Protocol: Primed T-Cell Activation: Antigen-Presenting Cell Co-Culture Method

Reagents

• Cell culture medium
• Dendritic cells from an appropriate source, such as murine bone marrow cells cultured in 10 mg/mL GM-CSF
• Antigen of interest, such as a synthetic peptide
• Lipopolysaccharide (LPS)
• Required when measuring proliferation:
  • CytoTrack Cell Proliferation Assay Kit (catalog 1351202, 1351203, 1351205) or CFDA-SE Cell Proliferation Assay Kit (1351201)
• Phosphate buffered saline, 10x (PBS) (BUF036A)
• Required when staining cell surface antigens:
  • Staining Buffer (BUF073)

Methods

1. Pulse dendritic cells with antigen (50–200 μg/mL) by incubating overnight in the presence of LPS (100 ng/mL). Antigen should be titrated to obtain reproducible results.
   Note: Non-pulsed dendritic cells should be included as a negative control.
2. Wash the dendritic cells twice in cell culture media. Resuspend cells in cell culture media and adjust to 1 x 10⁶ cells/mL.
3. Isolate T cells and resuspend in media at 1 x 10⁶ cells/mL.
4. Optional: For proliferation studies using CytoTrack Cell Proliferation Assay or CFDA-SE Cell Proliferation Assay, incubate cells with the dye following the recommended protocol, or see protocol FC18, Measuring Cell Proliferation Using CytoTrack, a Cell-Permeable Dye.
5. Co-culture the dendritic cells and T cells at increasing ratios, for example, 1:1, 1:5, and 1:10 (DC:T cells) to obtain a range of stimulation.
6. Incubate cells in a humidified 37°C, 5% CO₂ incubator for 6–96 hr as required.
7. Centrifuge at 300–400 x g for 5 min and discard supernatant.
8. Perform staining of cell surface antigens (protocol FC4, Direct Immunofluorescence Staining of Surface Epitopes of Cells and Whole Blood) or intracellular staining (protocol FC7, Direct Immunofluorescence Staining of Intracellular Antigens: The Leucoperm Method) as required along with associated wash steps.
   Note: Cytokine staining requires protein transport inhibitors such as brefeldin A and monensin. See protocol FC9, Direct Immunofluoresence Staining of Intracellular Cytokines in Whole Blood, for more details.
9. Acquire samples on a flow cytometer.

Notes

• Other types of antigen-presenting cells may be used but could require pulsing with different amounts of antigen and different incubation times
• All antibodies should be titrated prior to use to ensure that the optimal concentration is used
• Appropriate controls should be included; for example, an unstimulated control and incubation with non-pulsed dendritic cells