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The non-pulsed control population was labeled with a low concentration (0.25 mM) of CFSE. CFSE high-and CFSE low-labeled cells were mixed in a 1:1 ratio and injected i.v. into the mice 6 days after the immunization. The number of CFSE-positive cells remaining in the spleen after 19 hours was determined by flow cytometry, and the percentage of specific lysis was calculated as follows: % specific lysis = 100, vivo killing assay Mice were injected i.v. with PBS, 10 6 TUs IDLV-OVA, 100 mg OVA with 25 mg 5 0 ppp-dsRNA and 4 mL in vivo-jetPEI, 2007. ,
Ninety-six-well Multiscreen-HA sterile plates (Millipore, Molsheim, France) were coated with purified anti-IFN-g mAbs (Murine IFN-g ELISpot Pair from Diaclone). Plates were washed and blocked with complete culture medium for 2 hours before adding the cells. Various numbers of splenocytes from immunized and control mice were then added (2 to 4 3 10 5 per well) in the presence or absence of 10 mg/mL of OVA 257-264 peptide. The cells were cultured O/N at 37 C. They were then washed and the biotinylated mAbs were added (Murine IFN-g ELISpot Pair from Diaclone) in a solution of PBS 1% BSA. One hour and half later, the plates were washed and streptavidin-alkaline phosphatase (AKP) was added to the wells in a solution of PBS 1% BSA, ELISPOT assay Mice were injected i.v. with PBS, 10 6 TUs IDLV-OVA, 100 mg OVA with 25 mg 5 0 ppp-dsRNA and 4 mL in vivo-jetPEI, 2004. ,
For each mouse, the number of peptide-specific IFN-g-producing cells was determined by calculating the difference between the number of spots generated in the absence and in the presence of the OVA 257-264 peptide ,
, ) and then by FACS ARIA after labeling with anti-CD11c/eF450 (N418), anti-B220/PE (RA3-6B2), anti-CD317/ APC (eBio927) and anti-CD3e/FITC (17A2) antibodies. The purity was always > 96.3% of live cells. RNA from purified CD11c + cells was extracted from cell lysates with the RNeasy Plus microkit (QIAGEN, Courtaboeuf, France). cDNA was generated using a RT2 First Strand Kit and quantified using the RT2 Profiler Mouse antiviral responses and NF-kB signaling pathway PCR arrays (SABiosciences, QIAGEN, Courtaboeuf, France), according to the manufacturer's instructions. The results were analyzed using the SA Biosciences Data Analysis Web Portal, CD11c + CD3e-B220-CD317-splenic cells from immunized mice were first magnetically sorted with anti-CD11c-Beads
, Genotyping of mice with NEMO-deficient CD11c + cells To generate mice with a conditional depletion of the NF-kB pathway in CD11c + cells, offspring were genotyped using PCR for CD11cCre and for Nemo flox at each generation. DNA was extracted from biopsies of the offspring obtained after crossing CD11c-Cre mice with Nemo flox mice using the DNeasy Blood & Tissue Kit
, Reactions were performed with 2 mL of genomic DNA in a 10 mL total volume containing 1x reaction buffer (Invitrogen, Fisher Scientific distributor, Illkirch, France), 2 mM MgCl2 (Invitrogen), 0.2 mM of each dNTP
, The PCR conditions were as follows: 1 cycle of 3 min at 94 C, followed by 35 cycles of 30 s at 94 C, 1 min at 64 C, 1 min at 72 C with a final extension step of 2 min at 72 C in a CFX96 BioRad Thermocycler. PCR products were analyzed by gel electrophoresis on 2% agarose gels stained with ethidium bromide. The resulting PCR product sizes were 313 bp for the transgene
, mM MgCl2 (Invitrogen), 0.2 mM of each dNTP (Roche, Sigma Aldrich distributor, St Quentin Fallavier, France), 0.2 mM each of the primers and 1 unit of Platinum Taq polymerase (Invitrogen). PCR conditions were: 1 cycle of 2 min at 94 C, followed by 30 cycles of 30 s e5, Cell Reports, vol.2, pp.1242-1257, 2019.