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Product Information

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Contents: APC anti-mouse/rat Foxp3 Staining Set Catalog Number: 77-5775 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: FJK-16s Set Isotype: Rat IgG2a, κ

Mouse (BALB/c) splenocytes were surface-stained with FITC anti-mouse CD4 (RM4-5) (cat. 11-0042) and PE anti-mouse CD25 (PC61.5) (cat. 12-0251), and subsequently with 0.5μg APC anti-mouse Foxp3 (FJK-16s) or APC Rat IgG2a Iso Cntrl (cat. 17-4321) using the APC anti-mouse Foxp3 Staining Set. The dot plot on the left demonstrates co-staining of CD4 and FJK-16s, while the right plot demonstrates co-staining of CD25 and FJK-16s . Cells in the lymphocyte gate were used for analysis.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
71-5775	FITC anti-mouse/rat Foxp3 Staining Set	488	518	IC Flow
72-5775	PE anti-mouse/rat Foxp3 Staining Set	488	575	IC Flow
77-5775	APC anti-mouse/rat Foxp3 Staining Set	633	660	IC Flow

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

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Description

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The FJK-16s antibody reacts with mouse/rat 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.

Immunoblotting with FJK-16s antibody has mapped the epitope to amino acids 75-125 of the mouse Foxp3 protein. In the human, this region has been shown to be alternatively spliced at the mRNA level. Both the alternatively-spliced and non-spliced isoforms are present in the CD4+CD25+ subset of lymphocytes. Preliminary RT-PCR experiments have not revealed this alternatively-spliced isoform in mouse splenocytes, suggesting different gene regulation in the mouse and human.

Intracellular staining of mouse splenocytes with FJK-16s using the PE anti-mouse/rat Foxp3 Staining Set and protocol reveals approximately 2% of total cells in the C57Bl/6 strain and approximately 3-5% in the BALB/c mouse strain. Multicolor flow cytometric analysis demonstrates approximately 90% of the CD4+CD25+ cells and 4% of the CD4+CD25- cells staining with FJK-16s. B220+, CD11b+, CD11c+, and Ly6G/Gr-1+ cells do not show significant co-staining with FJK-16s.

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

FJK-16s cross-reacts with rat and canine Foxp3. This has been demonstrated by intracellular staining of Foxp3 and flow cytometry of rat splenocytes using the same method and reagents as used for mouse tissue.

Not included:
Fc Block (cat. 14-0161)
Flow Cytometry Staining Buffer (cat. 00-4222)
Rat IgG2a isotype Control (cat. 12-4321, 11-4321, or 17-4321)

Components:

1. eBioscience Fixation/Permeabilization Concentrate: 30 ml. Store at 4°C. 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 to make the Fixation/Permeabilization working solution. 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: 100 ml. Store at 4°C. The diluent is intended to be used in combination with the Fixation/Permeabilization Concentrate.
3. eBioscience Permeabilization Buffer (10X): 100 ml. Store at 4°C. 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. anti-mouse/rat Foxp3 (FJK-16s): 25 μg. Store at 4°C.

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Applications Reported

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For research use only, not for diagnostic or therapeutic use. This FJK-16s antibody has been reported for use in intracellular staining followed by flow cytometric analysis.

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Applications Tested

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This FJK-16s Set antibody has been tested . This can be used at less than or equal to 0.5 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.

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References

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Aswad, F., Kawamura, H., and G. Dennert. 2005. High Sensitivity of CD4+CD25+ Regulatory T Cells to Extracellular Metabolites Nicotinamide Adenine Dinucleotide and ATP: A Role for P2X7 Receptors. J Immunol. 175:3075-3083. (FJK-16s, Intracellular Staining for Flow Cytometry, Pubmed)

Beyersdorf, N., Gaupp, S., Balbach, K., Schmidt, J., Tokya, K.V., Lin, C.H., Hanke, T., Hunig, T., Kerkau, T., and R. Gold. 2005. Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis. J Exp Med. 202(3): 445-455. (FJK-16s, Intracellular Staining for Flow Cytometry in Rat, Pubmed)

Biller BJ, Elmslie RE, Burnett RC, Avery AC, Dow SW. Use of FoxP3 expression to identify regulatory T cells in healthy dogs and dogs with cancer. Vet Immunol Immunopathol. 2007 Mar 15;116(1-2):69-78 (FJK, IC Flow in canine, PubMed)

Fields, M.L., B.D. Hondowicz, M.H. Metzgar, S.A. Nish, G.N. Wharton, C.C. Picca, A.J. Caton, and J. Erikson. 2005. CD4+CD25+ Regulatory T cells inhibit the maturation but not the initiation of an autoantibody response. J. Immunol. 175: 4255-4264. (FJK-16s, Intracellular Staining for Flow Cytometry, PubMed)

Ko K., S. Yamazaki, K. Nakamura, T. Nishioka, K. Hirota, T. Yamaguchi, J. Shimizu, T. Nomura, T. Chiba, and S. Sakaguchi. 2005. Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+CD25+CD4+ regulatory T cells. J Exp Med 202: 885-91. (FJK-16s, Intracellular Staining for Flow Cytometry, PubMed)

Kohm AP, McMahon JS, Podojil JR, Begolka WS, Degutes M, Kasprowicz DJ, Ziegler SF, Miller SD. Cutting Edge: Anti-CD25 Monoclonal Antibody Injection Results in the Functional Inactivation, Not Depletion, of CD4+CD25+ T Regulatory Cells. J Immunol. 2006 Mar 15;176(6):3301-5. [FJK-16s; intracellular staining and IH/F, PubMed]

McGeachy, M.J., Stephens, L.A., and S.M. Anderton. 2005. Natural Recovery and Protection from Autoimmune Encephalomyelitis: Contribution of CD4+CD25+ Regulatory Cells within the Central Nervous System. J Immunol. 175:3025-3032. (FJK-16s, Intracellular Staining for Flow Cytometry, Pubmed)

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.

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Related Products

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Cat. 11-0042    FITC anti-mouse CD4 (L3T4) (clone RM4-5)
Cat. 12-0251    PE anti-mouse CD25 (Interleukin-2 Receptor alpha, IL-2 Receptor alpha, IL-2Ra, p55, IL2Ra) (clone PC61.5)
Cat. 17-4321    APC Rat IgG2a Isotype Control
Cat. 00-5523    eBioscience Foxp3 Staining Buffer Set

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Protocol for IC Staining

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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.

1. Add 100 µl of prepared cells (1x10⁶) to each tube.
2. Stain surface molecules such as CD4, CD8, CD25, etc. following the Surface Staining Protocol (http://www.ebioscience.com/ebioscience/appls/FCS.htm).
3. Wash in cold Flow Cytometry Staining Buffer (or cold PBS).
4. Resuspend cell pellet with pulse vortex and add 1 ml of freshly prepared Fixation/Permeabilization working solution to each sample. Pulse vortex again.
5. Incubate at 4°C between 30 minutes and 18 hours in the dark. (Comparable results, using the same donor, are obtained when incubated in the Fixation/Permeabilization Solution for varying times between 30 minutes and 18 hours.)
6. Wash once by adding 2 ml 1X Permeabilization Buffer (made from 10X Permeabilization Buffer) followed by centrifugation and decanting of supernatant.
7. Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
8. [OPTIONAL] Block with Fc block in 1X Permeabilization Buffer, in approximately 100 µl volume, at 4°C for 15 minutes.
9. 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.
10. Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
11. Repeat step 10.
12. 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.