Dulbecco modified eagle medium (dmem)

A549 and CTH^−/− cells were maintained at 37°C 5% CO₂ in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) and 1% Penicillin/Streptomycin (Sigma-Aldrich). For all experiments, 10⁵ cells were seeded in 24-well glass bottom plates (Cell Star, Greiner Bio-One) with supplemented DMEM and incubated at 37°C 5% CO₂ until confluency. Monolayers were challenged with 10⁵ spores/ml of A. fumigatus wild type and ΔmecB. Following co-incubation, monolayers were washed 3 times with PBS, and cells were fixed with 4% formaldehyde for 10 minutes and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 2 minutes. Finally, cells were stained with 300 nM DAPI for 5 minutes. Stained epithelial cells were imaged on the Nikon Te-2000 widefield microscope described above, using a 20× (0.75 NA) Nikon CFI Plan Apo Lambda objective lens and a 380-nm LED array module with a Nikon UV-2A for DAPI imaging. The number of epithelial cells remaining after infection was calculated with an in-house batch image processing and counting algorithm in Fiji [71 (link)]. The level of epithelial cell detachment for each condition was calculated relative to the number of cells attached in the negative controls with no fungi. All assays were carried out in biological triplicates.

The EAI assay was performed as described in detail elsewhere [9 (link)]. In brief, erythrocytes obtained from Balb/cJRj mice were purified using a Ficoll gradient (Ficoll-Paque, VWR, Cat#17-1440-02) and were stored for up to 2 weeks in DMEM (Gibco, Cat#61965) containing 10% FCS FCS (Amimed, Cat#2-01F30I) at 4°C prior to use.
Serial dilutions of sera were performed in 96-well U-bottom plates (Greiner, Cat#650161) in DMEM containing 10% FCS. 5x10⁵ cfu B. taylorii expressing GFP were added per well and the plates were incubated at 35°C, 5% CO₂ for 1h prior to the addition of 10⁶ red blood cells (multiplicity of infection, MOI = 0.5) in 100 μl DMEM containing 10% FCS. The next day, the supernatant was removed, the red blood cells were fixed using 1% PFA (EMS, Cat#EMS-15710) and 0.2% gluturaldehyde (EMS, Cat#16020) in PBS (BioConcept, Cat#3-05F29-I) for 10 min at 4°C in the dark. After quenching with 2% FCS in PBS, the cells were analysed for GFP signal by Flow Cytometry (BD Canto II using HTS autosampler).

HEK-Blue IL-33/IL-1β (HEK293-ST2L) cells were purchased from Invivogen (Toulouse, France) and cultivated according to the manufacturer's protocol in DMEM (Greiner bio-one, Frickenhausen, Germany) with 5% FCS (GE Healthcare, IL, USA), 2 mM l-glutamine, 100 IU/ml penicillin and 100 IU/ml streptomycin at 5% CO2, 37°C. 30 μg/ml blasticidin, 200 μg/ml hygromycin B Gold and 100 μg/ml zeocin were added after the second passage to maintain the plasmids encoding IL1R1, co-receptor IL-1RAcP, ST2L, and the gene for SEAP. To analyze IL-33 or IL-1β bioactivity, 5 × 10⁴ HEK293-ST2L cells were resuspended in 180 μl medium and were co-incubated with 20 μl of sample. In some experiments, 5 × 10⁴ HEK293-ST2L cells were resuspended in 160 μl and co-incubated with 20 μl sample and 20 μl serum or plasma for a total final volume of 200 μl. For inhibition of proteases, protease inhibitor cocktail (Sigma-Aldrich, Steinhein, Germany) was added in a dilution of 1:160 directly to the cell suspension. After a 22 h incubation period, 40 μl of medium supernatant were co-incubated with 160 μl QUANTI-blue substrate (Invivogen, Toulouse, France) and SEAP activity was measured photometrically at 635 nm for 2 h every minute. Cell viability of HEK293-ST2L cells was assessed by MTT assay to exclude cytotoxic effects mediated by the samples (Roche Life Sciences, Basel, Switzerland).