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PE Mouse anti-Human FoxP3
PE Mouse anti-Human FoxP3

Flow cytometric analysis of PE anti-human FoxP3 on resting PBMC. Human PBMC were stained with FITC anti-human CD4 (clone RPA-T4, Cat. No. 555346) and APC anti-human CD25 (clone M-A251, Cat. No. 555434 shown here or with clone 2A3, Cat. 340938, data not shown) simultaneously.  Cells were fixed and permeabilized (see recommended assay procedure) followed by intracellular staining with PE anti-human FoxP3 (clone 259D/C7; Cat No. 560046/560082). 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 PE anti-human FoxP3 on resting PBMC. Human PBMC were stained with FITC anti-human CD4 (clone RPA-T4, Cat. No. 555346) and APC anti-human CD25 (clone M-A251, Cat. No. 555434 shown here or with clone 2A3, Cat. 340938, data not shown) simultaneously.  Cells were fixed and permeabilized (see recommended assay procedure) followed by intracellular staining with PE anti-human FoxP3 (clone 259D/C7; Cat No. 560046/560082). 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_1645508
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 with R-PE under optimum conditions, and unconjugated antibody and free PE were removed.

Recommended Assay Procedures

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

          1.  Bring the buffers to RT (room temperature) before use. Prepare working solutions of the BD Pharmingen Human FoxP3 Buffer Set,

               Cat. No. 560098. (For Buffer A&C preparations, please see TDS of Cat. No. 560098 for details).

          2.  Prepare human PBMC. Dilute the cells with BD Pharmingen Stain Buffer (FBS)* to 1 X 10^7 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 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 cells, gently re-suspend pellet in residual volume of wash buffer and then add 0.5 ml of 1x working solution of

               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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  3. 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.
  4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560046 Rev. 3
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.

560046 Rev. 3
Format Details
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PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
560046 Rev.3
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
560046 Rev. 3

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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.