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Multicolor flow cytometric analysis of Foxp3 expression in mouse splenocytes. Balb/c splenic leucocytes were fixed and permeabilized using the Transcription Factor Buffer Set (Cat. No. 562574/562725). The cells were then stained with BD Horizon™ BV421 Rat Anti-Mouse CD4 (Cat. No. 740007) and either Alexa Fluor® 488 Rat IgG2a, κ Isotype Control (Cat. No. 557676; Left Plot) or Alexa Fluor® 488 Rat Anti-Mouse Foxp3 (Cat. No. 563487) antibodies at 0.5 µg/test. The bivariate pseudocolor density plot shows 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™ LSR II Flow Cytometer System and FlowJo™ software.

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 Alexa Fluor® 647 Rat Anti-Mouse CD4 (Cat. No. 557681) and Alexa Fluor® 488 Rat Anti-Mouse Foxp3 (Cat. No. 563487) 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 cytometry was performed using a BD™ LSR II Flow Cytometer System.
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BD Pharmingen™ Alexa Fluor® 488 Rat Anti-Mouse Foxp3

BD Pharmingen™ Alexa Fluor® 488 Rat Anti-Mouse Foxp3
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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.
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- 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.
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Companion Products



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.
Development References (5)
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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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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
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