This kit provides a complete reagent set for the in vitro directed differentiation of mouse naive CD4⁺ T cells into functional Th1 cells. Built on the classical IL-12-driven STAT4/T-bet differentiation pathway, it includes all components required for T cell activation, directed induction, and bypass blockade. The standardized, pre-optimized formulation is tailored for mouse splenic CD4⁺ T cells.

Product Components

Naive CD4⁺ T Cell Th1 Polarization Induction Protocol:

1. Coat anti-CD3ε/CD28 Antibodies for T Cell Activation

1.1 Dilute anti-mouse CD3ε and anti-mouse CD28 monoclonal antibodies to the working concentrations (10 μg/mL and 2 μg/mL, respectively) using sterile PBS.

1.2 Add 500 μL of the antibody mixture to each well of a 48-well cell culture plate, ensuring the liquid covers the well bottom.

1.3 Incubate the plate at 4°C for overnight coating (approximately 16-18 hours).

2. Seed Cells and Initiate Th1 Polarization

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

2.2 Resuspend mouse splenic CD4⁺ T cells (purity >95%) isolated using a CD4⁺ T Cell Isolation Reagent (Cat. No. 37657ES10) in complete Th1 polarization medium at a density of 4-5×10⁵ cells/mL.

Complete Th1 polarization medium: RPMI-1640 basal medium supplemented with 10% FBS, 55 μM β-mercaptoethanol, 5 ng/mL recombinant mouse IL-2, 30 ng/mL recombinant mouse IL-12, and 10 μg/mL anti-mouse IL-4 functional antibody.

2.3 Seed 500 μL of the cell suspension into each well of the pre-coated 48-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 Th1 polarization medium, adjust the cell density to 1×10⁶ 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 Th1 polarization medium 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 Th1 induction group, with the main difference being the medium used:

Th0 maintenance medium: RPMI-1640 basal medium supplemented with 10% FBS, 55 μM β-mercaptoethanol, 10 μg/mL anti-mouse IFN-γ, and 10 μg/mL anti-mouse IL-4 neutralizing antibody.

Note: No polarizing cytokines are added to this medium. Apart from this difference, cell seeding, medium exchange, density monitoring, and the day-5 restimulation are identical to those of the Th1 induction group.

Flow Cytometry Analysis

1. Cell collection: Collect the cells, wash once with PBS, discard the supernatant, and gently resuspend the 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 Mouse 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 Mouse Th1/Th2/Th17 Phenotyping Kit (BD 560758) (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.

Notes

1. For your safety and health, wear a lab coat and disposable gloves during operation. This product is for research use only.

2. Optional medium supplements: 1% sodium pyruvate, L-glutamine, HEPES, and penicillin-streptomycin.

Product Properties

Features

• Excellent differentiation efficiency with stable polarization rates.

• Low endotoxin and high activity.

• Stringent lot-to-lot quality control for excellent reproducibility.

Applications 

 

Figure 1. Flow cytometry analysis of IFN-γ expression in Th0 (left) and Th1 (right) polarized mouse CD4⁺ T cells. Th1 polarization efficiency: 53.6% (Th0: 0.011%).

Storage