Voie d'immunisation et séquence d'administration de l ... - TEL

tel-00827710, version 1 - 29 May 2013
3. The absolute number of multimer-positive T cells is the number of multimer-positive
cells within the « single, live, non-dump CD3 + CD8 + » T-cell gate present in the enriched
fraction. (see Note 23)
4. The frequency of circulating multimer-positive cells is defined as the absolute number of
multimer-positive T cells / absolute number of CD8 + T cells.
3.7 Intracellular Cytokine Staining (optimized for mice)
1. Restimulation of cells is performed in vivo. Three hours prior to leukocyte harvest, inject
mice intravenously with 5µg of CpG/DOTAP formulated as a mixture with 1µg specific
peptide (e.g. SIINFEKL peptide in the Ovalbumin model).
2. Perform the staining and enrichment as described in Sections 3.3 and 3.4 with addition
of BD GolgiPlug containing Brefeldin A during multimer and beads incubation steps
(final concentration 1/1000).
3. After elution from the column, resuspend enriched cells in MPB without azide, add
Aqua fluorescent reactive dye (final concentration 1/1000) to stain dead cells (see Note
12) and incubate 30 min at 4°C in the dark.
4. Spin 5 min at 300g at 4°C in 3 mL MPB without azide.
5. Resuspend cells in 100 µL MPB without azide; add the mix of antibodies for surface
staining, and incubate 20 min at 4°C in the dark.
6. Wash with 3 mL of MPB without azide and resuspend thoroughly cells with 250 µL of
Cytofix/Cytoperm reagent. Vortex and incubate 20 minutes at 4°C.
7. Wash with 1 mL of 1xPerm/wash buffer.
8. Incubate cells for 30 min at 4°C with anti-IFNγ antibody diluted in Perm/Wash buffer.
9. Wash cells once with Perm/Wash buffer and once with MPB without azide.
10. Resuspend in 300 μL PBS-2%FCS and acquire sample in flow cytometry (Figure 7).
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