This product is designed for the efficient in vitro differentiation of naive CD4⁺ T cells into canonical Tfh cells, generating a high proportion of Bcl6⁺CXCR5⁺PD-1⁺ICOS⁺ functional Tfh cells. It is suitable for mechanistic studies of humoral immunity, germinal center formation, T-B cell interactions, autoimmunity, vaccine responses, and antibody secretion regulation.

Product Components

Reconstitution Method

Naive CD4⁺ T Cell Tfh Polarization Induction Procedure:

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 Tfh 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 Tfh polarization medium at a density of 4-5×10⁵ cells/mL.

Complete Tfh polarization medium: RPMI-1640 basal medium supplemented with 10% 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 anti-mouse IFN-γ functional antibody, 10 μg/mL anti-mouse IL-2 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 Tfh 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 Tfh 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 Tfh induction group, with the main difference being the medium used:

1. 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.

2. No polarizing cytokines are added to this medium.

Apart from these differences, cell seeding, medium exchange, density monitoring, and the day-5 restimulation are identical to those of the Tfh 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, and resuspend the cells at 1×10⁷ cells/mL.

3. Cell blocking: Dispense 100 μL per well into 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 10% FBS in PBS per well with APC Rat Anti-Mouse PD-1 Antibody (refer to the antibody manual for the recommended amount).

5. Cell washing: Wash the cells with PBS to remove residual antibody, and resuspend the cells in PBS.

6. Viability dye staining: Add 7-AAD (BD 559925) per well and incubate at room temperature protected from light for 5 minutes.

7. Proceed to flow cytometry acquisition.

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 PD-1 expression in Th0 (left) and Tfh (right) polarized mouse CD4⁺ T cells. Tfh polarization efficiency: 94.2% (Th0: 9.35%).

Storage