Kit Summary

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

Key Benefits

  • Provides optimized reagents needed to induce Th2 differentiation
  • Yields ~80 x 106 CD4+ T cells, of which > 40% are Th2 polarized
  • Contains high quality bioactive proteins
  • Includes straightforward procedures
  • Does not require specialized instrumentation
 

 

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.

 

Kit Contents

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.

 

Data Examples
Intracellular Cytokine Staining of Differentiated Mouse Th2 Cells
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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.

Th2-differentiated Mouse CD4+ Cells Secrete High Levels of IL-4
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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).

Preparation and Storage
  • Stability & Storage
    Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Related Research Areas
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
 

 

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.

Coat the desired tissue culture plate or flask

Isolate mouse splenocytes.

Isolate mouse splenocytes

Isolate mouse naïve CD4+ T cells from splenocytes (e.g., using magnetic cell selection).

Perform a cell count

Perform a cell count.

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.

Suspend cells in media

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.

Harvest cells on Day 3

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

Verify Th2 cell differentiation

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