Before you start
- Coat plates with anti-CD3 the day before (or 2 h at 37 °C).
- Isolate naive CD4⁺ T cells (CD4⁺CD62L⁺CD44^lo CD25⁻).
Reagents and materials
Totals are for 8 wells (+10% extra). Per-well (1 mL, 24-well) amounts are in the middle column.
| Reagent | Per well (1 mL, 24-well) | Total |
|---|---|---|
| Anti-CD3ε coating solution (2–5 µg/mL in PBS)per well, coat overnight at 4 °C | 0.3 mL | 0.3 mL |
| cRPMI | 1 mL | 1 mL |
| Soluble anti-CD28 (final 1–2 µg/mL) | 2 µg | 2 µg |
| Naive CD4⁺ T cells (0.5–1 × 10⁶/mL) | 0.75 × 10⁶ cells | 0.75 × 10⁶ cells |
Buffers and recipes
Complete RPMI (cRPMI) per 500 mL
| RPMI 1640 | 440 mL |
| Heat-inactivated FBS (10%) | 50 mL |
| L-glutamine, 200 mM (2 mM) | 5 mL |
| Penicillin–streptomycin, 100× (1×) | 5 mL |
| 2-Mercaptoethanol, 55 mM (50 µM) | 0.45 mL |
Optional: 10 mM HEPES, 1 mM sodium pyruvate, 1× non-essential amino acids. Use within ~4 weeks.
Polarising conditions (final concentrations, mouse) per well
| Th1 | IL-12 10 ng/mL + anti-IL-4 10 µg/mL (+ IL-2 ~20 U/mL) |
| Th2 | IL-4 10–20 ng/mL + anti-IFN-γ 10 µg/mL (+ IL-2) |
| Th17 | TGF-β1 1–3 ng/mL + IL-6 20–30 ng/mL + anti-IFN-γ 10 µg/mL + anti-IL-4 10 µg/mL (optional IL-1β 10 ng/mL, IL-23 20 ng/mL) |
| iTreg | TGF-β1 2–5 ng/mL + IL-2 100 U/mL + anti-IFN-γ + anti-IL-4 (10 µg/mL each) |
| Th0 (control) | Anti-CD3/CD28 only, or with anti-IFN-γ/anti-IL-4 |
Ranges reflect common published conditions; titrate cytokines and check each new lot.
Equipment
- 24- or 48-well tissue-culture plates
- 37 °C, 5% CO₂ incubator
- Biosafety cabinet
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Wash the coated plate
Remove the anti-CD3 solution and wash wells twice with PBS.
Prepare cells and cytokines
Resuspend naive CD4⁺ T cells in cRPMI with soluble anti-CD28 and the cytokines/antibodies for each condition.
Plate
Add 1 mL cell suspension per well (0.5–1 × 10⁶ cells).
Culture
Incubate at 37 °C, 5% CO₂ for 3–5 days. Split or add fresh medium with the same cytokines if the medium turns yellow.
Read out
Restimulate for intracellular cytokines (IL-17A, IFN-γ, IL-4) or stain FoxP3; collect supernatants for ELISA if needed.
Critical points
- TGF-β1 dose decides Th17 vs Treg: low TGF-β with IL-6 favours Th17; higher TGF-β with IL-2 favours Treg.
- Serum lots contain TGF-β and vary — test lots for Th17/Treg work.
- Naive-cell purity strongly affects polarisation.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Low IL-17A | Use naive cells, check IL-6 and TGF-β activity, add IL-1β/IL-23, block IFN-γ and IL-4. |
| Low FoxP3 in iTreg | Increase TGF-β1, add IL-2, check anti-CD3 coating concentration (too strong reduces induction). |
| Poor viability | Reduce anti-CD3 coating, check 2-mercaptoethanol, avoid overcrowding. |
Safety
Primary mouse cells: BSL-1/2 practice. Recombinant cytokines: standard laboratory care.
Further reading
- Current Protocols in Immunology: in vitro differentiation of mouse T-helper subsets.
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.