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Product Information

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Contents: Phycoerythrin (PE) anti-mouse Foxp3 Catalog Number: 12-4771 Sizes: 25 ug, 100 ug 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: NRRF-30 Isotype: Rat IgG2a, κ

Mouse (BALB/c) splenocytes were surface-stained with CD4-FITC (clone RM4-5) (cat. 11-0042) and CD25-APC (clone PC61.5) (cat. 17-0251), and subsequently with 0.25μg/million cells PE anti-mouse Foxp3 (NRRF-30) (cat. 12-4771) or PE Rat IgG2a Iso Cntrl (cat. 12-4321) using the Foxp3 Staining Buffer Set (cat. 00-5523). The dot plot on the left demonstrates co-staining of CD4 and NRRF-30, while the right plot demonstrates co-staining of CD25 and NRRF-30 . Cells in the lymphocyte gate were used for analysis.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
12-4771	Phycoerythrin (PE) anti-mouse Foxp3	488	575	IC Flow
14-4771	Affinity Purified anti-mouse Foxp3	N/A	N/A	IH/F  WB

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 NRRF-30 antibody reacts with mouse 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 NRRF-30 antibody has mapped the epitope to amino acids 1-75 of the mouse Foxp3 protein.

Intracellular staining of mouse splenocytes with fluorochrome-conjugated NRRF-30 using the eBioscience Foxp3 Staining Buffers (cat. 00-5523) and corresponding staining protocol reveals approximately 3% of total cells in the C57Bl/6 strain and approximately 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 NRRF-30. Co-staining with FJK-16s (anti-mouse/rat Foxp3 cat. 71-5775), which has been mapped to amino acids 71-125, and NRRF-30 shows 100% correlation, indicating that the same cells are stained with both anti-mouse Foxp3 antibodies.

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

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Applications Reported

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For research use only, not for diagnostic or therapeutic use. This NRRF-30 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 NRRF-30 antibody has been tested by intracellular flow cytometric analysis of mouse splenocytes using the Foxp3 Staining Buffer Set (cat. 00-5523) and protocol for Foxp3. 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.

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Related Products

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Cat. 12-4321    PE Rat IgG2a Isotype Control
Cat. 12-5773    PE anti-mouse/rat Foxp3 (clone FJK-16s)
Cat. 14-5773    Affinity Purified anti-mouse/rat Foxp3 (clone FJK-16s)

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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.