C57bl 6 mice

Previously described IRAP^–/– mice on an Sv129 background obtained from S. Keller were back-crossed up to 10 times to C57BL/6 mice obtained from Janvier (St. Quentin-Fallavier, France). While for some experiments mice with a lower number of back-crosses were used, cross-presentation experiments were always performed with mice on an identical genetic background. Control mice were either mixed background or C57BL/6 mice bred in our facility or C57BL/6 mice purchased from Janvier. Recombination-activating gene (RAG)1-deficient OT-1 T cell receptor transgenic mice were obtained from Taconic (Germantown, NY, United States) and bred in our animal facility. Animal experimentation was approved by the Comité d’Éthique pour l’Expérimentation Animale Paris Descartes (no P2.LS.156.10). Murine bone marrow-derived DCs (BM-DCs) were produced in vitro by culturing cells extruded from large bones for 6–8 days in complete medium [Iscove’s modified Dulbecco’s medium (IMDM) complemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 50 mM β-mercaptoethanol supplemented with J558 supernatant containing 20 μg/ml granulocyte-macrophage colony-stimulating factor (GM-CSF)]. Bone marrow-derived DC differentiation and activation were checked by staining with CD11c and CD80 antibodies as described (Weimershaus and van Endert, 2013 (link)).

Male Nude and C57BL/6 mice (8 weeks old) were used as experimental animals (Janvier, France). Eight-week-old male Ccr2^−/− mice and their wild-type (WT) C57BL/6 littermates were purchased from the Jackson Laboratories. All animals were acclimated in the animal research facility at IRSN before irradiation at the age of 10 weeks. Mice were anesthetized by isoflurane inhalation and their right hindlimb was exposed to a single dose of 80 Gy irradiation using an Elekta Synergy Platform delivering 10-MV X-rays at 2.6 Gy/min (Elekta SAS, Boulogne-Billancourt, France). For chimerism assay, 10–12 weeks old GFP+ C57BL/6 mice were used as bone marrow donors (Jackson laboratories, Bar Harbor, ME). Recipient C57BL/6 mice (10 weeks old, Janvier) were exposed to 10 Gy whole body irradiation the day prior to GFP+ bone marrow transplant.

Adult (postnatal day 84-91) C57bl6 mice (all males from JanvierLabs) were injected subcutaneously with pentylenetatrazole (PTZ, 75 mg/kg, dissolved in saline), and recorded right after injection using video recordings. The procedure was made in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals and with the agreement of the Comité National de Réflexion Ethique sur l’Expérimentation Animale (#4018). The sampling of animals, as well as the experimental procedure and analysis of the data were determined based on previous published work. The animals to be used for PTZ vs. Control conditions were randomly chosen from the batch of C57bl6 mice delivered from JanvierLabs. After 20-35 min of observation, animals were killed by cervical dislocation, brains were rapidly extracted on ice and the cortex and the hippocampus were dissected, frozen in liquid nitrogen and stored at –80 °C until use for biochemistry. The first seizures were observed after 6–7 min of injection and a second sequence of seizures and/or abnormal gait were detected after 14–15 min. Two out of three animals injected with 75 mg/kg of PTZ had tonic-clonic seizures with rigid paw extension followed by death and one animal showed only partial clonus (as defined using Racine's scale).