Treg Polarization Kit, Mouse

Note:Some reagents may have leftovers.

Treg Polarization Induction of Naïve CD4⁺ T Cells

1 Coating with Anti-CD3ε/CD28 Antibodies for T Cell Activation
1.1 Dilute anti-mouse CD3ε monoclonal antibody and anti-mouse CD28 monoclonal antibody to final concentrations (10 µg/mL and 2 µg/mL) using sterile PBS.
1.2 Add 500 µL of the antibody mixture to each well of a 48-well cell culture plate, ensuring the bottom is fully covered.
1.3 Incubate the plate at 4°C overnight (approximately 16-18 hours) for coating.

2 Cell Seeding and Initiation of Treg Polarization

T cell polarization the cells should be resuspended in culture medium. Both the dilution and the cell density are important to assure optimal stimulation and cell growth. The following table lists culture plate sizes suitable for different cell numbers:

2.1 The next day, aspirate the coating solution and gently wash each well twice with ice-cold sterile PBS to remove unbound antibodies.
2.2 Resuspend the isolated naïve CD4⁺ T cells (from mouse spleen, purity >95% using CD4⁺ T cell selection beads) in Treg polarization complete medium at a density of 4-5×10⁵ cells/mL. (Or alternative: S0K1001 Mouse Naïve CD4 T Cell Isolation Kit)

Formula for Treg Polarization Complete Medium: RPMI-1640 basal medium, supplemented with 10% fetal bovine serum (FBS), 55 µM β-mercaptoethanol, 10 ng/mL mouse IL-2、2 ng/mL mouse TGF-β1, 10 µg/mL Invivo anti-mouse IFNγ Recombinant mAb, 10 µg/mL Invivo anti-mouse IL-4 Recombinant mAb.

2.3 Seed 500 µL of the cell suspension into each pre-coated well of the 48-well plate. Incubate the plate in a humidified cell culture incubator at 37°C with 5% CO₂.

3 Culture Maintenance and Cell Expansion
3.1 After 48 hours of culture, gently resuspend the cells, collect the suspension, and centrifuge at 300 × g for 5 minutes.
3.2 Discard the supernatant and resuspend the cell pellet in fresh Treg polarization complete medium. Adjust the cell density to 0.5- 1×10⁶ cells/mL and re-seed into a new plate for continued culture.
3.3 Subsequently, 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 change or split the cells into new wells at an appropriate ratio, supplementing with fresh Treg polarization complete medium to maintain optimal cell growth conditions.

4. Cell Capture

On day 5 of culture, harvest the cells and wash once with cold PBS.

Negative Control Group (Th0) Setup
To assess the specificity of polarization, a Th0 negative control group is established in parallel. The procedure is largely identical to the Treg induction group described above, with the following key differences:
1. Different Maintenance Medium: Use Th0 maintenance medium, which consists of RPMI-1640 basal medium supplemented with 10% FBS, 55 µM β-mercaptoethanol, 10 µg/mL anti-mouse IFN-γ antibody, and 10 µg/mL anti-mouse IL-4 antibody.
2. Absence of Polarizing Cytokines: This medium does not contain any polarizing cytokines.
Apart from the differences in medium composition, the step for cell seeding, medium changes, density monitoring are performed identically to the Treg induction group.

Flow Cytometry Detection
1.Cell Harvesting: Harvest the cells.Wash the cells once with phosphate-buffered saline (PBS). Discard the wash supernatant and gently resuspend any cells in a fresh volume of PBS by pipetting.

2.Cell Counting: Count the cells using an automated cell counter and calculate the total cell number. Centrifuge the cell suspension at 300 × g for 5 minutes at 4°C. Discard the supernatant and resuspend the cell pellet at a concentration of 1×10⁷ cells/mL in PBS.

3.Viability Staining: Add 100 µL of the diluted Fixable Viability Dye 452 (Cat# S0D0021) per 1×10⁶ cells. Refer to the product manual for specific dilution instructions. Incubate as recommended by the manufacture.

4.Cell Washing: Wash the cells by adding an excess volume of PBS. Centrifuge at 300 × g for 5 minutes at 4°C and carefully decant the supernatant to remove residual dye.

5.Cell Fixation: Transfer 100 µL of cell suspension (containing 1×10⁶ cells) to each well of a 96-well U-bottom plate. Centrifuge the plate at 300 × g for 5 minutes at 4°C and discard the supernatant by rapid inversion. Fix cells by adding 200 µL of 4% paraformaldehyde (PFA) per well. Incubate for 15 minutes at room temperature, protected from light.

6.Cell Washing: Centrifuge the plate at 300 × g for 5 minutes at 4°C and discard the supernatant. Add 200 µL of PBS to each well to wash. Repeat centrifugation and discard the supernatant to ensure complete removal of residual fixative.

7.Cell Permeabilization: Add 200 µL of BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution  to each well. Incubate for 30 minutes at 4°C. 

8.Cell Blocking: Add 100 µL of 1× BD Perm/Wash™ Buffer per well. Include an appropriate concentration of Mouse IgG (e.g., 0.5-1 µg/µL) in this buffer for Fc receptor blocking. Incubate for 10-15 minutes at 4°C. Centrifuge at 300 × g for 5 minutes at 4°C and discard the supernatant. Note: Step 8 can be performed concurrently with Step 7 by adding the blocking agent directly to the permeabilization solution.

9.Antibody Incubation: Add 100 µL of PBS containing 10% FBS per well/tube, along with the appropriately diluted PE anti-mouse FOXP3 Recombinant Antibody and CD25 Monoclonal Antibody (BC96), Brilliant Violet™ 421 as per the manufacturer's instructions.

10.Cell Washing: Centrifuge the plate at 300 × g for 5 minutes at 4°C and discard the supernatant to remove unbound antibody. Add 200 µL of PBS to each well for a final wash. Centrifuge again and discard the supernatant. Resuspend the final cell pellet in 100-200 µL of PBS or a dedicated flow cytometry staining buffer for acquisition.

11.Flow Cytometric Acquisition: Acquire samples on a flow cytometer.

store in separate containers to reduce the number of freeze-thaw cycles.