May 1, 2014 (Vol. 34, No. 9)

Flow Cytometry, Cell Activation, and Immunoregulatory Networks

Tools for understanding the development, phenotype, and function of specific T cell subsets are critical to the modern immunologist. Tonbo Biosciences provides a rapidly expanding portfolio of antibodies for flow cytometry (FCx-Pro™), antibodies for functional assays (In Vivo Ready™), and recombinant proteins for cell activation (RPx-Pro™). As described below, these products are provided as flexible phenotyping or activation panels for pursuing research focused on CD4+ T cell subsets and their immune regulatory pathways controlled by cytokines, chemokines or other growth factors.

Ready-Set Flow™ Panels for Th Cell Subset Phenotyping by Flow Cytometry

The simultaneous technical advances of flow cytometers with multiple lasers along with development of new dyes has led to routine utilization of multicolor panels of six or more antibody conjugates for cell analysis. A prime example is the ever expanding characterization and definition of specific CD4+ T helper (Th) cell subsets achieved through phenotyping and functional analysis with a set or panel of specificities. Delineation of a set of markers characteristic to a particular CD4+Th subset has resulted in the wide adoption of a consensus signature used to profile these cell populations.

CD4+ T cell subsets recognized through a specific phenotypic profile are also defined by specialized roles in shaping and regulating immune responses. Regulatory T cells, or Tregs, modulate and control other T cells by maintaining self-tolerance and also curtailing normal immune responses. A typical Treg phenotype profile would be CD4+CD25+FoxP3+CD127lo. Other Treg associated markers include GITR and TIGIT that are expressed on subsets of Treg cells. Current knowledge indicates a critical role for another CD4+ T cell subset termed Th17. Th17 cells function normally to provide anti-microbial immunity at epithelial/mucosal surfaces but also play a significant role in the inflammatory mediated pathogenesis of autoimmune diseases such as rheumatoid arthritis. In addition to secreting IL-17, these cells are often identified as CD4+RORγ(t)+ cells. Example panels are shown in Table 1A.

C57/Bl6 splenocytes were stained with CD3 FITC, CD4 violetFluor™ 450, CD25 APC, CD127 PerCPCy5.5, and Foxp3 PE. Left Panel: Expression of CD25 and CD127 is shown on CD3+CD4+ cells with gates drawn on CD25+CD127lo cells (blue) and CD25CD127high cells (red). Right Panel: Expression of Foxp3 in CD25+CD127lo cells (blue histogram) and CD25CD127high cells (red histogram) is shown. Isotype control staining in both populations is shown as the dashed histograms.

Along with experienced guidance, Tonbo Biosciences provides a variety of sizes and fluorochromes for dozens of key specific markers used for Th subset profiling. The flexibility to choose fluorochromes from violet to dark red emission ranges for myriad panels enables creation of unique panels, extension of standard panels, or facilitated integration into existing panels.

Recombinant Cytokine, Growth Factor and Antibody Activation Sets and Panels Driving CD4+ Th Cell Subset Differentiation

Advances enabled by T cell subset phenotyping using flow cytometry have been matched equally with the ability to instruct the differentiation of CD4+ T cell subsets using defined reagent sets or panels comprised of growth factors, cytokines, or functional antibodies. Knowledge of the composition and overall ratio of growth factors, cytokines, and chemokines in the cellular milieu, sometimes termed a T cell polarizing environment, serve to mimic the in vivo conditions in normal or disease sites.

Tonbo Biosciences provides a choice of In Vivo Ready (IVR) antibodies that are azide free and low endotoxin formats used to promote the activation and/or differentiation of T cells. Primary among these antibodies are CD3 and CD28, which drive polyclonal T cell proliferation by initiating TCR signaling events. Instruction of T cell differentiation to a distinct CD4+T cell subset is accomplished through the addition of blocking In Vivo Ready antibodies and recombinant proteins such as anti-IFNγ, anti-IL-12, rIL-2 and rIL-4 for Th2 cell expansion. RPx-Pro Recombinant Proteins and In Vivo Ready antibodies from Tonbo Biosciences offer flexibility and value for development of key polarizing and differentiation panels. Example panels are shown in Table 1B.

Table 1. Phenotyping and Activation Panel Examples

Equivalent Performance, Exceptional Value Enabling Development & Discovery

The need to remain on the cutting edge of T cell research requires optimal use of all the tools currently available, which often translates into use of more reagents per experiment. With an eye toward the budget constricted world of basic or applied research, Tonbo Biosciences guarantees all of our reagents in the product areas listed below perform in accordance with market standards and provide exceptional value—we offer the lowest prices on the market. Our product lines include:

  • FCx-Pro antibodies for flow cytometry
  • In Vivo Ready antibodies for functional assays
  • RPx-Pro recombinant proteins for cell activation

Enabling Portfolio for Th Cell Subsets and Immunoregulatory Networks

Filling the gap between required reagent panels and budget limitations is just one of the solutions Tonbo Biosciences has provided. The ability to design a specific phenotyping or activation panel allows the freedom to customize the experimental model enabling new discovery. Only Tonbo Biosciences offers the combination of flexibility, assured performance, and low pricing that bolsters our logo and tagline: Equivalent Performance, Exceptional Value

Taken together, phenotyping and activation panels from Tonbo Biosciences act as tool sets for researchers studying both development and function of Th subsets and their roles in immune responses. With a broader knowledge base comes the broader opportunity to discover new pathways, develop new immunoregulating drugs, or define new diagnostic markers.

Tonbo Biosciences

Jonathan Rosenberg
VP, Marketing
[email protected]

1. Campbell, DJ and MA Koch 2011 Nat. Rev. mmunol. 11: 119
2. Russ, BE, JE Prier, S Rao and SJ Turner 2013 Front. Genet. 4: 2018
3. Veldhoen, M, RJ Hocking, CJ Atkins, RM Locksley and B Stockinger 2006 Immunity 24: 179
4. Zhu, J and WE Paul 2008 Blood 112: 1557
5. Zhu, J, H Yamane and WE Paul 2010 Cell Res. 20:4

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