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Alexa Fluor® 647 Rat anti-Mouse Foxp3
Alexa Fluor® 647 Rat anti-Mouse Foxp3

Flow cytometric analysis of Alexa Fluor® 647 Rat anti-Mouse Foxp3 on mouse splenocytes. Balb/c mouse splenocytes were surface stained with FITC anti-mouse CD4 (clone RM4-5, Cat. No. 553047), then fixed and permeabilized using working solutions of Mouse Foxp3 Buffer Set (see recommended assay procedure, Cat. No. 560409) followed by intracellular staining with Alexa Fluor® 647 Rat anti-Mouse Foxp3 (0.03 µg/test). The dot plots were derived from the gated events based on light scattering characteristics of lymphocytes. Flow cytometry was performed on a BD FACSCalibur™ System.

Flow cytometric analysis of Alexa Fluor® 647 Rat anti-Mouse Foxp3 on mouse splenocytes. Balb/c mouse splenocytes were surface stained with FITC anti-mouse CD4 (clone RM4-5, Cat. No. 553047), then fixed and permeabilized using working solutions of Mouse Foxp3 Buffer Set (see recommended assay procedure, Cat. No. 560409) followed by intracellular staining with Alexa Fluor® 647 Rat anti-Mouse Foxp3 (0.03 µg/test). The dot plots were derived from the gated events based on light scattering characteristics of lymphocytes. Flow cytometry was performed on a BD FACSCalibur™ System.

Product Details
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BD Pharmingen™
Forkhead box p3; Forkhead box protein p3; JM2; Scurfin; Scurfy; Sf
Mouse (QC Testing)
Rat IgG2b
Mouse Foxp3 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_1645201
Aqueous buffered solution containing ≤0.09% sodium azide.
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 Conjugated Anti-Mouse Foxp3 Antibody

        1.   Prepare working solutions of the BD Pharmingen™ Mouse Foxp3 Buffer Set Cat. No. 560409 (for the buffer preparation, please see TDS

              Cat. No. 560409 buffer instructions for details).

        2.   Pre-warm Permeabilization buffer to 37°C before use. Keep Fixation buffer on ice.

        3.   Prepare a single-cell suspension from the peripheral lymphoid tissue of interest. Remove clumps of cells and/or debris by passing the

              suspension through a 70-µm nylon cell strainer. Use 1 × BD PharmLyse™ Lysing Buffer (Cat. No. 555899) to lyse red blood cells

              if necessary. Dilute the cells with BD Pharmingen™ Stain Buffer (FBS, Cat. No. 554656) to ten million cells/ml.

        4.   Pipette appropriate amount of CD4 or other surface staining reagent(s) to bottom of each 12 x 75 mm tube.

        5.   Add 100 µl of cells per tube, mix well. Incubate for 20 minutes at RT in the dark.

        6.   To wash cells, add 2 ml of BD Pharmingen Stain Buffer (FBS) to each tube. Centrifuge 250 x g for 10 minutes, and remove buffer.

        7.   To fix the cells, gently re-suspend pellet in residual volume of staining buffer and then add 2 ml of freshly prepared cold 1 x BD

              Pharmingen™ Mouse Foxp3 Fixation Buffer. Mix well. Incubate for 30 minutes at 4°C in the dark.

        8.   Centrifuge 500 x g for 5 minutes, and remove fixative.

        9.   To wash cells, re-suspend each pellet in 2 ml of freshly prepared pre-warmed 1 × BD Pharmingen Mouse Foxp3 Permeabilization

               buffer,  and centrifuge 500 x g for 5 minutes. Remove permeabilization buffer.

        10. To permeabilize the cells, gently re-suspend pellet in another 2 ml of freshly prepared pre-warmed 1 x BD Pharmingen Mouse Foxp3

              Permeabilization buffer. Incubate for 30 minutes at 37°C in the dark.

        11. Centrifuge 500 x g for 5 minutes, and remove buffer.

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

        13. Add 20 µl of conjugated Foxp3 antibody diluted with BD Pharmingen Stain Buffer (FBS) at appropriate concentrations (check the  

              figure legend from each format for the concentration) to re-suspend the pellet. Gently shake or vortex briefly.

        14. Incubate for 20 minutes at RT in the dark.

        15. Repeat wash step #12 two times.

        16. Resuspend the cells in 0.5 ml BD Pharmingen Stain Buffer (FBS) and analyze immediately.*

Note: We recommend using the BD Pharmingen™ Stain Buffer 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 cells, etc. before acquisition.

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

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  4. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  5. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  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. 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.
560401 Rev. 3
Antibody Details
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MF23

The MF23 monoclonal antibody specifically binds to mouse Foxp3. Foxp3 is a 50-55 kDa protein also known as Forkhead box p3, JM2, or IPEX. It is a member of the forkhead or winged helix family of transcription factors and is specifically expressed by T regulatory (Treg) cells. Foxp3 has been reported to be a key regulatory protein for Treg cell development and function. Ectopic expression of Foxp3 in conventional T cells is sufficient to induce suppressive activity, repress the production of cytokines such as IL2 and IFN-γ, and upregulate Treg cell-associated molecules such as CD25, CTLA4 and GITR. It has been found that the mutation of Foxp3 is responsible for "scurfy" mice. When overexpressed, Foxp3 leads to poor T cell proliferation and activation. Immunoblotting with MF23 antibody has confirmed it recognizes an epitope between 1-87 amino acids in the N-terminal domain.

560401 Rev. 3
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
560401 Rev.3
Citations & References
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Development References (3)

  1. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science. 2003; 299(5609):1057-1061. (Biology). View Reference
  2. Ono M, Yaguchi H, Ohkura N, et al. Foxp3 controls regulatory T-cell function by interacting with AML1/Runx1. Nature. 2007; 446(7136):685-689. (Biology). View Reference
  3. Zheng Y, Rudensky AY. Foxp3 in control of the regulatory T cell lineage. Nat Immunol. 2007; 8:457-462. (Biology). View Reference
560401 Rev. 3

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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.