BD Pharmingen™ Alexa Fluor® 647 Mouse anti-Human FoxP3

Name: Alexa Fluor® 647 Mouse anti-Human FoxP3
Brand: Alexa Fluor® 647 Mouse anti-Human FoxP3
SKU: 561184

Clone 236A/E7 (RUO)

Flow cytometric analysis of FoxP3 expressed in human lymphocytes. Human peripheral blood mononuclear cells (PBMC) were stained with PerCP-Cy5.5 anti-human CD4 (clone L200, Cat. No. 552838) and PE anti-human CD25 (Clone 2A3, Cat. No. 341009) simultaneously. The PBMC were fixed and permeabilized (see Recommended Assay Procedure) followed by intracellular staining with Alexa Fluor® 647 Mouse anti-Human FoxP3 (clone 236A/E7; Cat No. 561184). Two-color flow cytometric dot plots were derived from gated events based on the light scattering characteristics of lymphocytes and fluorescence characteristics of CD4+ or CD25+, respectively, shown as either FoxP3 versus CD25 (left panel) or CD4 versus FoxP3 (right panel). Flow cytometry was performed on a BD LSRII™ Flow Cytometer System.

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Product Details

Brand: BD Pharmingen™

Alternative Name: Forkhead box protein P3; Scurfin; AIID; IPEX; JM2; DIETER; PIDX; XPID

Reactivity: Human (QC Testing)

Isotype: Mouse BALB/c IgG1, κ

Immunogen: Human full-length FoxP3 Recombinant Protein

Application: Intracellular staining (flow cytometry) (Routinely Tested)

Vol. Per Test: 5 µl

RRID: AB_10584328

Storage Buffer: Aqueous buffered solution containing BSA and ≤0.09% sodium azide.

Regulatory Status: RUO

Preparation And Storage

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. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

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

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

Cat. No. 560098 (For the buffer preparation, please see the Technical Data Sheet of Cat. No. 560098 buffer instructions for details).

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

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

4. Add 100 µl 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 to pellet the cells and remove wash buffer.

6. To fix the cells, gently resuspend the cell pellet in residual volume of wash buffer and then add 2 ml 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, resuspend each cell pellet in 2 ml of BD Pharmingen Stain Buffer (FBS)*, and centrifuge 500 x g for 5 minutes. Remove

wash buffer.

9. To permeabilize the cells, gently resuspend 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 resuspend 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 300 µl of 1% formaldehyde in 1× 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.

Product Notices

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).
Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
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.
Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
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.
Source of all serum proteins is from USDA inspected abattoirs located in the United States.

Companion Products

Stain Buffer (FBS) RUO

Size 500 ML Cat No. 554656

Human FoxP3 Buffer Set RUO

Size 100 Tests Cat No. 560098

Alexa Fluor® 647 Mouse IgG1 κ Isotype Control RUO

Size 100 Tests Cat No. 557732

PE Mouse Anti-Human CD25 RUO

Size 100 Tests Cat No. 555432

PerCP-Cy™5.5 Mouse Anti-Human CD4 RUO

Size 50 Tests Cat No. 552838

PE Mouse Anti-Human CD25 RUO (GMP)

Size 50 Tests Cat No. 341009

View Details

561184 Rev. 1

Antibody Details

236A/E7

The 236A/E7 antibody reacts with the human FoxP3 (Forkhead box protein P3) transcription factor, a member of the forkhead or winged helix family of transcription factors. The expression of FoxP3, also known as Scurfin, IPEX and JM2, has been found to be associated with CD4+ CD25+ regulatory T cells and represents a specific marker for these cells. Flow cytometric analysis has shown that FoxP3 is expressed by the majority of CD4+ CD25+ (high) T cells in peripheral blood while less than half of 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 control of T cell mediated autoimmunity by suppressing the proliferation and cytokine production of other T cells. To support this hypothesis, it has been found that Foxp3 is mutated in scurfy (sf) mice. Cumulative evidence suggests that the 236A/E7 antibody recognizes epitopes from both isoforms of Exon 2 alternatively-spliced variants and is located in the region between Exon 2 and the Zn/LZ domains (aa 105-235).

561184 Rev. 1

Format Details

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 670-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.

Format: Alexa Fluor™ 647

Excitation Max: 653 nm

Emission Max: 669 nm

561184 Rev.1

Citations & References

View product citations for antibody "561184" on CiteAb

Development References (8)

Alvaro T, Lejeune M, Salvado MT, et al. Outcome in Hodgkin's lymphoma can be predicted from the presence of accompanying cytotoxic and regulatory T cells. Clin Cancer Res. 2005; 11(4):1467-1473. (Clone-specific: Immunohistochemistry). View Reference
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
Fox BC, Bignone PA, Brown PJ, Banham AH. Defense of the clone: antibody 259D effectively labels human FOXP3 in a variety of applications. Blood. 2008; 111(7):3897-3899. (Clone-specific: Western blot). View Reference
Lennon G, Auffray C, Polymeropoulos M, Soares MB. Consortium: an integrated molecular analysis of genomes and their expression. Genomics. 1996; 33(1):151-152. (Biology). View Reference
Roncador G, Brown PJ, Maestre L, et al. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur J Immunol. 2005; 35(6):1681-1691. (Immunogen: Flow cytometry). View Reference
Roncador G, Garcia JF, Maestre L, et al. FOXP3, a selective marker for a subset of adult T-cell leukaemia/lymphoma. Leukemia. 2005; 19(12):2247-2253. (Clone-specific: Immunohistochemistry). View Reference
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
Wolf D, Wolf AM, Rumpold H, et al. The expression of the regulatory T cell-specific forkhead box transcription factor FoxP3 is associated with poor prognosis in ovarian cancer. Clin Cancer Res. 2005; 11(23):8326-8331. (Biology). View Reference

View All (8)

561184 Rev. 1

Please refer to Support Documents for Quality Certificates

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.