15.2) mAbs or isotype-matched controls (all from eBiosciences). Fluorescence was analyzed on a FACSaria cytofluorometer (Becton Dickinson, Erembodegem, Belgium) and results were analyzed using the Flowjo software (Tree Star, Ashland, OR). Three days after irradiation, mice were injected s.c. with 500 μg BSA or OVA in the absence or presence 10 μg CpG-ODN, 1 μg GM-CSF and 1 μg sCD40L. For ex vivo experiments, spleen cells were isolated one day later and cocultured with OT-1 CD8+ T cells

for 18 h (cell ratio 1:2). T-cell activation was evaluated by quantifying IL-2 and IFN-γ by ELISA (BD Pharmingen, San Diego, CA) in the supernatants. For in vivo experiments, mice were injected i.v. one day later with 2×106 CFDA-SE-labeled check details OT-1 CD8+ T cells. Spleen and draining LN cells were collected two days later and the proliferation OT-1 CD8+ T cells was determined by evaluating CFDA-SE staining

Bortezomib ic50 by FACS. To evaluate in vitro the cross-presentation activity of microglia, CD11b+ CNS cells were isolated three days after irradiation, incubated for 8 h with 100 μM BSA or OVA. Then, 1×105 CD11b+ CNS cells were cocultured with 2×105 OT-1 CD8+ T cells for 18 h. T-cell activation was evaluated by quantifying IL-2 and IFN-γ by ELISA in the supernatants. To evaluate ex vivo and in vivo cross-presentation activity of microglia, mice were intracranially injected with 200 μg OVA or BSA (+/−10 μg CpG-ODN, 1 μg GM-CSF and 1 μg sCD40L), three days after irradiation. For ex vivo assay, CD11b+ CNS cells were magnetically sorted the day after and incubated with OT-1 CD8+ T cells

(cell ratio 1:2) for 18 h. T-cell activation was evaluated by quantifying IL-2 and IFN-γ by heptaminol ELISA in the 24 h culture supernatants. For the in vivo assay, mice were additionally injected the day after with 2×106 CFDA-SE-labeled OT-1 CD8+ T cells in the brain. CNS cells were collected two days later for FACS analysis. CD11b+ cells were analyzed for CD11b, H2-Kb, I-Ab, CD80 and CD86 staining. OT-1 CD8+ T-cell proliferation was evaluated by FACS analysis of CFDA-SE labeling. OT-1 CD8+ T-cell activation was evaluated by quantifying IFN-γ production, using the mouse IFN-γ secretion assay kit (Myltenyi Biotec). Briefly, brain cells were incubated 3 h with the OVA peptide SIINFEKL (Affiland), 10 min on ice in the presence of mouse IFN-γ catch reagent, before additional 45 min incubation at 37 °C in RPMI medium. Cells were then labelled for 10 min on ice with the allophycocyanin IFN-γ detection reagent. Cell flourescence was analyzed by flow cytometry. Data are shown as mean ± SD and were analyzed by the Student’s t test to reveal significant differences (*p < 0.05; **p < 0.005; ***p < 0.0005). GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA) was used for all statistical analyses.