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Foxp3 Antibodies Optimized for Flow Cytometry, IHC, WB For Mouse, Human, Rat, Non-Human Primates & Canine
Tregs & Foxp3: the Suppression Phenomenon
Until recently, definitive flow cytometric analysis of CD4+CD25+ regulatory T cells, expressing Foxp3, has been hindered due to lack of suitable Foxp3 antibodies. Using eBioscience Foxp3 antibodies PCH101, FJK-16s and NRRF-30, Foxp3 can now be identified at the single-cell level (see figures and table below). Mouse Foxp3
Immunological Identity, Specificity and Validation of Foxp3 AntibodiesAs has been done with many cell surface and intracellular (e.g., cytokine) proteins, corroboration of staining pattern using antibodies to distinct epitopes from the same protein allows researchers to confirm staining patterns and hence specificity. To this end, eBioscience utilized protein deletion constructs to epitope-map a panel of Foxp3 antibodies. The Figure below shows the schematic representation of the Foxp3 protein with functional domains and also localizes the regions recognized by each of eBioscience's Foxp3 antibodies.
In the figures below, co-staining experiments indicate that several Foxp3 antibodies stain the same cells. The anti-mouse Foxp3 antibodies, FJK-16s and NRRF-30 recognize different epitopes in the amino terminus, with the former recognizing the region corresponding to the human splice variant. These two antibodies identify the same population of lymphocytes, and co-staining experiments demonstrate 100% correlation (see figure below). These data suggest that these two antibodies, which recognize different regions of recombinant Foxp3, bind the same protein expressed in the same cells. Additionally, the same approach was taken for the human Foxp3 antibodies. |
Co-staining of eBioscience foxp3 antibodies that recognize different epitopes. Left: Mouse splenocytes were co-stained with FJK-16s and eBio7979. Right: NRRF-30 co-staining with FJK-16s. |
Existence of splice variant in human, not mouse: immunoblotting of PBMC (Lane A), BALB/c splenocyte (Lane B), or recombinant human Foxp3 fusion protein (Lane C); probed with eBio7979 (cat. 14-7979). |
Co-staining of eBioscience Foxp3 antibodies that recognize different epitopes. Left: Human PBMCs were co-stained with PCH101 and 236A/E7. Right: Co-staining of PCH101 and eBio7979. |
The expanding panel of antibodies reactive with distinct epitopes of Foxp3 is useful for investigating the complete Foxp3 protein expression profile at the single-cell level in both human and mouse, particularly with regards to its relationship to functionally-defined Regulatory T cells. In addition, the presence of an alternatively spliced transcript in human adds another layer of complexity to the Foxp3 expression pattern and functional significance. Currently this phenomenon appears to be unique to human since both co-staining at the single-cell level using several different combinations of antibodies to different domains and immunoblotting data suggests that only one isoform exists in the mouse. (See flow analysis and immunoblot data above.) In the human, two isoforms can be present; the anti-mouse/rat Foxp3 antibody, FJK-16s, crossreacts to human Foxp3 but recognizes the region that can be alternatively spliced in some transcripts. Therefore, the FJK-16s antibody labels the Foxp3 protein isoform that has not been spliced, while PCH101 and 236A/E7 antibodies recognize both isoforms of the protein. Using these reagents together will allow researchers to investigate the function of these two isoforms of human Foxp3 protein in the same cell population.
The Foxp3 antibodies have been validated for intracellular staining with flow cytometric analysis. The protocols and staining buffers have been developed for optimal and consistent staining Foxp3 staining while preserving surface antigen staining. Furthermore, the protocol was designed for robustness, reproducibility, ease-of-use, as well as flexibility, to allow researchers to stain at their convenience. In addition, the antibodies have been reported useful for immunohistological staining (both frozen and paraffin sections) and western blotting of endogenous protein from total human PBMCs or mouse splenocytes, without the need for enrichment of Tregs.
C57BL6 spleen cryosections were stained with anti-mouse/rat Foxp3 antibody, FJK-16s (cat. 14-5773) and revealed with colorimetric methods. Image courtesy of Cintia De Paiva. |
Staining of human tonsillar tissue section with PCH101 (cat. 14-4776). Image courtesy of Roger Sutmuller.
