CellXVivo Human Th2 Cell Differentiation Kit

Kit Summary

For the differentiation of Th2 cells from a preparation of naïve CD4+ T cells.

Why Differentiate Th2 Cells In Vitro?

T helper type 2 (Th2) cells are a lineage of CD4⁺ effector T cells that provide host protection against intestinal helminths and extracellular bacteria in addition to 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 IL17E/IL-25. The CellXVivo™ Human Th2 Cell Differentiation Kit contains all necessary components to differentiate human naïve CD4⁺ T cells into Th2 polarized cells.

This kit contains the following optimized proteins and reagents to drive efficient differentiation of naïve CD4⁺ T cells into Th2 polarized cells.

• Mouse Anti-Human CD3 Antibody
• Human Th2 Differentiation Supplements
• Reconstitution Buffers
• Wash Buffer (20X)

Provides sufficient reagents for the differentiation of one 24-well plate or 125 tests in a 96-well format.

Stability and Storage

Store the unopened kit at 2 to 8°C. After opening the kit, Mouse Anti-Human CD3 Antibody and Human Th2 Reagents 1-4 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.

Scientific Data Examples for CellXVivo Human Th2 Cell Differentiation Kit

Detection of IL-5, IFN-gamma, and IL-17 in Differentiated Human Th2 Cells.  Human peripheral blood naïve CD4+T cells were differentiated for 13 days using reagents included in the Human Th2 Cell Differentiation Kit. On day 13, cell culture supernatant was collected and cytokine secretion was determined using the Human IL-5 Quantikine®ELISA Kit ( Catalog # D5000B), the Human IFN-gamma Quantikine ELISA Kit (Catalog # DIF50), and the Human IL-17 Quantikine ELISA Kit (Catalog # D1700).

Intracellular Cytokine Staining of Differentiated Human Th2 Cells.  Flow cytometry data showing human peripheral blood naïve CD4+T cells without (A, C) and with (B, D) a 13 day differentiation using reagents included in the Human Th2 Cell Differentiation Kit. On day 13 of differentiation, the cells were re-stimulated with mitogens and stained with a PerCP-conjugated Mouse Anti-Human IL-17 Monoclonal Antibody (Catalog # IC3171C), 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). Quadrants were set based on isotype control-stained samples.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, human CD4+ T cells can be differentiated into Th2 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 Th2 Differentiation Media for 13 days
• Refresh the Human Th2 Differentiation Media every 3-4 days
• Analyze culture media and cells for a Th2-specific cytokine production profile

View Graphic Procedure

 

 

Reagents Provided

Reagents Supplied in the CellXVivo^™ Human Th2 Cell Differentiation Kit (Catalog # CDK002):

• Mouse Anti-Human CD3 Antibody
• Human Th2 Reagents
• Reconstitution Buffers
• Wash Buffer (20X)

 

Other Supplies Required

Reagents

• MagCellect™ Human Naïve CD4⁺ T Cell Isolation Kit (Catalog # MAGH115 or equivalent)
• Monensin
• PMA
• Ficoll-Hypaque™
• X-VIVO™ 15 Chemically Defined, Serum-free Hematopoietic Cell Medium (Lonza, or equivalent)
• Sterile deionized water
• Ionomycin
• Penicillin-Streptomycin

Equipment

• Tissue culture flasks and/or plates
• Hemocytometer
• 37 °C and 5% CO₂ humidified incubator
• Centrifuge
• Pipettes and pipette tips

 

Procedure Overview

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 magnetic cell selection).

Perform a cell count.

Suspend 1-2 x 10⁵ naïve CD4⁺ T cells/mL in Human Th2 Differentiation Media.

Culture the cells on plates pre-coated with CD3 Antibody for 13 days.

Refresh the Diferentiation Media every 3-4 days.

Stimulate the cells with mitogens.

Verify Th2 cell differentiation by analyzing cytokine expression using flow cytometry.

The Th2 cells are now ready for further downstream applications.