Protocol for the Differentiation and Characterization of Human Th17 Cells
T helper type 17 (Th17) cells are a subset of CD4+ effector T cells that promote cell-mediated immune responses against extracellular bacteria and fungi. Th17 cells secrete TNF-alpha, IL-6, IL-9, IL-17A, IL-17F, IL-21, IL-22, and (human) IL-26/AK155.
Differentiation into the Th17 lineage is promoted by cytokines such as TGF-beta and IL-6, while their survival and expansion are dependent on IL-21 and IL-23. Their development is controlled by the transcription factor RORgammaT. Th17 polarized cells are present in low abundance in normal human peripheral blood. In vitro differentiation of Th17 cells from the larger CD4+ T cell population provides increased numbers of Th17 cells to facilitate research into their functions.
The CellXVivo™ Human Th17 Cell Differentiation Kit (Catalog # CDK003) contains R&D Systems high quality cytokines needed for the differentiation of Th17 cells. Alternatively, individual cytokines or user-defined combinations of reagents can be used to generate these cells. The following protocol describes the use of the CellXVivo kit.
Human Th17 Differentiation Media
Reconstitute Human Th17 Reagent 1, 2, and 3 each with 250 µL of Reconstitution Buffer 1. This yields 400X stock solutions.
Reconstitute Human Th17 Reagent 4 and Reagent 5 each with 250 µL of Reconstitution Buffer 2. This yields 400X stock solutions.
Add 62.5 μL of Human Th17 Reagent 1, 2, 3, 4, and 5 to 24.6 mL of cell culture media (X-VIVO 15 medium, 100 units/mL Penicillin, 100 µg/mL Streptomycin).
Human CD3 and CD28 Antibodies
Reconstitute the Mouse Anti-Human CD3 and CD28 antibodies each with 100 µL of Reconstitution Buffer 2. This yields 40X stock solutions.
Add 10 mL of 20X Wash Buffer to 190 mL of sterile deionized water to prepare 200 mL of 1X Wash Buffer.
Just before plate coating, dilute the 40X antibody stock solutions 40-fold with 1X Wash Buffer.
Protocol for Th17 Differentiation
Coat the wells of a tissue culture plate with Mouse Anti-Human CD3 and CD28 antibodies. Please see our website for a graphic outline of this procedure.
For a 24-well plate, add 125 µL/well of diluted anti-CD3 antibody and 125 µL/well of diluted anti-CD28 antibody. For a 96-well plate, add 25 µL/well of diluted anti-CD3 antibody and 25 µL/well of diluted anti-CD28 antibody.
Incubate at 2-8 °C overnight.
Wash the plate with 1X Wash Buffer twice before use.
Isolate human peripheral blood mononuclear cells (PBMCs) from human blood using Ficoll-Hypaque density gradient centrifugation. See Leukocyte Preparation Protocol.
Isolate human naïve CD4+ T cells from human PBMCs using the MagCellect Human Naïve CD4+ T Cell Isolation Kit (Catalog # MAGH115) or a Human CD4+ T Cell Enrichment Column (Catalog # HCD43 or Catalog # HCD4C-1000). Cick here for graphic illustrations of MagCellect and T cell column assay principles.
Suspend human naïve CD4+ T cells at 1-2 x 105 cells/mL in Human Th17 Differentation Media.
Add the cells to an anti-human CD3 antibody and anti-human CD28 antibody-coated plate. For a 24-well plate, add 1 mL/well. For a 96-well plate, add 0.2 mL/well.
Incubate the cells in a 37 °C, 5% CO2 humidified incubator for 10 days. Refresh the Human Th17 Differentiation Media every 2-3 days according to step 7.
Refresh the Human Th17 Differentiation Media by removing 900 µL of the media from each well of a 24-well plate or 180 µL of the media from each well of a 96-well plate and replenishing with the same volume of fresh Human Th17 Differentiation Media every 2-3 days.
* Note: If the cell culture media turns yellow or the cell density reaches 1.5 x 106 cells/mL, the cells need to be split 1:2.
Verify successful Th17 cell differentiation by analysis of the cells with flow cytometry and analysis of the cell culture media with multi-analyte or single-analyte assays.
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.
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.
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.
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.
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. 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. 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.
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.