Unprimed T Cell Activation – Antibody Stimulation Methods

Reagents

Phosphate buffered saline (PBS, cat. #BUF036A)

Anti-CD3 and CD28 antibodies - see Table 1

Cell media

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 4^oC or 1–2 hr at 37^oC. For the unstimulated control add 50–100 µl of sterile PBS.

3. Remove supernatant and wash the plate with PBS to remove unbound antibody.

4. Isolate PBMC using the appropriate methods, for example, for human peripheral blood refer to protocol FC2 Preparation of Human Peripheral Blood Mononuclear Cells, for mouse and rat spleen refer to protocol FC3 Preparation of Peritoneal Macrophages, Bone Marrow, Thymus and Spleen Cells.

5. Resuspend in the appropriate media and adjust to 1 x 10⁶ cells/ml.

Optional step

For proliferation studies using CytoTrack^™ Cell Proliferation Assay or CFSE, incubate cells with the dye following the recommended protocol, or see protocol FC18 Measuring Cell Proliferation Using Cell Permeable Dyes.

6. Add 1 x 10⁵ cells to each well.

7. Add CD28 antibody at 1–5 µg/ml and incubate cells in a humidified 37^oC, 5% C0₂ incubator for 6–96 hrs as required.

8. Harvest cells and perform surface or intracellular staining as required. Note: cytokine staining requires golgi inhibitors such as brefeldin A and monensin, see protocol FC9 Direct Immunofluoresence Staining of Intracellular Cytokines in Blood for more details.

9. Acquire by flow cytometry.

Notes

Appropriate controls should be carried out for flow cytometry, consider including the following:

• Unstained cells (should always be included to monitor autofluorescence)
• Unstimulated control