flow
14-7979	Affinity Purified anti-mouse/human Foxp3	N/A	N/A	IH/F IHC(Paraffin) IP WB
51-7979	Alexa Fluor® 647 anti-mouse/human Foxp3	633	668	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 and mouse 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 PCH101 (cat. 72-5776) and 236A/E7 (cat. 12-4777). Additional antibodies to mouse Foxp3 include FJK-16s (cat. 72-5775) and NRRF-30 (cat. 12-4771).

The eBio7979 antibody reacts with human and mouse 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.

Please see the following link for FAQ regarding the usage of eBioscience Foxp3 reagents: http://www.ebioscience.com/ebioscience/Foxp3FAQs.htm

Applications Reported

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

Applications Tested

This eBio7979 antibody has been tested intracellular 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.

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.

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

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

Related Products

Cat. 51-4714    Alexa Fluor® 647 Mouse IgG1, K Isotype Control
Cat. 51-4774    Alexa Fluor® 647 anti-mouse/human/rat Foxp3 (clone 150D/E4)
Cat. 17-4776    APC anti-human Foxp3 (clone PCH101)
Cat. 51-4776    Alexa Fluor® 647 anti-human Foxp3 (clone PCH101)
Cat. 17-4777    Allophycocyanin (APC) anti-human Foxp3 (clone 236A/E7)
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.