CellXVivo Mouse Th2 Cell Differentiation Kit

For the differentiation of Th2 cells from a preparation of CD4+ T cells isolated from mouse splenocytes.

Why Expand and Differentiate Th2 Cells Ex Vivo?

T helper type 2 (Th2) cells are a lineage of CD4⁺ effector T cells that provide host protection against intestinal parasites and extracellular bacteria. In addition, they provide support for B cell-dependent humoral responses. Pathological Th2 cell activity is a hallmark of allergic inflammation and asthma. Differentiation of CD4⁺ effector cells into the Th2 lineage is promoted by cytokines such as IL-4 in combination with either IL-2, IL-7, or TSLP. Th2 cells secrete IL-4, IL-5, IL-9, IL-13, and IL-17E/IL-25. In vitro differentiation of mouse Th2 cells from the larger naïve CD4⁺ T cell population provides increased numbers of Th2 cells to facilitate downstream research. The CellXVivo^™ Mouse Th2 Cell Differentiation Kit contains all necessary components to differentiate mouse naïve CD4⁺ T cells into Th2 polarized cells.

This kit contains the following reagents for the ex vivo differentiation of mouse Th2 cells.

• Hamster Anti-Mouse CD3, Th2
• Rat Anti-Mouse CD28, Th2
• Mouse Th2 Reagent 1
• Mouse Th2 Reagent 2
• Mouse Th2 Reagent 3
• Reconstitution Buffer 1
• Reconstitution Buffer 2
• 20X Wash Buffer

The quantity of the components in the kit is sufficient to differentiate approximately 4 x 10⁶ naïve CD4⁺ T cells, and generate 80 x 10⁶ T cells, of which >40% are IL-4⁺ Th2 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.

Scientific Data Examples for CellXVivo Mouse Th2 Cell Differentiation Kit

Th2-differentiated Mouse CD4+ Cells Secrete High Levels of IL-4. 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 stimulated with Anti-Mouse CD3 and Anti-Mouse CD28 overnight. Cell culture supernatant was collected and cytokine secretion determined using the Mouse IL-4 Quantikine®ELISA Kit (Catalog # M4000B), the Mouse IFN-gamma Quantikine®ELISA Kit (Catalog # MIF00), and the Mouse IL-17 Quantikine®ELISA Kit (Catalog # M1700).

Intracellular Cytokine Staining of Differentiated Mouse Th2 Cells.  Flow cytometry data of mouse naïve CD4+T cells (A, C) and differentiated Th2 cells (B, D) generated using reagents included in this kit. After 6 days of differentiation, naïve CD4+cells or differentiated Th2 cells were stimulated with Cell Activation Cocktail (Tocris®, Catalog # 5476) and stained with conjugated Anti-Mouse IL-4 (Clone 11B11), Anti-Mouse IFN-gamma (Catalog # IC485P), 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 Th2 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 Th2 cell expansion after 6 days in culture

View Graphic Procedure

 

 

Reagents Provided

Reagents Supplied in the CellXVivo^™ Mouse Th2 Cell Differentiation Kit (Catalog # CDK019):

• Hamster Anti-Mouse CD3, Th2
• Rat Anti-Mouse CD28, Th2
• Mouse Th2 Reagent 1
• Mouse Th2 Reagent 2
• Mouse Th2 Reagent 3
• Reconstitution Buffer 1
• Reconstitution Buffer 2
• 20X Wash Buffer

 

Other Supplies Required

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

 

Procedure Overview

Protocol for mouse Th2 cell differentiation using the CellXVivo^™ Mouse Th2 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 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 Th2 Differentiation Media. Culture the cells on plates pre-coated with Hamster Anti-Mouse CD3 antibody.

Harvest cells on day 3.

Dilute cells 1:20 with fresh Mouse Th2 Differentiation Media.

Culture cells in a new flask for an additional 3 days.

Verify Th2 cell differentiation by analyzing cytokine expression via flow cytometry or ELISA (optional).