CellXVivo Human Th1 Cell Differentiation Kit
R&D Systems, part of Bio-Techne | Catalog # CDK001
Key Product Details
Summary for CellXVivo Human Th1 Cell Differentiation Kit
For the differentiation of Th1 cells from a preparation of naïve CD4+ T cells.
Key Benefits
- Yields a highly enriched population of Th1 polarized T cells
- Provides optimized reagents needed to induce Th1 differentiation
- Involves validated and straightforward procedures
- Does not require specialized instrumentation
Why Differentiate Th1 Cells In Vitro?
T helper type 1 (Th1) cells are a subset of CD4+ effector T cells that promote cell-mediated immune responses against intracellular viral and bacterial pathogens. Differentiation into the Th1 lineage is promoted by cytokines such as IL-12 and IFN-gamma. Th1 cells are characterized by the secretion of IFN-gamma, IL-10, and TNF-alpha. Although Th1 cells are critical for the clearance of intracellular pathogens, exaggerated Th1 responses are associated with autoimmune diseases including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and type 1 diabetes. Th1 polarized cells are present in low abundance in normal human peripheral blood. In vitro differentiation of Th1 cells from the larger naïve CD4+ T cell population provides increased numbers of Th1 cells to facilitate downstream research.
This kit contains the following optimized reagents to drive efficient differentiation of naïve CD4+ T cells into Th1 polarized cells.
- Mouse Anti-Human CD3 Antibody
- Human Th1 Differentiation Supplements
- Reconstitution Buffers
- Wash Buffer (20X)
Provides sufficient reagents for the differentiation of one 24-well plate.
Stability and Storage
Store the unopened kit at 2 to 8 °C. After opening the kit, Mouse Anti-Human CD3 Antibody, Human Th1 Reagent 1, and Human Th1 Reagent 2 may be stored at 2-8 °C under sterile conditions for up to 30 days or at -20 °C to -70 °C in a manual defrost freezer for up to 3 months. Reconstitution Buffer 1, Reconstitution Buffer 2, and 20X Wash Buffer may be stored under sterile conditions for up to 12 months at 2 to 8 °C. Do not use beyond the expiration date of the kit.
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Verification of Th1 Cell Identity using Flow Cytometry. Human peripheral blood naïve CD4+T cells without (A, C) and with (B, D) a 5 day differentiation using the reagents included in the Human Th1 Cell Differentiation Kit. (A, B) The cells were stained with an APC-conjugated Mouse Anti-Human IFN-gamma Monoclonal Antibody (Catalog # IC285A) and a PE-conjugated Mouse Anti-Human IL-4 Monoclonal Antibody (Catalog # IC204P). (C, D) The cells were stained with an APC-conjugated Mouse Anti-Human IFN-gamma Monoclonal Antibody (Catalog # IC285A) and a PerCP-conjugated Mouse Anti-Human IL-17 Monoclonal Antibody (Catalog # IC3171C). Control cultures were used to place the quadrants. |
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Detection of IFN-gamma, IL-4, and IL-17 in Human Th1 Cells. Human peripheral blood naïve CD4+T cells were cultured for 5 days using reagents included in the Human Th1 Cell Differentiation Kit. After fixation of cells in the wells, cytokine expression was determined using the Human IFN-gamma Quantikine ELISA Kit (Catalog # DIF50), the Human IL-4 Quantikine ELISA Kit (Catalog # D4050), and the Human IL-17 Quantikine ELISA Kit (Catalog # D1700). |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, human CD4+ T cells can be differentiated into Th1 cells using the following procedure:
- Coat a plate with Mouse Anti-Human CD3 Antibody
- Isolate human naïve CD4+ T cells from a PBMC preparation
- Culture naïve CD4+ T cells in Human Th1 Differentiation Media for 5 days
- Stimulate cells with monensin
- Analyze culture media and cells for a Th1-specific cytokine production profile
Reagents Supplied in the CellXVivo™ Human Th1 Cell Differentiation Kit (Catalog # CDK001):
- Mouse Anti-Human CD3 Antibody
- Human Th1 Reagents
- Reconstitution Buffers
- Wash Buffer (20X)
Reagents
- MagCellect™ Human Naïve CD4+ T Cell Isolation Kit (Catalog # MAGH115 or equivalent)
- Monensin
- PMA
- Ficoll-Hypaque™
- RPMI 1640
- L-Glutamine
- Ionomycin
- Penicillin
- Streptomycin
- Fetal Bovine Serum (FBS)
- beta-Mercaptoethanol
Equipment
- Tissue culture flasks and/or plates
- Hemocytometer
- 37 °C and 5% CO2 humidified incubator
- Centrifuge
- Pipettes and pipette tips
Coat wells of a 24-well plate with Mouse Anti-Human CD3 Antibody.
Isolate PBMCs from human blood (e.g., using Ficoll-Hypaque™ density centrifugation).
Isolate human naïve CD4+ T cells from PBMCs (e.g., using R&D Systems MagCellect™ Human Naïve CD4+ T Cell Isolation Kit).
Perform a cell count.
Suspend 1-2 x 105 naïve CD4+ T cells/mL in media supplemented with Human Th1 Reagent 1 and Human Th1 Reagent 2.
Culture the cells on plates pre-coated with Mouse Anti-Human CD3 Antibody for 5 days.
Refresh the Diferentiation Media every 3-4 days.
Stimulate the cells with mitogens.
Verify Th1 cell differentiaiton by analying cytokine expression via flow cytometry.
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