T75 culture flask

Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Basel, Switzerland) and cultured at 37 °C, 5% CO₂, and 20% O₂ in endothelial growth medium 2 (EGM-2, Lonza) supplemented with 5% heat-inactivated fetal bovine serum in T75 culture flasks (Greiner Bio-one). Culture medium was replaced three times a week. HUVEC were used at passage 3–6.

HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (PAN-Biotech), supplemented with 10% fetal bovine serum (FBS) (PAN-Biotech), 1 × MEM Non-Essential Amino Acids (NEAA) (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific). For transfection, 5 × 10⁶ HEK293 cells were plated into T75 culture flasks (Greiner Bio-One). 24 h after plating the cells, cells were transfected with 10-µg plasmid using jetPrime (Polyplus) according to the manufacturer's instructions. Medium was changed four hours after transfection. 24 h after transfection, cells were harvested for further analysis. For immunocytochemistry experiments, 2 × 10⁵ HEK293 cells were plated into a 12-well plate on 12 mm cover slips (YX03.1, Roth). Cover slips were coated with 0.1% poly-lysine (P2636, Sigma-Aldrich) 4 h before plating the cells. 24 h after plating, the cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature (RT).

A. fumigatus conidia were spread onto diluted Sabouraud dextrose agar in T75 culture flasks (Greiner Bio-One, Germany) and incubated at 37 °C for 7 d. Diluted Sabouraud dextrose agar was selected to promote sporulation. Conidia were harvested via immersion in 30 mL phosphate buffered saline (PBS) (Thermo Fisher Scientific, UK) containing 0.05% Tween-80 (Thermo Fisher Scientific, UK). Conidial suspensions were passed through a sterile 40 µm strainer to remove hyphal fragments, washed twice using PBS and then counted using a Neubauer improved haemocytometer (Petrikkou et al., 2001 (link)). For all experiments, suspensions were diluted as required in RPMI (RPMI 1640 + Glutamax, Fisher Scientific, UK).

Primary microglia cells were isolated by the method of (Giulian and Baker, 1986 (link)). Briefly, brains from neonatal mice (P0-P3; mixed gender) were stripped of the meninges and dissociated using mechanical shearing and trypsin (Life Technologies, Carlsbad, CA, USA). Cells of two brains were plated on poly-L-lysine (PLL, Sigma-Aldrich by Merck KGaA, Darmstadt, Germany) coated T75 culture flasks (Greiner bio-one, Kremsmünster, Austria) and cultivated in DMEM (GIBCO by Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS; GIBCO) and 1% penicillin/streptomycin (P/S; GIBCO). On the next day, cells were washed three times with DPBS (GIBCO) to remove cellular debris and cultured with DMEM supplemented with 10% FCS, 1% P/S and 1% L929 conditioned medium as a source of growth factors. After 7-10 days loosely attached mature microglia were shaken off the astrocytic layer with a repetition of the harvesting procedure all two to three days for up to three times. For experiments, primary microglia were seeded into well plates and allowed to adhere overnight in DMEM complemented with 1% N-2 supplement (GIBCO) before experiments were performed.

A. fumigatus conidia were spread onto Sabouraud dextrose agar in T75 culture flasks (Greiner Bio-One, Germany) and incubated at 37 °C for 7 d. Conidia were harvested via immersion in phosphate buffered saline (PBS) (Thermo Fisher Scientific, UK) containing 0.05% Tween-80 (Thermo Fisher Scientific, UK). Conidial suspensions were filtered using a 40 µm strainer, washed twice using PBS and counted using a Neubauer improved haemocytometer.

CD14⁺ monocytes were purified from the buffy coat of each donor sample by centrifugation through Ficoll-Paque (Amersham, GE Healthcare, Little Chalfont, UK) and then isolated by immunomagnetic separation 48 h after sampling, as previously described.^{74 (link)} Briefly, PBMCs were suspended in PBS containing 0.5% BSA and 2 mmol·L⁻¹ EDTA, and CD14⁺ cells were purified using antihuman CD14 magnetic particles (BD Biosciences, San Jose, CA, USA) according to the supplier’s instructions. Cells were seeded at a density of 5 × 10⁶ cells/T75 culture flasks (Greiner, Frickenhauser, Germany) in αMEM (Invitrogen, Carlsbad, CA, USA) containing 10% FBS (Sigma-Aldrich, St. Louis, MO, USA) and 25 ng·mL⁻¹ M-CSF (R&D System, Abingdon, UK). Cells were differentiated into mature OCs over nine days with M-CSF and RANKL (R&D System), as previously described.^{45 ,75 (link)} After nine days of maturation, 12 systematic and evenly distributed images of OCs from each donor were taken using a CKX41 microscope with an SC30 camera (Olympus Corporation, Shinjuku, Tokyo, Japan). These images were used to manually count the number of OCs (with ≥2 nuclei) and the mean number of nuclei/OC. A representative example from three different donors is displayed in Supplementary Information 3A.

Non-immortalized HBMEC originating from adult human brain cortex (Cell Systems, Kirkland, WA, USA, ACBRI 376) were cultivated in gelatin (Serva Electrophoresis, Heidelberg, Germany) coated T-75 culture flasks (Greiner Bio-One, Frickenhausen, Germany). Cells were propagated in RPMI-1640 medium (Sigma-Aldrich), supplemented with 10% fetal calf serum (FCS) (Thermo Fisher Scientific, Waltham, MA, USA), 10% Nu-Serum (BD Biosciences, San Jose, CA, USA), 2 mM L-glutamine (Thermo Fisher), 1 mM sodium pyruvate (Thermo Fisher), 1% minimum essential medium non-essential amino acids (Thermo Fisher), 5 U/ml heparin (Biochrom, Berlin, Germany), and 0.3% endothelial cell growth supplement (Cell Systems). Cultures were kept in a humid atmosphere at 37 °C with 5% CO₂. Confluent monolayers were expanded as described previously [19 (link)], and experiments were coherently conducted with recently thawed cells at passage 8. Basic endothelial cell attributes of HBMEC (characteristic spindle-shaped growth pattern and expression of the endothelial marker CD31) as well as inducibility of intercellular adhesion molecule 1 had been confirmed in preliminary experiments [19 (link)].