Protocol for the Differentiation and Characterization of Human Th17 Cells

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 shown in the table below. 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 monensin 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.

Sample Data for Th17 cells

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Figure 1. Intracellular Cytokine Staining of Differentiated Human Th17.Flow cytometry data showing human peripheral blood naïve CD4+ T cells without (A, C) and with (B, D) a 10 day differentiation using reagents included in the Human Th17 Cell Differentiation Kit (Catalog # CDK003). On day 10 of differentiation, the cells were re-stimulated with mitogens and stained with APC-conjugated Mouse Anti-Human IL-17 Monoclonal Antibody (Catalog # IC317A), PE-conjugated Mouse Anti-Human IFN-gamma Monoclonal Antibody (Catalog # IC285P) and a Fluorescein-conjugated Mouse Anti-Human IL-4 Monoclonal Antibody (Catalog # IC204F). Quadrants were placed based on isotype control-stained samples.

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.

Click here for data examples of the analysis of secreted human, mouse, or rat cytokines.

Proteome Profiler Antibody Arrays for Cytokine Detection

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.

• Largest Luminex Multiplex Available: simultaneously analyze up to 100 analytes
• Flexible Analyte Selection: choose from over 175 analytes
• Unique Analytes Offered: over 50 analytes are exclusively available from R&D Systems
• Rapidly Expanding Menu: new analytes are released monthly
• Polystyrene or Magnetic Options: all analytes are available in either the polystyrene or magnetic microparticle format

Luminex Performance Assays from R&D Systems are designed to maximize assay accuracy and precision while preserving the benefits of multiplexing.

• Accurate and Reproducible Results: panel development and validation testing are similar to R&D Systems gold-standard Quantikine^® ELISA assays
• Polystyrene or Magnetic Options: select panels are available in either the polystyrene or magnetic microparticle format
• User-Defined Analyte Selection: choose analytes from established panels and select “premixed” or “end-user mixed” options
• User-selected combinations of analytes can be assembled with our Luminex Assay Ordering Tool. The ordering tool walks you step-by-step through choices of screening or performance assays, polystyrene or magnetic bead formats, species options, and target analytes of interest.
• Our list of analytes for Screening Assays numbers more than 200 and is continuously growing. Please see our Luminex Assay Customization Tool for the current selection.

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.

Figure 2

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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.

Figure 3

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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 Performance Assay Panels

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 analytes to confirm assay specificity. This stringent validation testing is used to optimize assay performance 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.