Primed T Cell Activation - Antigen Presenting Cell Co-Culture

Reagents

Phosphate buffered saline (PBS, cat. #BUF036A)

Cell media

Lipopolysaccharide (LPS)

Methods

1. Prepare dendritic cells from an appropriate source, for example, murine bone marrow cultured in 10 ng/ml GM-CSF.

2. Incubate dendritic cells with antigen (50-200 mg/ml) overnight in the presence of LPS (100 ng/ml). Antigen should be titrated to obtain reproducible results. Non pulsed dendritic cells should be included as a negative control.

3. Wash the dendritic cells twice in cell culture media. Count and resuspend at 1 x 10⁶ /ml.

4. Isolate T cells and resuspend in media at 1 x 10⁶ cells/ml.

Optional step

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

5. Co-culture the dendritic cells and T cells at increasing ratios, for example, 1:1, 1:5 and 1:10 (DC:T cell) to obtain a range of stimulation.

6. Incubate cells in a humidified 37^oC, 5% C0₂ incubator for 6-96 hrs as required.

7. 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.

8. Acquire by flow cytometry.

Notes

Appropriate controls should always be carried out, for flow cytometry the following should be considered;

• Unstained cells should always be included in the experimental set-up to monitor autofluorescence.
• Unstimulated control.
• Fully stimulated control using PMA and ionomycin or PHA, see FC15 Unprimed T Cell Activation – Pharmacologic Means.