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The antibody was purified by affinity chromatography, and conjugated with PE under optimal conditions. The solution is free of unconjugated PE and unconjugated antibody.
Concentration:
0.2 mg/ml
Storage & Handling:
The antibody solution should be stored undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Each lot of this antibody is quality control tested by immunofluorescent intracellular staining with flow cytometric analysis. For immunofluorescent staining, the suggested use of this reagent is ≤1.0 µg per million cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application.
COA:
Application Notes:
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: The FOXP3 Fix/Perm buffer (4X) must be freshly diluted by diluting one (1) part FOPX3 Fix/Perm buffer (4X) with three (3) parts PBS. The FOXP3 Perm buffer (10X) should be diluted by diluting one (1) part FOXP3 Perm buffer (10X) with nine (9) parts of 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, 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 with clone 150D.
Application References:
1. Ono M, et al.:Nature 2007 446:685 2. Hori S, et al. 2003. Science 299:1057 3. Fontenot JD, et al. 2003 Nature Immunol 4:330 4. Fallarino F, et al. 2009. J. Immunol. 183:6033. PubMed 5. Barber A, et al. 2009 J. Immunol. 183:6939. PubMed 6. Nakashima H, et al. 2010. J. Immunol. 184:4637. PubMed
C57BL/6 splenocytes surface stained with CD4 (GK1.5) APC and then intracellularly stained MF-14 PE
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 150D monoclonal antibody reacts with human, mouse and rat FOXP3. The 150D antibody recognizes FOXP3 epitope encoded by exon 2.
Other Names:
Forkhead box protein P3, Scurfin, JM2, IPEX, Zinc finger protein JM2
Master regulatory gene in Treg cell development, crucial for immune homeostasis.
Regulation:
Present at high level in T reg cells. Induced by T cell activation.
Interaction:
Interacts with DNA
Antigen References:
1. Ono M, et al.:Nature 2007 446:685 2. Hori S, et al. 2003. Science 299:1057 3. Fontenot JD, et al. 2003 Nature Immunol 4:330 4. Fallarino F, et al. 2009. J. Immunol. 183:6033. 5. Barber A, et al. 2009 J. Immunol. 183:6939. 6. Nakashima H, et al. 2010. J. Immunol. 184:4637.
*These products may be covered by one or more Limited Use Label Licenses (see the BioLegend Catalog or our website, www.biolegend.com/terms). BioLegend products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without written approval of BioLegend. By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use.
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-mouse FOXP3
Western blot analysis of extracts from untransfected 293T cells (Lane 1) or 293T cells transfected with a plasmid encoding mouse FOXP3 protein (Lane 2), using anti-FOXP3, clone MF-14.
PE anti-mouse FOXP3
C57BL/6 splenocytes surface stained with CD4 (GK1.5) APC and then intracellularly stained MF-14 PE
Alexa Fluor® 488 anti-mouse FOXP3
C57BL/6 splenocytes were surface stained with CD4 (GK1.5) APC and MF-14 Alexa Fluor® 488
Alexa Fluor® 647 anti-mouse FOXP3
C57BL/6 splenocytes surface stained with CD4 (GK1.5) PE and then intracellularly stained with MF-14 Alexa Fluor® 647
Pacific Blue™ anti-mouse FOXP3
C57BL/6 splenocytes surface stained with CD4 (GK1.5) PE and then intracellularly stained with MF-14 Pacific Blue™
