CD4+ T cell subsets exert diverse functions due to their expression of characteristic cytokines. Regulatory T (Treg) cells constitute one subset of CD4+ effector T cells that limit the development and progression of immune responses, oppose some functions of Th17 cells, and suppress the development of autoimmunity. Treg cells are characterized by the secretion of IL-9, IL-10, IL-35, and TGF-beta 1.
The development of the Treg subset of CD4+ T cells is induced by distinct extracellular signals (e.g. IL-2 in combination with TGF-beta) and is controlled by distinct transcription factors (e.g. FoxP3). Treg cells are present in low abundance in normal human peripheral blood. In vitro differentiation of Treg cells from the larger CD4+ T cell population provides increased numbers of Treg cells to facilitate research into their functions.
|Products for the Differentiation of Regulatory T Cells
T Cell Subsets Poster
CD4+ T Cell Subsets Brochure
Regulatory T Cells Brochure
Following cell differentiation, flow cytometry can be used to verify the expression of established cell surface or intracellular markers of CD4+ T cell subsets. Intracellular markers include transcription factors that control CD4+ T cell differentiation as well as signature cytokines as they traffic through secretory organelles.
Determine the percentage of differentiated cells by flow cytometry using the suggested reagents. To prepare cells for flow cytometry, wash the cells once with RPMI and resuspend them in 1 mL of RPMI, 2 mM L-glutamine, 10 units/mL penicillin, 10 µg/mL streptomycin, 10% FBS, 50 ng/mL PMA, and 1 µg/mL calcium ionomycin. Incubate the cells in a 37 °C, 5% CO2, humidified incubator for 1 hour. Then add monesin to a final concentration of 3 µM and incubate for an additional 3 hours.
For protocols to stain membrane-associated molecules or intracellular molecules (using either detergents or alcohol to permeabilize the cell membrane), please visit Flow Cytometry Protocols on our website.
Transcription Factor Antibodies
Cell Surface Protein Antibodies
Flow Cytometry Supplemental Reagents
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|Detection of FoxP3 in Human Natural Tregs by Flow Cytometry..Natural Tregs from human peripheral blood mononuclear cells (PBMC) were surface stained with (A) Fluorescein-conjugated Mouse Anti-Human CD4 Monoclonal Antibody (Catalog # FAB3791F) and (B) APC-conjugated Mouse Anti-Human CD25/IL-2 R alpha Monoclonal Antibody (Catalog # FAB1020A) followed by intracellular staining using PE-conjugated Rabbit Anti-Human/ Mouse FoxP3 Monoclonal Antibody (Catalog # IC8214P). To facilitate intracellular staining, cells were fixed and permeabilized with FlowX FoxP3 Fixation & Permeabilization Buffer Kit (Catalog # FC012).Cells were gated on lymphocytes.
The culture medium from CD4+ T cell differentiation procedures should be analyzed to confirm that the cells are secreting cytokines relevant to the desired cell subset. Multiplex detection techniques enable efficient screening for many cytokines simultaneously. Both Proteome Profiler™ Antibody Arrays and Luminex®-based Flow Cytometry Assays are optimized for maximum specificity and sensitivity of analyte detection.
Proteome Profiler™ Antibody Arrays allow for the measurement of up to 119 proteins in a single sample. These arrays require no specialized equipment and eliminate the need for multiple Western blot experiments. Antibody array kits contain buffers, detection antibodies, and membranes spotted in duplicate with high quality capture antibodies. The arrays utilize chemiluminescence for detection, and membranes can be assessed for protein levels in the same manner as traditional Western blots. Select arrays are also suitable for use with the LI-COR® detection system. Please see our website for a generalized protocol or video on how to use these antibody arrays. Detailed array-specific instructions are provided in the booklet for each array kit.
Luminex Screening and Performance Assays utilize color-coded polystyrene or superparamagnetic beads coated with analyte-specific antibodies. Beads recognizing different target analytes are mixed together and incubated with the sample. Captured analytes are subsequently detected using a cocktail of biotinylated detection antibodies and a streptavidin-phycoerythrin conjugate.
The Luminex Assay Customization Tool is designed to help you build the right Luminex assay for your research.
Polystyrene beads are designed for use with the Luminex 100™, Luminex 200, or Bio-Rad® Bio-Plex® dual-laser analyzers. One laser in the instrument determines the color of each bead while the second laser determines the magnitude of the PE-derived signal, which is directly proportional to the amount of analyte bound.
Magnetic beads are compatible with Luminex MAGPIX®, Luminex 100™, Luminex 200, and Bio-Rad® Bio-Plex® analyzers. The Luminex MAGPIX® analyzer utilizes a magnet to hold the superparamagnetic microparticles in a monolayer while light emitting diodes illuminate and a CCD camera images each well.
Luminex Screening Assays from R&D Systems are designed to maximize multiplexing capacity and flexibility while maintaining assay specificity.
Luminex Performance Assays from R&D Systems are designed to maximize assay accuracy and precision while preserving the benefits of multiplexing.
Data from the measurement of 158 analytes secreted by unstimulated and stimulated NK cells.
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|Figure 1. A) Heat map showing the tested biomarkers. Colors correspond to high, mid, and low abundance. B) Quantitative comparison of biomarker concentrations between stimulated and unstimulated NK cells.
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|Figure 2. A) Heat map showing the tested biomarkers. Colors correspond to high, mid, and low abundance. B) Quantitative comparison of biomarker concentrations between stimulated and unstimulated NK cells. *The increased IL-12 levels in stimulated cells was due to addition of IL-12 during the stimulation procedure.
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|Figure 3. A) Heat map showing the tested biomarkers. Colors correspond to high, mid, and low abundance. B) Quantitative comparison of biomarker concentrations between stimulated and unstimulated NK cells.
Luminex Screening and Performance Assays Specialty Catalog
R&D Systems offers a wide variety of sandwich ELISA kits that are designed to provide high levels of specificity, accuracy, precision, and sensitivity in analyte quantification. They are available in a range of formats including colorimetric, fluorescence, and chemiluminescence-based kits for measuring intracellular and extracellular proteins.
Quantikine® ELISA kits have been exhaustively tested for superior quality and reproducibility. Kit performance relies heavily on the selection of high quality antibody pairs and rigorous in-house testing throughout the development process. This includes component and kit stability, sensitivity, linearity, recovery, intra- and inter-assay precision, as well as cross-reactivity and interference testing with related an¬alytes to confirm assay specificity. This stringent validation testing is used to optimize assay per¬formance and verify that each kit will provide reproducible results both well-to-well and lot-to-lot.
DuoSet® ELISA Development Systems contain the basic components required to develop an immunoassay. They offer an economical alternative to buying separate antibodies and proteins.
|ELISA Immunoassays for Regulatory T Cells
ELISA Development Troubleshooting Guide