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The FOXP3 antibody was purified by affinity chromatography, and conjugated with Alexa Fluor® 488 under optimal conditions. The solution is free of unconjugated Alexa Fluor® 488 .
Storage & Handling:
The FOXP3 antibody solution should be stored undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Each lot of this FOXP3 antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For immunofluorescent staining, the suggested use of this reagent is 5 µl per 106 cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application.
* Alexa Fluor® 488 has a maximum emission of 519 nm when it is excited at 488 nm. ** Alexa Fluor® is a registered trademark of Molecular Probes, Inc. Alexa Fluor® dye antibody conjugates are sold under license from Molecular Probes, Inc. for research use only, except for use in combination with microarrays and high content screening, and are covered by pending and issued patents.
Additional reported applications (for the relevant formats) include: Western blotting1, and immunohistochemical staining1 of acetone-fixed frozen sections and formalin-fixed paraffin-embedded sections. The 259D antibody gives strong positivity on paraffin and frozen sections and the antibody stains some epithelial cells. The binding of 206D to FOXP3 can be partially blocked by 259D, but 206D does not show significant blocking effect on 259D binding.
Surface Staining & FOXP3 Buffer Preparation:
Centrifugation steps: perform at 250Xg for 5min Incubation steps: perform at room temperature
1. Perform cell surface staining if necessary (See protocol: Cell Surface Immunofluorescence Staining Protocol). 2. Prepare 1x buffer solutions of FOXP3 Fix/Perm buffer and FOXP3 Perm buffer in PBS.
NOTE: The FOXP3 Perm buffer (10X) may have crystalization or precipitation observed when it is stored at 2-8°C, however, it is normal and does not affect the buffer performance. If there is a heavy precipitation observed after diluting to 1X working solution, it may be clarified by filtering. Caution: The FOXP3 Fix/Perm buffer contains paraformaldehyde, which is toxigenic and mutagenic. Please handle with caution and wear gloves, lab coat and necessary protection to avoid direct body contact.
FOXP3 Intracellular Staining Procedures:
3. Add 1 ml of 1x Biolegend's FOXP3 Fix/Perm solution to each tube, resuspend the cells (gentle vortex) and incubate in the dark for 20 minutes, then centrifuge and remove the supernatant. The cell pellet will now be translucent and difficult to see; take care not to dislodge and accidentally aspirate cells at all later stages of staining protocol. 4. Wash: resuspend cells in cell staining buffer (Cat. No. 420201); centrifuge, then discard the supernatant. 5. Wash: resuspend in 1ml 1X BioLegend's FOXP3 Perm buffer; centrifuge, then discard the supernatant. 6. Resuspend cells in 1ml 1X BioLegend's FOXP3 Perm buffer, incubate in the dark for 15 minutes; centrifuge, then discard the supernatant. Resuspend the pellet in 100 µl of 1X BioLegend's FOXP3 Perm buffer. 7. Add appropriate amount of flurochrome conjugated anti-FOXP3 antibody and incubate in the dark for 30 minutes. 8. Wash twice with cell staining buffer (see step 4) then resuspend in 0.5ml cell staining buffer. Analyze with flow cytometer using appropriate instrument settings.
NOTE: BioLegend's FOXP3 Fix/Perm buffer set (Cat. No. 421403) is specifically developed and formulated for intracellular staining FOXP3 with minimum effect on surface fluorochrome staining and is highly recommended for optimal result of FOXP3 intracellular immunofluorescence staining.
Application References:
1. Roncador G, et al. 2005 Eur. J. Immunol. 35:1681. 2. Yang ZZ, et al. 2006. Blood 107:3639. PubMed 3. Gavin MA, et al. 2006. P. Natl. Acad. Sci. USA 103:6659. PubMed 4. Groh V, et al. 2006. Nature Immunology 7:755. PubMed 5. Tran DQ, et al. 2007. Blood doi:10.1182/blood-2007-06-094656 PubMed 6. Fox BC, et al. 2008. Blood 111:3897. PubMed 7. Monti P,et al. 2008. Diabetes. 57:2341. PubMed 8. Torgerson TR, et al. 2009. J. Immunol. 183:907. PubMed 9. Vonderheide RH, et al. 2010. Clin Cancer Res. 16:3485. PubMed 10. Rapoport AP, et al. 2011. Blood 117:788. PubMed 11. Tresoldi, E., et al. 2011. Haematologica. 96:1357. PubMed.
