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

tel-00827710, version 1 - 29 May 2013
2) Magnetic enrichment
Leukocytes were harvested from 15 lymph nodes (2 inguinal, 2 axillary, 2 brachial, 4
cervical- deep and superficial, 2 peri-aortic, and the mesenteric chain) and the spleen. Organs
were mashed and cells transferred in a tube after filtering the cell suspension with a 70µm cell
strainer. Cells were Fc-blocked with anti-CD16/CD32 antibody and stained with PE-labeled
K b -SIINFEKL tetramers in PBS containing 2% FCS and 0.1% of Sodium azide for 30min at
4°C. This was followed by an incubation with anti-PE magnetic microbeads (Miltenyi
Biotech). Cells were passed over a magnetic LS column to enrich tetramer-positive cells.
Bound cells were eluted (“enriched fraction”). A 5µl aliquot was collected for precise
counting of the bound fraction using the Accucheck beads (Invitrogen).
3) Flow Cytometry
Cells were stained with a mixture of antibodies (anti-CD11c, CD11b, CD4, NK1.1, F4/80,
B220, CD3, CD8) to exclude cells that are not of interest (DUMP gate) and focus the
subsequent analysis on CD8 + T cells (Table 8). Prior to analysis, DAPI (Invitrogen) was
added to label dead cells. Cells were analyzed using a FACS Canto II (BD Biosciences). Live,
non-clumped, CD3 + CD8 + tetramer-positive cells were gated. The percentage of tetramer-
positive cells in each sample was multiplied by the total number of cells in the enriched
fraction in order to obtain the absolute number of tetramer-positive CD8 + T cells.
Table 8. Antibodies used for flow cytometry experiments
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