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Alexa Fluor® 647 Mouse anti-Human FoxP3
Alexa Fluor® 647 Mouse anti-Human FoxP3

Flow cytometric analysis of FoxP3 on resting PBMC. Human PBMC were stained with FITC Mouse Anti-Human CD4 (Cat. No. 555346) and PE Mouse Anti-Human CD25 (Cat. No. 555432) simultaneously.  Cells were fixed and permeabilized (see recommended assay procedure) followed by intracellular staining with Alexa 647 anti-human FoxP3 (Cat No. 560045). The dot plots were derived from the gated events based on light scattering characteristics of lymphocytes and fluorescence characteristics of CD4+ or CD25+ respectively, shown as either FoxP3 vs CD25 (left panel) or FoxP3 vs CD4 (right panel). Flow cytometry was performed on a BD FACSCalibur™ System.

Flow cytometric analysis of FoxP3 on resting PBMC. Human PBMC were stained with FITC Mouse Anti-Human CD4 (Cat. No. 555346) and PE Mouse Anti-Human CD25 (Cat. No. 555432) simultaneously.  Cells were fixed and permeabilized (see recommended assay procedure) followed by intracellular staining with Alexa 647 anti-human FoxP3 (Cat No. 560045). The dot plots were derived from the gated events based on light scattering characteristics of lymphocytes and fluorescence characteristics of CD4+ or CD25+ respectively, shown as either FoxP3 vs CD25 (left panel) or FoxP3 vs CD4 (right panel). Flow cytometry was performed on a BD FACSCalibur™ System.

Product Details
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BD Pharmingen™
scurfin; IPEX; JM2; AIID; XPID; DIETER; PIDX
Human (QC Testing), Rhesus, Cynomolgus, Baboon (Tested in Development)
Mouse BALB/c IgG1
FoxP3
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645411
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.

Recommended Assay Procedures

Cell Preparation and Staining Procedures for Conjugated Anti-Human FoxP3 Antibody

          1.  Bring the buffers to RT before use. Prepare working solutions of the BD Pharmingen Human FoxP3 Buffer Set

               Cat. No. 560098 (For the buffer preparation, please see TDS Cat. No. 560098 buffer instructions for details).

          2.  Prepare human PBMC. Dilute the cells with BD Pharmingen Stain Buffer (FBS)* to ten million cells/ml.

          3.  Pipette appropriate amount of surface staining reagent to bottom of each 12 x 75 mm tube.

          4.  Add 100µl of cells per tube, vortex, incubate for 20 minutes at RT protected from light.

          5.  Add 2 ml of wash buffer. Centrifuge 250 x g for 10 minutes, and remove wash buffer.

          6.  To fix the cells, gently re-suspend pellet in residual volume of wash buffer and then add 2ml of 1x Human FoxP3 Buffer A. Vortex.

                Incubate for 10 minutes at RT in the dark.

          7.  Centrifuge 500 x g for 5 minutes, and remove fixative. Caution: Be aware the pellet is buoyant.

          8.  To wash cells, re-suspend each pellet in 2ml of BD Pharmingen Stain Buffer (FBS)*, and centrifuge 500 x g for 5 minutes. Remove

               wash buffer.

          9.  To permeabilize the cells, gently re-suspend pellet in residual volume of wash buffer and then add 0.5 ml of 1x working solution

               Human FoxP3 Buffer C to each tube. Vortex.  Incubate for 30 minutes at RT protected from light.

        10.  To wash cells, add 2 ml of BD Pharmingen Stain Buffer (FBS)* to each tube, centrifuge 500 x g for 5 minutes at RT.  Remove buffer

                and repeat wash step.  Remove buffer.

        11.  Add conjugated FoxP3 antibody at appropriate concentrations to re-suspend the pellet. Gently shake or vortex.

        12.  Incubate for 30 minutes in the dark at RT.

        13.  Repeat wash step #10.

        14.  Resuspend in wash buffer and analyze immediately.

                Optional  Add 300µl of 1% formaldehyde in 1x PBS and store at 4°C.  Analyze cells within 24 hours.

*   We recommend using the BD Pharmingen Stain Buffer (FBS; Cat No. 554656) for all wash steps and covering tubes during incubation steps with caps or parafilm.  We also recommend optimizing forward scatter and side scatter voltages to visualize lymphocytes as separate from debris, red cell ghosts and/or platelets before acquisition.

**  Acquire at least 15,000 to 25,000 CD4 positive lymphocytes.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  7. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  8. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  9. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560045 Rev. 5
Antibody Details
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259D/C7

The 259D/C7 monoclonal antibody specifically recognizes human Forkhead box protein P3 (FoxP3) that is also known as Scurfin. FoxP3 is encoded by FOXP3 (Forkhead box P3), likewise known as IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-Linked) and JM2, that belongs to the forkhead/winged-helix family of transcriptional regulators. FoxP3 is expressed in CD4+ regulatory T cells (Treg) and represents a specific marker for these cells. Flow cytometric analyses have shown that FoxP3 is expressed by the majority of CD4+CD25+high T cells in peripheral blood while less than half of the CD4+CD25+intermediate cell population are FoxP3 positive. Approximately 5-10% of peripheral CD4+ cells are CD4+CD25+ T regulatory cells. T regulatory cells are thought to play a critical role in the regulation of T cell-mediated immunity and to protect against autoimmunity by suppressing the proliferation and cytokine production of other T cells. In support of this hypothesis, it has been found that Foxp3 is mutated in Scurfy (sf) mice that have defective T cell tolerance leading to an X-linked lymphoproliferative disease. The 259D/C7 antibody recognizes all currently-identified isoforms of human FoxP3 and is crossreactive with FoxP3 from Cynomolgus, Rhesus and Baboon primates.

560045 Rev. 5
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
560045 Rev.5
Citations & References
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Development References (3)

  1. Brunkow ME, Jeffery EW, Hjerrild KA, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. 2001; 27(1):68-73. (Biology). View Reference
  2. Giovanna Roncador et al. Analysis of Foxp3 protein expression in human CD4+CD25+ regulatory Tcells at a single cell level. Eur J Immunol. 2005; 35(Immunogen).
  3. Wildin RS, Ramsdell F, Peake J, et al. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet. 2001; 27(1):18-20. (Biology). View Reference
560045 Rev. 5

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.