Protocol: Unprimed T-Cell Activation: Pharmacological Methods

Reagents

• Cell culture medium
• Required when measuring proliferation:
  • CytoTrack Cell Proliferation Assay Kit (catalog 1351202, 1351203, 1351205) or CFDA-SE Cell Proliferation Assay Kit (1351201)
• Appropriate stimulation reagents; see Table 1
• Phosphate Buffered Saline, 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• Required when staining cell surface antigens:
  • Staining Buffer (BUF073)

Methods

1. Prepare cells appropriately using sterile techniques. Refer to protocol FC1 for information on preparation of cells from tissue culture cell lines, FC2 for isolation of peripheral blood mononuclear cells from whole blood, and FC3 for preparation of cells from tissues.
2. Resuspend cells in the appropriate cell culture medium and adjust to 1 x 10⁶ cells/mL.
3. Optional: For proliferation studies using CytoTrack Cell Proliferation Assay or CFDA-SE Cell Proliferation Assay kits, incubate cells with the dye following the recommended protocol or see protocol FC18: "Measuring Cell Proliferation Using CytoTrack, a Cell-Permeable Dye".
4. Add the pharmacological agent to each well; see Table 1 for a list of reagents and suggested concentrations.
   Note: Prepare an unstimulated control by adding an equivalent amount of the solution in which the stimulation reagent(s) were prepared (vehicle control).
5. Incubate cells in a humidified 37ᵒC, 5% CO₂ incubator for 6–72 hr depending on the experimental needs.
6. Centrifuge at 300–400 x g for 5 min and discard supernatant.
7. Resuspend cells at 1 x 10⁷ cells/mL in cold (4ᵒC) PBS/BSA.
8. Add 100 µL of the stimulated and unstimulated control cells into as many 5 mL tubes as required.
9. Perform staining of cell surface antigens (protocol FC4) or intracellular staining (protocol FC7), as required, along with associated wash steps.
   Note: Cytokine staining requires protein transport inhibitors such as brefeldin A and monensin; for more details, see protocol FC9: "Direct Immunofluorescence Staining of Intracellular Cytokines in Whole Blood".
10. Acquire samples on a flow cytometer.

Notes

• As optimal stimulation conditions will differ depending on the species and origin of your cells as well as the experimental requirements, optimization of conditions for each of your experiments is recommended
• All antibodies should be titrated prior to use to ensure the optimal concentration is used
• Appropriate controls should be carried out for flow cytometry; for example, an unstimulated control is important to include in a stimulation assay