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

tel-00827710, version 1 - 29 May 2013
thus giving a positive signal in one fluorescent channel, which will be referred to as the
« dump channel » in our gating strategy (see Section 3.5 and Figure 5A)
4. Viability marker that will specifically stain dead cells (e.g., DAPI Nucleic Acid Stain,
Invitrogen) (see Note 10)
5. Fluorescently labeled mAbs including at least an anti-CD8 antibody. Others will be
chosen depending on the desired phenotypic characterization of target T cells (see Note
11)
6. For Intracellular Cytokine Staining (ICS) in mice: CpG formulated with DOTAP, and
specific peptide for in vivo restimulation
7. For ICS, BDCytofix/Cytoperm Fixation/Permeabilization solution kit with BD
GolgiPlug containing Brefeldin A (BD biosciences)
8. For ICS, LIVE/DEAD fixable dead cell stain kit such as Aqua (Invitrogen) (see Note
12)
3. Methods
Please note that we describe in this section both mice and human protocols, which suppose
the reader to be careful to specific human or mice reagents and buffers.
For human, you will start with HLA typing (Section 3.2), then stain with multimer(s)
(Section 3.3), optionally pursue by enrichment (Section 3.4), and finally acquire your
samples on flow cytometer (Section 3.5) and evaluate precursor frequency (Section 3.6).
For mice, you will start with mice dissection (Section 3.1), then stain with multimer(s)
(Section 3.3), and optionally pursue the experiment by enrichment (Section 3.4), and/or
intracellular cytokine staining (Section 3.7). In all cases you will acquire your samples on
flow cytometer (Section 3.5), and evaluate precursor frequency (Section 3.6).
3.1 Mice dissection
1. Harvest 15 lymph nodes (2 inguinal, 2 axillary, 2 brachial, 4 cervical- deep and
superficial, 2 peri-aortic, and the mesenteric chain) and the spleen in a 60mm-Petri dish
containing 2 mL of Mice R-10 buffer.
2. Mash the organs and transfer the cells into a Falcon 15mL tube after filtering the cell
suspension with a 70µm cell strainer.
3. Wash the well with 3x1 mL Mice R-10 to recover the maximum of cells.
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