Protocol: Unprimed T-Cell Activation: Antibody Stimulation Method

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)
• CD3 and CD28 antibodies (see Table 1)
• Sterile phosphate buffered saline, 10x (PBS) (BUF036A)
• PBS containing 1% bovine serum albumin (PBS/BSA)
• Required when staining cell surface antigens:
  • Staining Buffer (BUF073)

Table 1. Anti-CD3 and CD28 antibodies.

Methods

1. Prepare a 5–10 µg/mL solution of anti-CD3 in sterile PBS.
2. Add 50–100 µL to each well of a 96-well plate, seal, and incubate overnight at 4ᵒC or 1–2 hr at 37°C.
   Note: Prepare wells for unstimulated controls by adding 50–100 µL of sterile PBS.
3. Remove supernatant and wash the wells with sterile PBS to remove unbound antibody.
4. Prepare cells appropriately using sterile techniques. Refer to protocol FC1, Preparation of Tissue Culture Cell Lines for Flow Cytometry, for information on preparation of tissue culture cell lines; FC2, Preparation of Peripheral Blood Mononuclear Cells for Flow Cytometry, for preparation of peripheral blood mononuclear cells; and FC3, Preparation of Peritoneal Macrophages, Bone Marrow, Thymus, and Spleen Cells for Flow Cytometry, for preparation of peritoneal macrophages, bone marrow, thymus, and spleen cells.
5. Resuspend cells in the appropriate cell culture media and adjust to 1 x 10⁶ cells/mL.
6. 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.
7. Add 100 µL cells to each well.
8. Add CD28 antibody at 1–5 µg/mL and incubate cells in a humidified 37°C, 5% CO₂ incubator for 6–96 hr as required.
9. Centrifuge at 300–400 x g for 5 min and discard supernatant.
10. 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.
11. Acquire samples on a flow cytometer.

Notes

• Optimal stimulation conditions will differ depending on the species and origin of your cells and the experimental requirements, so it is recommended to optimize conditions for each of your experiments
• 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 is important to include in a stimulation assay