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

tel-00827710, version 1 - 29 May 2013
- forward TCCATCGGCGCAGCAAGCATGGAA
- reverse ATGTCTAGAAGGGGAAACACATCTGCC
3) Flow cytometry
To analyze remaining injected splenocytes at different time points post-immunization, CD45.
1 mice were immunized with a mixture of 3 types of CD45.2 splenocytes labeled with 3
different concentrations of CFSE: WTmOVA 5µM CFSE, K bm1 mOVA 1,5µM CFSE and
β2m -/- mOVA 0,5µM CFSE. At different time points post-immunization the spleen and lymph
nodes were harvested and cells were labeled for CD45.2. The 3 populations of injected
splenocytes were identified based on the differing degrees of CFSE staining.
V. CYTOKINE ANALYSIS
Plasma samples were harvested and conserved at -80°C for analysis. Mouse IFNa level was
quantified by ELISA following manufacturer’s instructions (PBL biomedical).
VI. LISTERIA INFECTION
The Ovalbumin-expressing Listeria monocytogenes is a kind gift from N. Glaichenhaus. Mice
were infected i.v. with 5x10 5 colony forming units (CFU). Two days later, the spleen and the
liver were harvested and homogenized in 0.2% NP-40 in water, and serial dilutions were
plated to determine the CFU per organ.
VII. STATISTICAL ANALYSIS
These analyses were performed in collaboration with Sandrine Leroy
Data is plotted with bars representing median values. Non-parametric (two-tailed) Mann-
Whitney tests were used to compare the distributions between two conditions. In some
instances, selective comparisons between two groups within a multi-parameter experiment
were also performed using a non-parametric Mann-Whitney test. Continuous measurements
were studied over time or according to the peptide concentration unsing general linear
modeling. Statistical analysis was performed using Stata 11 software (StataCorp, College
Station, TX USA) and Prism 5 (Graphpad Software Inc., La Jolla, CA USA).
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