Mouse Regulatory T Cell Staining Kit #1

Also known as: Forkhead Box P3, Scurfin, JM2, Treg, PE Foxp3 FJK-16s PE; CD4 FITC, CD25 APC

RUO: For Research Use Only

mouse CD4/Foxp3 antibody, Cytokine Kits conjugate

BALB/c splenocytes were surface stained with Anti-Mouse CD4 FITC (cat. 11-0042) and Anti-Mouse CD25 APC (cat. 17-0251), then fixed and permeabilized using the Foxp3 Staining Buffers and subsequently stained with 0.5 µg of Rat IgG2a κ Isotype Control PE (cat. 12-4321) or 0.5 µg of Anti-Mouse/Rat Foxp3 PE. Cells in the lymphocyte gate were used for analysis.

Contents: Mouse Regulatory T Cell Staining Kit #1
Catalog Number: 88-8111
Formulation: Phosphate buffer pH 7.2,
150 mM NaCl, 0.09% NaN₃
Storage Conditions: Store at 2-8°C. Light sensitive material. Use within 6 months of receipt or by date indicated on the bottle.
Clone: FJK-16s
Host/Isotype: Rat IgG2a

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Available Formats
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications	Regulatory Status
88-8111	Mouse Regulatory T Cell Staining Kit #1	N/A	N/A	IC Flow	RUO
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 Alexa Fluor® 647 conjugated products with eFluor® 660 conjugated products.

Description

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 mouse Foxp3 staining sets and protocol reveals approximately 2% of total splenocytes 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. These data are consistent with a recent report which follows expression of Foxp3, using a GFP knock-in (Fontenot et al, 2005).

FJK-16s cross-reacts with rat 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. Please note that the CD4 and CD25 antibodies included in this kit only recognize the mouse antigens. For staining rat tissue, please use (CD4 FITC cat. 11-0400 and CD25 cat. 13-0390)

The anti-mouse Foxp3 Staining Set has been formulated and optimized for the staining of mouse splenocytes with the FJK-16s monoclonal antibody

Not included:
Isotype controls for anti-CD4 (rat IgG2a, cat. 11-4321) and anti-CD25 (rat IgG1, cat. 17-4301)

Components

FITC anti-mouse CD4 (RM4-5): 50 μg. Store at 4°C in the dark.
APC anti-mouse CD25 (PC61.5): 50 μg. Store at 4°C in the dark.
PE anti-mouse/rat Foxp3 (FJK-16s): 25 μg. Store at 4°C in the dark.
PE Rat IgG2a isotype control: 50 μg. Store at 4°C in the dark.
Affinity Purified anti-mouse CD16/32 (Fc Block): 50 μg
eBioscience Flow Cytometry Staining Buffer: 200 ml
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. Use within 6 months of receipt. 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.
eBioscience Fixation/Permeabilization Diluent: 100 ml. Store at 4°C. The diluent is intended to be used in combination with the Fixation/Permeabilization Concentrate.
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.

Applications Reported

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

References

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)

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.

Protocol

To view protocol, please download the TDS by clicking the PDF button above.

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