Tfh Polarization Kit, Mouse

Note:Some reagents may have leftovers.

Tfh 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. 2 Cell Seeding and Initiation of Tfh 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 Tfh polarization complete medium at a density of 4-5×10⁵ cells/mL. (Or alternative: S0K1001 Mouse Naïve CD4 T Cell Isolation Kit)
Formula for Tfh Polarization Complete Medium: RPMI-1640 basal medium, supplemented with 10% fetal bovine serum (FBS), 55 µM β-mercaptoethanol, 100 ng/mL recombinant mouse IL-6, 50 ng/mL recombinant mouse IL-21, 30 ng/mL recombinant mouse TGF-β1, 10 µg/mL Invivo anti-mouse IFNγ Recombinant mAb, 10 µg/mL Invivo anti-mouse IL-4 Recombinant mAb and 10 µg/mL a Invivo anti-mouse IL-2 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 Tfh polarization complete medium. Adjust the cell density to 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 Tfh polarization complete medium to maintain optimal cell growth conditions.

4. Cell Restimulation and Intracellular Cytokine Capture
On day 5 of culture, harvest the cells and wash once with ice-cold PBS. Resuspend the cells in RPMI-1640 complete medium containing 10 ng/mL PMA, 1 µg/mL Ionomycin, and 10 µg/mL Brefeldin A. Adjust the cell density to 1×10⁶ cells/mL and incubate in a humidified incubator at 37°C with 5% CO₂ for 5 hours in the dark for restimulation.

3. 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 Tfh 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 Invivo anti-mouse IFNγ Recombinant mAb, 10 µg/mL Invivo anti-mouse IL-4 Recombinant mAb.

2. Absence of Polarizing Cytokines: This medium does not contain any polarizing cytokines and Invivo anti-mouse IL-2 Recombinant mAb.
Apart from the differences in medium composition, the step for cell seeding, medium changes, density monitoring, and the restimulation step on day 5 are performed identically to the Tfh 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.Cell Blocking: Transfer 100 µL of cell suspension per well/tube to a 96-well plate or flow cytometry tube. Add Mouse IgG (Mouse FcR Blocking Reagent) and incubate at 4°C for 30 minutes. Centrifuge at 300 × g for 5 minutes and discard the supernatant.

4.Antibody Incubation: Add 100 µL of PBS containing 10% FBS per well/tube, along with the appropriately diluted APC Rat Anti-Mouse PD-1 Antibody (Cat# S0B5654) (refer to the product manual for the recommended amount). Incubate at 4°C in the dark for 30 minutes.

5.Cell Washing: Add PBS to wash the cells, centrifuge at 300 × g for 5 minutes, and discard the supernatant to remove unbound antibody. Repeat the wash step once. Finally, resuspend the cells in 100-200 µL of PBS.

6.Viability Staining: Add 7-AAD to each sample. Incubate at room temperature in the dark for 5-10 minutes. 

7.Flow Cytometric Acquisition: Acquire the samples on a flow cytometer immediately.

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