Contents: Biotin anti-mouse/rat Foxp3
Catalog Number: 13-5773
Formulation: Phosphate buffer pH 7.2,
150 mM NaCl, 0.09% NaN₃
Storage Conditions: Store at 4°C.
DO NOT FREEZE.
Clone: FJK-16s
Isotype: Rat IgG2a, κ

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13-5773-80

25 ug

13-5773-82

100 ug

BALB/c splenocytes were surface-stained with FITC anti-mouse CD4 (RM4-5) (cat. 11-0042) and APC anti-mouse CD25 (PC61.5) (cat. 17-0251) and subsequently with 0.25 μg Biotin anti-mouse Foxp3 (clone FJK-16s) or Biotin Rat IgG2a Iso Cntrl (cat. 13-4321) followed by SAV-PE (cat. 12-4317) using the Foxp3 Staining Buffer Set (cat. 00-5523). Quadrant lines demarcate the isotype control. Cells in the lymphocyte gate were used for analysis.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
14-5773	Affinity Purified anti-mouse/rat Foxp3	N/A	N/A	IH/F IHC(Paraffin) WB
13-5773	Biotin anti-mouse/rat Foxp3	N/A	N/A	IC Flow IH/F WB
11-5773	FITC anti-mouse/rat Foxp3	488	518	IC Flow IH/F
12-5773	PE anti-mouse/rat Foxp3	488	575	IC Flow
15-5773	Phycoerythrin-Cy5 (PE-Cy5) anti-mouse/rat Foxp3	488	670	IC Flow
17-5773	Allophycocyanin (APC) anti-mouse/rat Foxp3	633	660	IC Flow
25-5773	Phycoerythrin-Cy7 (PE-Cy7) anti-mouse/rat Foxp3	488	760	IC Flow
51-5773	Alexa Fluor® 647 anti-mouse/rat Foxp3	633	668	IC Flow
53-5773	Alexa Fluor® 488 anti-mouse/rat Foxp3	488	519	IC Flow
56-5773	Alexa Fluor® 700 anti-mouse/rat Foxp3 (APC-Cy5.5 Equivalent)	633	723	IC Flow
45-5773	PerCP-Cy5.5 anti-mouse/rat Foxp3 (PE-Cy5.5 replacement)	488	690	IC Flow
48-5773	eFluor^TM 450 anti-mouse/rat Foxp3 (Pacific Blue® replacement)	405	450	IC Flow
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 Pacific Blue® and APC-Alexa Fluor® 750 conjugated products with eFluor™ 450 and APC-eFluor™ 780 conjugated products, respectively.

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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 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. Please note that FJK-16s has been optimized for use with the mouse Foxp3 Staining Set (cat. 72-5775 or 71-5775 or 77-5775). The use of other fixation and staining buffers is not recommended.

Applications Reported

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 and immunohistology on frozen tissue sections.

Applications Tested

This FJK-16s antibody has been tested by intracellular staining and flow cytometric analysis of mouse splenocytes. 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.

References

Habicht A, Dada S, Jurewicz M, Fife BT, Yagita H, Azuma M, Sayegh MH, Guleria I. A link between PDL1 and T regulatory cells in fetomaternal tolerance. J Immunol. 2007 Oct 15;179(8):5211-9. (FJK-16s, IH/F, PubMed)

Heimesaat MM, Fischer A, Siegmund B, Kupz A, Niebergall J, Fuchs D, Jahn HK, Freudenberg M, Loddenkemper C, Batra A, Lehr HA, Liesenfeld O, Blaut M, Göbel UB, Schumann RR, Bereswill S. Shift towards pro-inflammatory intestinal bacteria aggravates acute murine colitis via Toll-like receptors 2 and 4. PLoS ONE. 2007 Jul 25;2(7):e662. (FJK-16s, IHC paraffin, 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-16s, IC Flow in canine, PubMed)

Leithäuser F, Meinhardt-Krajina T, Fink K, Wotschke B, Möller P, Reimann J. Foxp3-expressing CD103+ regulatory T cells accumulate in dendritic cell aggregates of the colonic mucosa in murine transfer colitis. Am J Pathol. 2006 Jun;168(6):1898-909. (FJK-16s, IHC paraffin, 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)

Suvas S, Azkur AK, Rouse BT.Qa-1b and CD94-NKG2a interaction regulate cytolytic activity of herpes simplex virus-specific memory CD8+ T cells in the latently infected trigeminal ganglia. J Immunol. 2006 Feb 1;176(3):1703-11. (FJK-16s, Immunofluorescence, PubMed)

Fields ML, Hondowicz BD, Metzgar MH, Nish SA, Wharton GN, Picca CC, Caton AJ, Erikson J. CD4+CD25+ Regulatory T cells inhibit the maturation but not the initiation of an autoantibody response. J Immunol. 2005 Oct 1;175(7):4255-64. (FJK-16s, IC Flow, PubMed)

Ko K, Yamazaki S, Nakamura K, Nishioka T, Hirota K, Yamaguchi T, Shimizu J, Nomura T, Chiba T, Sakaguchi S. Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+CD25+CD4+ regulatory T cells. J Exp Med. 2005 Oct 3;202(7):885-91. (FJK-16s, IC Flow, PubMed)

Aswad F, Kawamura H, Dennert G. High Sensitivity of CD4+CD25+ Regulatory T Cells to Extracellular Metabolites Nicotinamide Adenine Dinucleotide and ATP: A Role for P2X7 Receptors. J Immunol. 2005 Sep 1;175(5):3075-83. (FJK-16s, IC Flow, PubMed)

Beyersdorf N, Gaupp S, Balbach K, Schmidt J, Toyka KV, Lin CH, Hanke T, Hünig T, Kerkau T, Gold R. Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis. J Exp Med. 2005 Aug 1;202(3):445-55. (FJK-16s, IC Flow in Rat, PubMed)

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

Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science. 2003 Feb 14;299(5609):1057-61.

Related Products

Cat. 12-4317  Streptavidin-PE (Phycoerythrin)
Cat. 13-4321  Biotin Rat IgG2a Isotype Control
Cat. 00-5523  eBioscience Foxp3 Staining Buffer Set
Cat. 71-5775  FITC anti-mouse/rat Foxp3 Staining Set (clone FJK-16s Set)
Cat. 72-5775  PE anti-mouse/rat Foxp3 Staining Set (clone FJK-16s Set)
Cat. 72-5776  PE anti-human Foxp3 Staining Set (preferentially stains CD4+CD25hi) (clone PCH101 Set)

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. (Comparable results, using the same donor, are obtained when incubated in the Fixation/Permeabilization Solution for varying times between 30 minutes and 18 hours.)
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 in 1X Permeabilization Buffer, in approximately 100 µl volume, at 4°C for 15 minutes.
Without washing after blocking step, add biotinylated anti-Foxp3 antibody 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.
Add pre-titered SAV-fluorochrome in 1X Permeabilization Buffer (such as SAV-PE, Cat. No. 12-4317, or SAV-APC, Cat. No. 17-4317). Incubate for 20 minutes.
Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
Repeat step 13.
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.