Th2 cells are a key CD4⁺ T cell subset that mediates humoral immunity, anti-parasitic defense, and allergic diseases such as asthma. They rely on the GATA-3/STAT6 pathway to secrete signature cytokines including IL-4, IL-5, and IL-13. The Human Th2 Polarization Kit provides the cytokines and antibodies required for directed induction, enabling the rapid, stable, and high-yield generation of functional Th2 cells.

Product Components

Human Th2 Polarization Kit Operation Procedure:

1. Coat anti-human CD3 mAb for T Cell Activation

1.1 Dilute anti-human CD3 mAb to the working concentration (5 μg/mL) using sterile PBS.

1.2 Add the diluted anti-human CD3 mAb at 1 mL per well to a 24-well cell culture plate for coating. Incubate at 37°C for 2 hours or at 4°C overnight.

2. Seed Cells and Initiate Th2 Polarization

2.1 The next day, aspirate the coating solution and gently wash the plate 2-3 times with pre-chilled sterile PBS to remove unbound antibodies.

2.2 Resuspend naive CD4⁺ T cells isolated using a CD4⁺ T Cell Isolation Reagent (CD4 Nanobeads, Cat. No. 37605ES10) in complete Th2 polarization medium at a density of 1×10⁶ cells/mL.

Complete Th2 polarization medium: RPMI-1640 basal medium supplemented with 10% FBS, 55 μM β-mercaptoethanol, 40 ng/mL IL-2, 30 ng/mL IL-4, 10 μg/mL Anti-Human IFN-γ mAb, and 1 μg/mL anti-human CD28 mAb.

2.3 Seed 1 mL of the cell suspension into each well of the pre-coated 24-well plate. Incubate at 37°C, 5% CO₂.

3. Culture Maintenance and Cell Expansion

3.1 After 48 hours of culture, gently pipette to collect the cell suspension and centrifuge at 300×g for 5 minutes.

3.2 Discard the supernatant, resuspend the cells in fresh complete Th2 polarization medium, adjust the cell density to 3×10⁵ cells/mL, and reseed into new plates for continued culture.

3.3 Thereafter, monitor cell density and morphology under an inverted microscope every 12 hours. If the cell density becomes too high (e.g., >2×10⁶ cells/mL), perform a half-medium exchange or split the cells into new wells at an appropriate ratio, supplementing with fresh complete Th2 polarization medium (without additional anti-human CD28 mAb) to maintain optimal cell growth.

4. Cell Restimulation and Intracellular Cytokine Capture

On day 5 of culture, collect the cells and wash once with pre-chilled PBS. Resuspend the cells in complete RPMI-1640 medium containing 10 ng/mL PMA, 1 μg/mL ionomycin, and 10 μg/mL brefeldin A, adjust the density to 1×10⁶ cells/mL, and incubate protected from light at 37°C, 5% CO₂ for 5 hours of co-stimulation.

Negative Control (Th0) Setup

To assess polarization specificity, a Th0 negative control should be set up in parallel. The procedure is essentially identical to that of the Th2 induction group, with the main difference being the medium used:

1. Th0 maintenance medium: RPMI-1640 basal medium supplemented with 10% FBS, 55 μM β-mercaptoethanol, 10 μg/mL anti-human IL-4 mAb, 10 μg/mL anti-human IFN-γ mAb, 40 ng/mL IL-2, and 1 μg/mL anti-human CD28 mAb.

2. No polarizing cytokines are added to this medium.

Apart from these differences, cell seeding, medium exchange, and density monitoring are identical to those of the Th2 induction group.

Flow Cytometry Analysis

1. Cell collection: After induction, discard the culture supernatant, wash the cells once with PBS, discard the supernatant, and gently resuspend the adherent cells in PBS by pipetting.

2. Cell counting: Count the cells using an automated cell counter to determine the total cell number, centrifuge at 300×g for 5 minutes, and discard the supernatant.

3. Viability dye staining: Add the diluted Fixable Viability Dye 452 at 100 μL per 1×10⁶ cells (refer to the product manual for dilution instructions).

4. Cell washing: Wash the cells with PBS, centrifuge at 300×g for 5 minutes, and remove residual viability dye.

5. Cell fixation: Resuspend the cells in PBS at 1×10⁷ cells/mL, dispense 100 μL per well into a 96-well plate, centrifuge at 300×g for 5 minutes, and discard the supernatant. Add 200 μL of 4% PFA per well and incubate at room temperature protected from light for 15 minutes.

6. Cell washing: Centrifuge at 300×g for 5 minutes and discard the supernatant. Add 200 μL of PBS per well and centrifuge at 300×g for 5 minutes to wash the cells and remove residual fixative.

7. Cell permeabilization: Add 200 μL of fixation/permeabilization solution (BD 554714) per well and incubate at 4-8°C for 30 minutes.

8. Cell blocking: Add 100 μL of 1× BD Perm/Wash™ Buffer per well with Human IgG for blocking (steps 7 and 8 may be performed simultaneously), then centrifuge at 300×g for 5 minutes and discard the supernatant.

9. Antibody incubation: Add 100 μL of 1× BD Perm/Wash™ Buffer per well with PE/Dazzle™ 594 anti-human IL-4 Antibody (Biolegend 500831) (refer to the antibody manual for the recommended amount).

10. Cell washing: Centrifuge at 300×g for 5 minutes and discard the supernatant. Add 200 μL of PBS per well and centrifuge at 300×g for 5 minutes to wash the cells and remove residual antibodies, then resuspend the cells in 100-200 μL.

11. Proceed to flow cytometry acquisition.

Product Properties

Features

• Precisely optimized formulation with high directed differentiation efficiency.

• Fully human-origin components with excellent cell viability.

• Stringent lot-to-lot quality control for stable experimental reproducibility.

Applications

Figure 1. Flow cytometry analysis of IL-4 expression in Th0 (left) and Th2 (right) polarized human CD4⁺ T cells. Th2 polarization efficiency: 22.8% (Th0: 0.011%).

Storage