Two-color flow cytometric analysis of Foxp3 expression in mouse splenocytes. Mouse splenic leucocytes were fixed and permeabilized using appropriately-diluted solutions from the Transcription Factor Buffer Set (Cat. No. 562574/562725). The cells were then stained with FITC Rat Anti-Mouse CD4 (Cat. No. 553047/553046/561835) and Alexa Fluor® 647 Rat Anti-Mouse Foxp3 (Cat. No. 563486) antibodies. The two-color flow cytometric dot plot shows the correlated expression patterns of CD4 versus Foxp3 for gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Multicolor flow cytometric analysis of Foxp3 expression in mouse splenic lymphocytes. BALB/c mouse splenic leucocytes were fixed and permeabilized using Transcription Factor Buffer Set (Cat. No. 562574/562725). The cells were then stained with PE Rat Anti-Mouse CD4 (Cat. No. 553048/553049/561837) either Alexa Fluor™ 647 Rat IgG2a, κ Isotype Control (Cat. No. 557690; Left Plot) or Alexa Fluor™ 647 Rat Anti-Mouse Foxp3 antibody (Cat. No. 563486; Right Plot) at 0.06 µg/test. The bivariate pseudocolor density plots show the correlated expression patterns of CD4 versus Foxp3 (or Ig Isotype control staining) for gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis was performed using a BD LSRFortessa™ Cell Analyzer System and FloJo™ software. Data on this Technical Data Sheet are not lot-specific.
Two-color flow cytometric analysis of Foxp3 expression in mouse splenocytes. Mouse splenic leucocytes were fixed and permeabilized using appropriately-diluted solutions from the Transcription Factor Buffer Set (Cat. No. 562574/562725). The cells were then stained with FITC Rat Anti-Mouse CD4 (Cat. No. 553047/553046/561835) and Alexa Fluor® 647 Rat Anti-Mouse Foxp3 (Cat. No. 563486) antibodies. The two-color flow cytometric dot plot shows the correlated expression patterns of CD4 versus Foxp3 for gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Multicolor flow cytometric analysis of Foxp3 expression in mouse splenic lymphocytes. BALB/c mouse splenic leucocytes were fixed and permeabilized using Transcription Factor Buffer Set (Cat. No. 562574/562725). The cells were then stained with PE Rat Anti-Mouse CD4 (Cat. No. 553048/553049/561837) either Alexa Fluor™ 647 Rat IgG2a, κ Isotype Control (Cat. No. 557690; Left Plot) or Alexa Fluor™ 647 Rat Anti-Mouse Foxp3 antibody (Cat. No. 563486; Right Plot) at 0.06 µg/test. The bivariate pseudocolor density plots show the correlated expression patterns of CD4 versus Foxp3 (or Ig Isotype control staining) for gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis was performed using a BD LSRFortessa™ Cell Analyzer System and FloJo™ software. Data on this Technical Data Sheet are not lot-specific.
Product Details
BD Pharmingen™
Forkhead box P3; IPEX; Forkhead box protein P3; JM2; Scurfin; Scurfy; Sf
Mouse (QC Testing)
Rat IgG2a, κ
Foxp3 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_2738235
Aqueous buffered solution containing ≤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 the dye under optimum conditions and unreacted dye was removed.
Recommended Assay Procedures
BD® CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- This product is provided under an intellectual property license between Life Technologies Corporation and BD Businesses. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for any other use, contact Life Technologies Corporation, Cell Analysis Business Unit Business Development, 29851 Willow Creek Road, Eugene, OR 97402, USA, Tel: (541) 465-8300. Fax: (541) 335-0504.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Alexa Fluor™ is a trademark of Life Technologies Corporation.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Lysing Buffer RUO
Size
100 mL
Cat No.
555899
Transcription Factor Buffer Set RUO
Size
100 Tests
Cat No.
562574
Transcription Factor Buffer Set RUO
Size
25 Tests
Cat No.
562725
563486 Rev. 2
Antibody Details
R16-715
The R16-715 monoclonal antibody specifically binds to mouse Foxp3. Foxp3 is a 50-55 kDa protein also known as Forkhead box P3, JM2, IPEX, Scurfin, and Sf. It is a member of the forkhead or winged helix family of transcription factors and is specifically expressed by T regulatory (Treg) cells. Foxp3 is 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.
563486 Rev. 2
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 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
563486 Rev.2
Citations & References
View product citations for antibody "563486" on CiteAb
Development References (8)
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Angiari S, Runtsch MC, Sutton CE, et al. Pharmacological Activation of Pyruvate Kinase M2 Inhibits CD4 + T Cell Pathogenicity and Suppresses Autoimmunity. Cell Metab. 2020 ; 31(2):391-405. (Clone-specific: Flow cytometry). View Reference
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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
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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
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Huang L, Li Y, Du Y, et al. Mild photothermal therapy potentiates anti-PD-L1 treatment for immunologically cold tumors via an all-in-one and all-in-control strategy. Nat Commun. 2019; 10(1):4871. (Clone-specific: Flow cytometry). View Reference
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Jianqin Lu, Xiangsheng Liu, Yu-Pei Liao, et al. Nano-enabled pancreas cancer immunotherapy using immunogenic cell death and reversing immunosuppression. Nat Commun. 2017; 8(1):1811. (Clone-specific: Flow cytometry). View Reference
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Jinushi M, Sato M, Kanamoto A, et al. Milk fat globule epidermal growth factor-8 blockade triggers tumor destruction through coordinated cell-autonomous and immune-mediated mechanisms. J Exp Med. 2009; 206(6):1317-1326. (Biology). View Reference
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Vasconcellos R, Carter NA, Rosser EC, Mauri C. IL-12p35 subunit contributes to autoimmunity by limiting IL-27-driven regulatory responses. J Immunol. 2011; 187(6):3402-3412. (Biology). View Reference
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Zheng Y, Rudensky AY. Foxp3 in control of the regulatory T cell lineage. Nat Immunol. 2007; 8:457-462. (Biology). View Reference
563486 Rev. 2
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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.