CellXVivo Mouse Th1 Cell Differentiation Kit
R&D Systems, part of Bio-Techne | Catalog # CDK018
Key Product Details
Summary for CellXVivo Mouse Th1 Cell Differentiation Kit
For the differentiation of Th1 cells from a preparation of CD4+ T cells isolated from mouse splenocytes.
Key Benefits
- Provides optimized reagents needed to induce Th1 differentiation
- Yields ~80 x 107 CD4+ T cells, of which 70-90% are IFN-γ+ Th1 polarized
- Contains high quality bioactive proteins
- Includes straightforward procedures
- Does not require specialized instrumentation
Why Expand and Differentiate Mouse Th1 and Th2 Cells Ex Vivo?
T helper type 1 (Th1) cells are a lineage of CD4+ effector T cells that promote cell-mediated immune responses against intracellular viral and bacterial pathogens. Enhanced Th1 responses are associated with autoimmune diseases including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and type-1 diabetes. Differentiation of CD4+ effector T cells into the Th1 lineage is promoted by cytokines such as IL-12 and IFN-gamma. Th1 cells secrete IFN-gamma, IL-10, and TNF-alpha. The CellXVivo™ Mouse Th1 Cell Differentiation Kit contains optimized reagents for Th1 differentiation from naïve CD4+ cells.
This kit contains the following reagents for the ex vivo differentiation of mouse Th1 cells.
- Hamster Anti-Mouse CD3, Th1
- Mouse Th1 Reagent 1
- Mouse Th1 Reagent 2
- Mouse Th1 Reagent 3
- Mouse Th1 Reagent 4
- Reconstitution Buffer 1
- Reconstitution Buffer 2
- 20X Wash Buffer
The quantity of components in this kit is sufficient to differentiate approximately 8 x 106 naïve CD4+ T cells, and generate 8 x 107 CD4+ cells of which 70-90% are IFN-gamma+ Th1 polarized cells.
Note: Results may vary due to strain, age, and/or the health of the mice used for isolation.
Stability and Storage
Store the unopened kit at ≤-20 °C. Do not use past the kit expiration date.
Limitations
- FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
- The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
- This reagent should not be used beyond the expiration date indicated on the label.
- Results may vary due to variations among cells derived from different donors.
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Th1-differentiated Mouse CD4+ Cells Secrete High Levels of IFN-gamma. Mouse naïve CD4+ T cells were differentiated for 6 days using the reagents included in this kit. On day 6 of differentiation cells were harvested and re-stimulated with Anti-Mouse CD3 and Anti-Mouse CD28 overnight. The cell culture supernatant was collected and cytokine secretion was determined using the Mouse IFN-gamma Quantikine®ELISA Kit (Catalog # MIF00), the Mouse IL-4 Quantikine®ELISA Kit (Catalog # M4000B), and the Mouse IL-17 Quantikine® ELISA Kit (Catalog # M1700). |
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Upregulation of T-bet, TNF-alpha, and IL-18R alpha in Th1-polarized Mouse CD4 Cells. Mouse CD4 cells were isolated and differentiated into Th1 cells using the CellXVivo™ Mouse Th1 Cell Differentiation Kit. Differentiated mouse Th1 cells (A, B, C) and naïve mouse CD4 cells (D, E, F) were analyzed by flow cytometry for (A, D) T-bet expression using Human T-bet/TBX21 Alexa Fluor®488-conjugated Antibody (Catalog # IC53851G), (B, E) TNF-alpha expression using Mouse TNF-alpha PE-conjugated Antibody (Catalog # IC410P), and (C, F) IL-18R alpha expression using Mouse IL-18 R alpha /IL-1 R5 APC-conjugated Antibody (Catalog # FAB1216A). Cells were also gated on CD4 expression using the Mouse CD4 Alexa Fluor®700-conjugated Antibody (Catalog # FAB554N). Differentiated mouse Th1 cells were stimulated with Cell Activation Cocktail (Tocris®, Catalog # 5476) for 4 hours prior to antibody staining. Quadrants were determined by naïve CD4 T cells expression levels. |
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Intracellular Cytokine Staining of Differentiated Mouse Th1 Cells. Flow cytometry data of mouse naïve CD4+T cells (A, C) and differentiated Th1 cells (B, D) generated using reagents included in this kit. After 6 days of differentiation, naïve CD4+cells or differentiated Th1 cells were stimulated with Cell Activation Cocktail (Tocris®, Catalog # 5476) and stained with conjugated Anti-Mouse IFN-gamma (R&D Systems, Catalog # IC485), Anti-Mouse IL-4 (Clone 11B11), and Anti-Mouse IL-17A (Novus Biologicals, Catalog # NBP1-72027) Monoclonal Antibodies. Quadrants were set based on isotype-stained controls. |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, CD4+ T cells isolated from mouse splenocytes are differentiated ex vivo into the Th1 phenotype with reagents provided in this kit and using the following procedure:
- Isolate CD4+ T cells from mouse splenocytes
- Culture CD4+ T cells in reagents provided in kit
- Verify Th1 cell expansion after 6 days in culture
This kit contains the following reagents for the ex vivo differentiation of mouse Th1 cells.
- Hamster Anti-Mouse CD3, Th1
- Mouse Th1 Reagent 1
- Mouse Th1 Reagent 2
- Mouse Th1 Reagent 3
- Mouse Th1 Reagent 4
- Reconstitution Buffer 1
- Reconstitution Buffer 2
- 20X Wash Buffer
Reagents
- Laboratory mice
- X-VIVO™ 15 Chemically Defined, Serum-free Hematopoietic Cell Medium (Lonza, or equivalent)
- MagCellect™ Mouse Naïve CD4+ T Cell Isolation Kit (R&D Systems, Catalog # MAGM205, or equivalent)
- Penicillin/Streptomycin (optional)
- Cell Activation Cocktail 500X (Tocris®, Catalog # 5476)
Equipment
- Tissue culture plates and/or flasks
- Sterile deionized water
- Pipettes and pipette tips
- Inverted microscope
- Hemocytometer
- 37 °C, 5% CO2 incubator
- Centrifuge
Protocol for mouse Th1 cell differentiation using the CellXVivo™ Mouse Th1 Cell Differentiation Kit.
Note: Results may vary due to strain, age, and/or the health of the mice used for isolation.
Coat the desired tissue culture plate with Hamster Anti-Mouse CD3,
Th1 antibody.
Isolate mouse splenocytes.
Isolate mouse naïve CD4+ T cells from splenocytes (e.g., using magnetic cell selection).
Perform a cell count.
Suspend 1 x 106 naïve CD4+ T cells/mL in Mouse Th1 Differentiation Media. Culture the cells on plates pre-coated with Hamster Anti-Mouse CD3 antibody.
Harvest cells on day 3.
Dilute cells 1:10 with fresh Mouse Th1 Differentiation Media.
Culture cells in a new flask for an additional 3 days.
Verify Th1 cell differentiation by analyzing cytokine expression via flow cytometry or ELISA (optional).
