Immunofluorescence staining of cells in combination with PI staining of cells for cell cycle analysis

Primed T Cell Activation - Antigen Presenting Cell Co-Culture

Primed T Cell Activation – Antigen Presenting Cell Co-Culture

Primed T Cell Activation – Antigen Presenting Cell Co-Culture

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This method provides a general procedure for activating T cells prior to staining for intracellular cytokines, activation markers or cell proliferation using dendritic cells that have been pulsed with antigen. Other types of antigen presenting cells may require different amounts of antigen and incubation times.

This protocol is for use with the majority of Bio-Rad reagents. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with the product and batch specific information provided with each vial. A certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques; these are guidelines only and may need to be adjusted for particular applications.

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 37oC, 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;