小鼠Th2极化套装

Note: Somereagents may have leftovers.

Induction of Th2 Polarization in CD4⁺ T Cells

1 Coating with anti-CD3ε/CD28 antibodies to activate T cells

1.1 Using sterile PBS, dilute the anti-mouse CD3ε monoclonal antibody and the anti-mouse CD28 monoclonal antibody separately to their working concentrations (10 µg/mL and 2 µg/mL).

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

1.3 Place the culture plate in a 4°C refrigerator and allow it to coat overnight (approximately 16–18 hours).

2 Seeding cells and initiating Th2 polarization

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

2.2 Mouse splenic CD4⁺ T cells, sorted using CD4⁺ T cell selection magnetic beads (Cat. No.: S0K0003) and with a purity >95%, were resuspended in Th2-polarizing complete medium at a concentration of 4–5×10⁵cells/mL.

Th2-polarizing complete medium formulation: RPMI-1640 basal medium supplemented with 10% fetal bovine serum, 55 µM β‑mercaptoethanol, 10 ng/mL recombinant mouse IL-2, and 25 ng/mL recombinant mouse IL-4.

2.3 Aliquots of 500 µL cell suspension were added to each well of a coated 48‑well plate. The plates were then placed in a cell culture incubator at 37°C with 5% CO₂ for culture.

3 Culture Maintenance and Cell Expansion

3.1 After 72 hours of culture, gently pipette and collect the cell suspension, then centrifuge at 300 ×g for 5 minutes.

3.2 Discard the supernatant, resuspend the cells in fresh Th2-polarizing complete medium, and adjust the cell density to 1×10⁶cells/mL, then reseed them into a new plate for continued culture.

3.3 Thereafter, observe the cell density and condition under an inverted microscope every 12 hours. If the cell density becomes too high (e.g., >2×10⁶cells/mL), perform a half-medium change or split the cells into new wells at an appropriate ratio, supplementing with fresh Th2-polarizing complete medium to maintain optimal cell growth.

4. Cell Collection

On day 6 of culture, collect the cells,After washing once with pre-cooled PBS, resuspend the cells in RPMI-1640 complete medium containing 10 ng/mL PMA, 1 µg/mL Ionomycin, and 2 μM Monensin, adjust the cell density to 1×10⁶cells/mL, and incubate under light‑protected co‑stimulation conditions at 37°C and 5% CO₂ for 5 hours.

Th0 negative control group setup

To evaluate the specificity of polarization, a parallel Th0 negative control group is established. Its procedure is basically the same as that of the above‑mentioned Th2 induction group, with the main difference being:

1. Use different maintenance culture media: Use Th0 maintenance medium, which consists of RPMI-1640 basal medium supplemented with 10% fetal bovine serum, 55 µM β-mercaptoethanol, 2 ng/mL recombinant mouse IL-2, 10 µg/mL anti-mouse IFN-γ, and 10 µg/mL anti-mouseIL-4 functional antibodybody.

Apart from the above differences in culture media, cell seeding, medium changes, and density monitoring are all the same as in the Th2 induction group.

Flow cytometry detection1. Collect cells: Harvest the cells, wash them once with PBS, discard the supernatant, and gently resuspend the cells in PBS by pipetting;

2. Count: Use a cell counter to count and calculate the total number of cells; centrifuge at 300×g for 5 minutes, then discard the supernatant.

3. Live/dead dye staining: Add the diluted Fixable Viability Dye 452 (S0D0021) at a concentration of 100 μl per 1 million cells (refer to the manual for dilution instructions);

4. Wash the cells: Wash the cells with PBS, centrifuge at 300×g for 5 minutes, and remove any remaining live/dead dye;

5. Cell fixation: Resuspend the cells in PBS at a concentration of 10 million cells per milliliter, add 100 μl per well to a 96-well plate, centrifuge at 300×g for 5 minutes, and discard the supernatant; then add 200 μl of 1× Fixation/Permeabilization solution to each well and incubate in the dark at room temperature for 15 minutes.

6. Wash the cells: Centrifuge at 300×g for 5 minutes, discard the supernatant; add 200 μl of PBS to each well, centrifuge at 300×g for 5 minutes to wash the cells and remove residual fixative.

7. Permeabilize the cells: Add 200 μl of 1× Permeabilization Buffer to each well, and incubate in a refrigerator at 4–8°C for 30 minutes.

8. Block the cells: Add 100 μl of 1× BD Perm/Wash™ Buffer to each well, and include Mouse FcR Blocking Reagent (CAT: S0B0599) for cell blocking (steps 7 and 8 can be performed simultaneously). Then centrifuge at 300×g for 5 minutes and discard the supernatant.

9. Incubate with the antibody: Add 100 μl of PBS containing 10% FBS to each well, and then add the APC-conjugated anti-mouse IL-4 antibody (refer to the product manual for the recommended volume);

10. Wash the cells: Centrifuge at 300×g for 5 minutes, discard the supernatant; add 200 μl of PBS to each well, centrifuge again at 300×g for 5 minutes to wash the cells and remove residual antibody, then resuspend the cells in 100–200 μl;

11. Analyze on the flow cytometer.

Store in separate containers to minimize freeze-thaw cycles.