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The specificity of all Foxp3 monoclonal antibodies has been confirmed by numerous publications. (see reference list below) A recent manuscript by Tran et al (Blood 2007) suggests that monoclonal antibiody PCH101 is not specific on activated/cultured Tregs. Leading scientists in the Regulatory T Cell field have shown PCH101 is an excellent antibody for examining Foxp3 expression in resting and cultured cells. The data in the Tran paper suggested that PCH101 staining is found on all cells when cultured with TGF-β or with neutralizing antibodies to TGF-β. The cells cultured with TGF-β clearly show 2 distinct populations albeit shifted higher off the axis. It is the opinion of eBioscience that the 2 populations consist of a positive Foxp3 population and a negative population. This interpretation would then be consistent with all the subsequent findings (RT-PCR and siRNA). The siRNA can only knock down the brightest staining population but not the dim population, since the dim population is in fact negative. The shift/increase in background staining is not seen in other publications nor in any eBioscience data comparing PCH101 to any other Foxp3 antibody. The cause for the shift seen by Tran et al is not known.
Several articles recently published utilizing PCH101 in cultured/activated Tregs and confirming specificity:
Ahmadzadeh et al J Immunother 2007;30:294–302
Pillai et al Clin Immunol. 2007 Apr;123(1):18-29
Wang et al Eur. J. Immunol. 2007. 37: 129–138
In particular the Wang paper shows costaining of 236A and PCH101 suggesting the antibodies, that are known to recognize different epitopes, see the same cell population. This is additional evidence confirming specificity of PCH101 for Foxp3. Additionally please see the following link for a letter from the authors with additional supporting data.
The transient expression pattern of Foxp3 in in vitro cultured cells shown by Pillai et al further supports the specificity of PCH101 for Foxp3. The figure below illustrates a distinct population of Foxp3 negative and positive population an be identified in activated Treg cultures.
CD4+CD25- T-cells upregulate FOXP3 transiently following activation. Bead sorted ‘untouched’ CD3+CD25- T-cells or CD4+CD25- T-cells were stained with CFSE and activated with either allogeneic T-cell-depleted PBMC. CD25 and FOXP3 expression and CFSE dilution were monitored over time. Color-coded contour plots of gated CD4+ T-cells from allostimulated cultures are shown, with CFSE on the x-axis and CD25 (top row) or FOXP3 (bottom row) on the y-axis. The vertical lines separate dividing (CFSElow) from non-dividing (CFSEhigh) cells. Courtesy of Pillai V, Ortega SB, Wang CK, Karandikar NJ. University of Texas Southwestern Medical Center
The anti-human Foxp3 antibodies, PCH101 and 236A/E7, crossreact to several non-human primate species in intracellular staining studies. Additionally, antibodies to CD4 (OKT4) and CD25 (BC96) also stain rhesus and cynomolgus lymphocytes, making characterization of natural Tregs in non-human primates more comprehensive.
It has been confirmed that FJK-16s crossreacts with canine Foxp3 protein (Biller BJ, Elmslie RE, Burnett RC, Avery AC, Dow SW. Use of FoxP3 expression to identify regulatory T cells in healthy dogs and dogs with cancer. Vet Immunol Immunopathol. 2007 Mar 15;116(1-2):69-78, pubmed).
As researchers learn more about the importance of Foxp3 as a master control of immune regulation, the commitment of eBioscience to providing critically-important, well-validated reagents becomes clear. The Foxp3 antibodies are available in a variety of fluorochromes (including tandem dyes and Alexa Fluors®*) that is constantly expanding to meet your needs. In combination with our vast offering of fluorochrome-conjugated antibodies reactive with cell surface antigens, Foxp3 staining can be analyzed against more than 6 colors allowing for the intricate analysis of cell populations and subsets of populations.
