Analysis of DC activation markers and cytokine production

DCs (10⁶/well) were co-incubated with viable or UV-inactivated Aspergillus conidia (MOI = 2) in 6 well plates. For short-term activation studies cells were analysed after 6 h of co-incubation, whereas in long-term interaction studies amphotericin B (2 μg/ml) was added after 6 h to inhibit extracellular fungal growth. Cells were collected at 24 h and blocked for 10 min at RT with Fc receptor blocking solution (Human TruStain FcX^TM). DCs (2–3 × 10⁵ cells) were treated with 1 μg/ml FITC-anti-human CCR7, APC-anti-human CD54, PE-anti-human CD80 or PerCP-anti-human HLA-DR (MHCII) monoclonal antibodies (BioLegend) diluted by FACS staining buffer (dPBS supplemented with 1% heat-inactivated FBS) and incubated for 30 min in the dark at 4 °C. After two washes in FACS staining buffer, cells were fixed in 1% paraformaldehyde and subjected to flow cytometry (LSRII). For preparation of intracellular cytokine staining, Brefeldin A (5 μg/ml, Fluka) was added 6 h before harvest to block intracellular protein transport. Cells were collected at 30 h and treated with Fc receptor blocking solution for 10 min at RT. Cells were fixed with 4% paraformaldehyde for 20 min at 4 °C. To make cell membranes permeable, fixed cells were washed with perm wash buffer (FACS staining buffer containing 0.1% saponin). The cells were stained at 4 °C for 30 min with 1 μg/ml of either PE-anti-human TNF-α, FITC-anti-human IL-1β, PE-anti-human IL-12/IL-23 p40, PE-anti-human IL-4, PE-anti-human TGF-β or APC-anti-human IL-10 monoclonal antibodies (BioLegend) prepared in perm wash buffer. After washing with perm wash buffer, the cells were left in 1% paraformaldehyde at 4 °C in the dark before analysed by flow cytometry. Mean fluorescence intensity (MFI) was analysed by FlowJo software and normalised to mock-infected DCs.