Regulatory T cells (Treg) are a central CD4⁺ T cell subset that maintains immune tolerance and suppresses excessive inflammatory responses, and they are essential for research on autoimmunity, transplant immunology, tumor immunity, and inflammatory regulation. The Mouse Treg Polarization Kit is designed for the in vitro directed induction of mouse naive CD4⁺ T cells, providing a standardized polarization system for the efficient, stable, and reproducible generation of high-purity functional iTreg cells.

Product Components

Treg Polarization Induction Protocol for CD4⁺ T Cells:

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 Treg 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. 37607ES10) in complete Treg polarization medium at a density of 4-5×10⁵ cells/mL.

Complete Treg polarization medium: RPMI-1640 basal medium supplemented with 10% FBS, 55 μM β-mercaptoethanol, 10 ng/mL recombinant mouse IL-2, 2 ng/mL recombinant mouse TGF-β1, 10 μg/mL anti-mouse IFN-γ functional antibody, 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 Treg 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 Treg polarization medium to maintain optimal cell growth.

4. Cell Collection

On day 5 of culture, collect the cells and wash once with pre-chilled PBS.

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 Treg 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, 2 ng/mL recombinant mouse IL-2, 10 μg/mL anti-mouse IFN-γ, and 10 μg/mL anti-mouse IL-4 functional antibody.

Note: Apart from the difference in medium, cell seeding, medium exchange, and density monitoring are identical to those of the Treg 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 1× Fixation/Permeabilization solution 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 1× Permeabilization Buffer 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 10% FBS in PBS per well with PE anti-mouse FOXP3 Recombinant Antibody and CD25 Monoclonal Antibody (BC96), Brilliant Violet™ 421 (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.

3. Aliquot into individual containers to minimize freeze-thaw cycles.

Product Properties

Features

• Classical gold-standard combination with high polarization efficiency.

• High-activity quality control with stable lot-to-lot performance.

• Streamlined operation with a short experimental cycle.

• Optimized ratios for convenient and reliable use.

Applications

 

Figure 1. Flow cytometry analysis of Foxp3 and CD25 expression in non-induced (Treg−, left) and induced (Treg⁺, right) mouse CD4⁺ T cells. Treg polarization efficiency (CD25⁺Foxp3⁺): 86.3% (Treg−: 4.12%).

Storage