In Vitro Human T Cell Activation Using anti-CD3/anti-CD28 Antibodies

Materials Needed

Cells & media

• Primary human PBMCs or purified human T cells
• Complete T-cell culture medium (e.g., RPMI + serum + L-glutamine + antibiotics)
• Optional cytokines (e.g., IL-2) depending on downstream goals

Antibodies

• Purified anti-human CD3 antibody (In Vivo Star™ Functional Antibodies, clones OKT3, UCHT1, SP34-2, or 12F6)
• Purified anti-human CD28 antibody (In Vivo Star™ Functional Antibodies, clones 28.2, 15E8, or 9.3)

Plasticware, consumables, and equipment

• Tissue culture-treated plates (24- or 96-well commonly used)
• Sterile PBS (without calcium/magnesium)
• Biological safety cabinet
• CO₂ incubator (37°C)
• Centrifuge
• Hemocytometer or automated cell counter

Protocol for Plate-Bound Activation

1. Plate coating
   1. Prepare antibody coating solution by diluting anti-CD3 antibody in sterile PBS to the desired working concentration (0.5-10 μg/ml range; 96-well plates typically use a lower concentration range, 0.5-5 μg/ml, while 24-well plates use a higher range at 1-10 μg/ml. Optimize empirically.)
   2. Dispense enough solution to fully cover the culture surface of each well.
   3. Incubate plate for 1–2 hours at 37°C or overnight at 4°C to allow adsorption.
   4. Wash by removing the coating solution and gently rinsing wells with sterile PBS to remove unbound antibody.
2. Cell preparation
   5. Obtain PBMCs or purified T cells using your preferred isolation method.
   6. Wash cells and resuspend in complete culture medium.
   7. Adjust cell concentration to the desired seeding density (optimize for your assay, typically 0.5-2 x 10⁶/ml).
3. T-cell activation
   8. Add the prepared cells into anti-CD3-coated wells. (200 μl for 96 well plates or 1-2 ml for 24 well plates.)
   9. Add soluble anti-CD28 antibody directly to the culture medium at the empirically determined working concentration. (Standard co-stimulation is typically 1-5 μg/ml. For strong co-stimulation, use 5-10 μg/ml.)
   10. Add cytokines (e.g., IL-2) if expansion or survival support is desired.
   11. Culture cells in a 37°C, 5% CO₂ incubator.
4. Culture maintenance
   12. Monitor cells daily for clustering, proliferation, and viability.
   13. Refresh medium and cytokines depending on cell density and duration of culture.
   14. Harvest cells or supernatant at desired timepoints for downstream assays (flow cytometry, cytokine analysis, functional studies, etc.).

Tips and Tricks

• Empirical optimization is critical - antibody concentrations, cell density, and cytokine supplementation dramatically influence activation strength. Consider setting up a matrix to assess dilutions of both antibodies in a single assay.
• Antibody clone matters - Different clones have different potencies. Start at mid-range and adjust.
• Cell purity matters - contaminating cell types can alter activation kinetics.
• Avoid over-stimulation - excessive signaling can lead to activation-induced cell death.
• Cluster formation is a good sign - visible aggregation often correlates with effective activation.
• Include proper controls - unstimulated and single-signal controls help interpret downstream data.
• Gentle handling preserves viability - activated T cells can be fragile.
• Timepoints matter - early signaling events occur within hours, while proliferation peaks days later. Activation markers (CD25, CD69) can be assessed after 48-72 h.
• Edge wells evaporate faster - consider filling unused wells with PBS or avoid using edge wells, especially for 96-well plates.