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Product Information

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Contents: Mouse Regulatory T cell Staining Kit #2 (w/ APC Foxp3 FJK-16s, FITC CD4, PE CD25; Treg Kit) Catalog Number: 88-8118 Formulation: Phosphate buffer pH 7.2, 150 mM NaCl, 0.09% NaN3 Storage Conditions: Store at 4°C. Clone: FJK-16s Isotype: Rat IgG2a

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 (cat. 77-5775). 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
88-8118	Mouse Regulatory T cell Staining Kit #2 (w/ APC Foxp3 FJK-16s, FITC CD4, PE CD25; Treg Kit)	N/A	N/A	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 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.

The anti-mouse/rat 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. 12-4301)

Components:

1. FITC anti-mouse CD4 (RM4-5): 50 μg. Store at 4°C in the dark.
2. PE anti-mouse CD25 (PC61.5): 50 μg. Store at 4°C in the dark.
3. APC anti-mouse/rat Foxp3 (FJK-16s): 25 μg. Store at 4°C in the dark.
4. APC Rat IgG2a isotype control: 50 μg. Store at 4°C in the dark.
5. Affinity Purified anti-mouse CD16/32 (Fc Block): 50 μg
6. eBioscience Flow Cytometry Staining Buffer: 200 ml
7. 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 4 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.
8. eBioscience Fixation/Permeabilization Diluent: 100 ml. Store at 4°C. The diluent is intended to be used in combination with the Fixation/Permeabilization Concentrate.
9. 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.

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Applications Reported

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

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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)

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. 88-8111    Mouse Regulatory T cell Staining Kit (w/ PE Foxp3 FJK-16s, FITC CD4, APC CD25; Treg Kit) (clone FJK-16s)

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Protocol for IC Staining

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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.

1. Add 100 µl of prepared cells (1x10⁶) to each tube.
2. Stain surface molecules as usual. Use approximately 0.125 µg/test of CD4 and 0.06 µg/test of CD25 antibodies in about 100 μl Flow Staining Buffer. It is best to titer these reagents for your particular application. Incubate at 4°C for approximately 30 minutes. Fc block can be added before incubation with surface antibodies.
3. Wash in cold Flow Cytometry Staining Buffer. (Centrifuge and decant.)
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 for 30 minutes - 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 cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
7. Repeat step 6.
8. [OPTIONAL] Block with 1-2 µg/test Fc block in 1X Permeabilization Buffer, in approximately 100 µl volume, at 4°C for 15 minutes.
9. Without washing after blocking step, add 0.5 µg/test anti-mouse/rat Foxp3 (FJK-16s) 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.