Human PBMCs were surface stained with CD4-APC, CD25-PE and intracellular stained with 259D Alexa 488.
FOXP3 is a 50-55 kD transcription factor, also known as Forkhead box protein P3, Scurfin, JM2, or IPEX. It is proposed to be a master regulatory gene and more specific marker of T regulatory cells than most cell surface markers (such as CD4 and CD25). Transduced expression of FOXP3 in CD4+/CD25- cells has been shown to induce GITR, CD103, and CTLA4 and impart a T regulatory cell phenotype. FOXP3 is mutated in X-linked autoimmunity-allergic dysregulation syndrome (XLAAD or IPEX) in humans and in "scurfy" mice. Overexpression of FOXP3 has been shown to lead to a hypoactive immune state suggesting that this transcriptional factor is a central regulator of T cell activity. In human, unlike in mouse, two isoforms of FOXP3 have been reported: one (FOXP3) corresponding to the canonical full-length sequence; the other (FOXP3 δ2) lacking exon 2. The 259D antibody recognizes human FOXP3 epitope in the region of amino acids 105-235.
Other Names:
Forkhead box protein P3, Scurfin, JM2, IPEX, Zinc finger protein JM2
Structure:
Forkhead/winged-helix transcription factor family, approximately 50 kD, contains zinc finger and forkhead domains
Distribution:
Nuclear; expressed in T regulatory cells
Function:
Transcription factor proposed to be a master regulatory gene in T regulatory cell development and a critical factor for immune homeostasis
Regulation:
FOXP3 is present at high levels in T regulatory cell can also be induced by T cell activation
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This data display is provided for general comparisons between formats. Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors. If you need assistance with selecting the best format contact our expert technical support team.
Purified anti-human FOXP3
Cell extract from HEK293T cells transfected with human FoxP3 cDNA was resolved by electrophoresis, transferred to nitrocellulose, and probed with monoclonal anti-FoxP3 antibody (clone 259D). Proteins were visualized using a goat anti-mouse secondary conjugated to HRP and a chemiluminescence detection system.
Formalin-fixed, paraffin-embedded Cynomolgus kidney was treated with EDTA pH 8.0 using a high pressure cooker prior to staining. Staining was carried out with monoclonal anti-FoxP3 (clone 259D) at 10 µg/ml followed by biotinylated goat anti-mouse and streptavidin HRP. Staining was visualized with DAB substrate and the tissue counterstained with hematoxylin.
PE anti-human FOXP3
Human peripheral blood lymphocytes surface stained with CD4 FITC, then intracellular stained with 259D PE. Quadrant markers were set based on staining with PE mouse IgG1, κ isotype control
Alexa Fluor® 488 anti-human FOXP3
Human PBMCs were surface stained with CD4-APC, CD25-PE and intracellular stained with 259D Alexa 488.
Alexa Fluor® 647 anti-human FOXP3
Human peripheral blood lymphocytes surface stained with CD4 FITC, then intracellular stained with 259D Alexa Fluor® 647. Quadrant markers were set based on staining with Alexa Fluor® 647 mouse IgG1, κ isotype control
Pacific Blue™ anti-human FOXP3
Human peripheral blood lymphocytes stained with CD4 APC and then intracellular stained with Pacific Blue™ anti-human FOXP3 (clone 259D). The quadrant markers were set based on Pacific Blue™ mIgG1, k isotype control
Human Treg Flow™ Kit (FOXP3 Alexa Fluor® 488/CD4 PE-Cy5/CD25 PE)
Human peripheral blood mononuclear cells stained with BioLegend's Human Treg Flow™ Kit (FOXP3 Alexa Fluor® 488/CD4 PE-Cy5/CD25 PE) and analyzed on lymphocyte population