Foxp3 Reagents | ||||
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Description | Clone | Application | Format | Catalog No. |
Human Foxp3 | PCH101 | Flow (IC), WB, IH (paraffin & frozen) | Purified | 14-4776 |
(reacts w/ Monkey) | Flow (IC), WB, IH (paraffin & frozen) | Biotin | 13-4776 | |
Flow (IC), IH (paraffin & frozen) | FITC | 11-4776 | ||
Flow (IC) | FITC Staining Set | 71-5776 | ||
Flow (IC) | PE | 12-4776 | ||
Flow (IC) | PE Staining Set | 72-5776 | ||
Flow (IC) | PE-Cy5 | 15-4776 | ||
Flow (IC), IH (paraffin & frozen) | APC | 17-4776 | ||
Flow (IC) | APC Staining Set | 77-5776 | ||
Flow (IC), IH (paraffin & frozen) | Alexa Fluor® 488 | 53-4776 | ||
Flow (IC) | Alexa Fluor® 488 Staining Set | 73-5776 | ||
Flow (IC) | Alexa Fluor® 647 | 51-4776 | ||
Flow (IC) | Alexa Fluor® 700 | 56-4776 | ||
Flow (IC) | eFluor® 450 | 48-4776 | ||
Flow (IC) | Staining Buffer Set | 00-5523 | ||
Flow (IC) | Treg Staining Kit (w/ PE Foxp3 PCH101) |
88-8999 | ||
Flow (IC) | Treg Staining Kit #2 (w/ APC Foxp3 PCH101) |
88-8998 | ||
Flow (IC) | Treg Staining Kit #3 (w/ PE-Cy5 Foxp3 PCH101) |
88-8995 | ||
Flow (IC) | Human/NHP Treg Staining Kit (w/ PE Foxp3 PCH101) |
88-4999 | ||
Flow (IC) | Human/NHP Treg Staining Kit #2 (w/ APC Foxp3 PCH101) |
88-4994 | ||
NEW! | Flow (IC) | Human Foxp3 Whole Blood Staining Kit | 88-8996 | |
Human Foxp3 | 236A/E7 | Flow (IC), IHC (paraffin & frozen), WB | Purified | 14-4777 |
(reacts w/ Monkey) | Flow (IC), IH (paraffin & frozen) | Biotin | 13-4777 | |
Flow (IC) | FITC | 11-4777 | ||
Flow (IC) | PE | 12-4777 | ||
Flow (IC) | PE Staining Set | 72-5774 | ||
Flow (IC) | APC | 17-4777 | ||
Flow (IC) | APC Staining Set | 77-5774 | ||
Flow (IC), IH (paraffin & frozen) | Alexa Fluor® 488 | 53-4777 | ||
Flow (IC) | Alexa Fluor® 488 Staining Set | 73-5774 | ||
Flow (IC) | Alexa Fluor® 647 | 51-4777 | ||
Flow (IC) | eFluor® 450 | 57-4777 | ||
Flow (IC) | Staining Buffer Set | 00-5523 | ||
Human/Mouse Foxp3 | eBio7979 | WB, IH (paraffin) | Purified | 14-7979 |
Flow (IC) | PE | 12-7979 | ||
Flow (IC) | Alexa Fluor® 488 | 53-7979 | ||
Flow (IC) | Alexa Fluor® 647 | 51-7979 | ||
Flow (IC) | Staining Buffer Set | 00-5523 | ||
Mouse/Rat Foxp3 | FJK-16s | Flow (IC), WB, IH (frozen) | Purified | 14-5773 |
Flow (IC), WB, IH (frozen) | Biotin | 13-5773 | ||
Flow (IC) | FITC | 11-5773 | ||
Flow (IC) | FITC Staining Set | 71-5775 | ||
Flow (IC) | PE | 12-5773 | ||
Flow (IC) | PE Staining Set | 72-5775 | ||
Flow (IC) | PE-Cy5 | 15-5773 | ||
Flow (IC), IH (frozen) | APC | 17-5773 | ||
Flow (IC) | APC Staining Set | 77-5775 | ||
Flow (IC), IH (frozen) | Alexa Fluor® 488 | 53-5773 | ||
Flow (IC) | Alexa Fluor® 647 | 51-5773 | ||
Flow (IC) | Alexa Fluor® 700 | 56-5773 | ||
Flow (IC) | eFluor® 450 | 57-5773 | ||
Flow (IC) | Staining Buffer Set | 00-5523 | ||
Flow (IC) | Treg Staining Kit (w/ PE Foxp3 FJK-16s) |
88-8111 | ||
Flow (IC) | Treg Staining Kit #2 (w/ APC Foxp3 FJK-16s) |
88-8118 | ||
Flow (IC) | Treg Staining Kit #3 (w/ PE-Cy5 Foxp3 FJK-16s) |
88-8115 | ||
Mouse Foxp3 | NRRF-30 | WB, IH (frozen) | Purified | 14-4771 |
Flow (IC) | PE | 12-4771 | ||
Flow (IC) | Staining Buffer Set | 00-5523 | ||
Mouse/Human Rat Foxp3 | 150D/E4 | Flow (IC), WB, IH | Purified | 14-4774 |
Flow (IC) | PE | 12-4774 | ||
Flow (IC) | Alexa Fluor® 488 | 53-4774 | ||
Flow (IC) | Alexa Fluor® 647 | 51-4774 |
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