PE anti-human FOXP3 Flow Kit

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 ≤0.3 µg per million cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application.

ICFC - Quality tested

Materials Provided:
1. PE anti-human FOXP3, 25 tests
2. PE Mouse IgG1, k isotype control (ICFC), 25 tests
3. FOXP3 Fix/Perm buffer set, 100 tests (Cat. No. 421403)
Materials not included:
Cell Staining Buffer (Cat. No. 420201)

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 intracellularly staining FOXP3 with minimum effect on surface fluorochrome staining and is highly recommended for optimal results of FOXP3 intracellular immunofluorescence staining.

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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 206D antibody recognizes human FOXP3 epitope in the region of amino acids 105-235.