S1620

Primary BMDMs were grown for 6 days in IMDM (12440–053, Gibco) supplemented with 10% FBS (S1620, lot number 221C16, Biowest), 30% L929-conditioned medium and 1% penicillin and streptomycin. BMDMs (1 × 10⁶) were seeded in 12-well cell culture plates (3513, Costar) in DMEM (11995–065, Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin before stimulation with ligands. For infection granulocyte macrophage colony-stimulating factor (GM-CSF)–derived bone marrow cells (denoted as BMDMs) were prepared as previously described and infected with A. fumigatus conidia at a multiplicity of infection (MOI) of 10 for 18 h. For priming, BMDMs were incubated with 100 ng/mL of LPS (tlrl-smlps, Invivogen) for 3 h and washed before infecting with A. fumigatus as described above.
For stimulation with translation inhibitors, BMDMs were incubated with 1 μg/mL of Pam3CSK4 (tlrl-pms, Invivogen) for 4 h, washed, and incubated with 25 μg/mL of anisomycin (11308, Cayman Chemical), 50 μg/mL of cycloheximide (01810, Sigma-Aldrich) or 50 μg/mL of puromycin (ant-pr-1, Invivogen).

Primary BMDMs were cultured for 6 days in Iscove's modified Dulbecco's medium (Thermo Fisher Scientific, 12440-053) supplemented with 10% FBS (Biowest, S1620), 30% L929-conditioned medium, 1% nonessential amino acids (Thermo Fisher Scientific, 11140-050), and 1% penicillin and streptomycin (Thermo Fisher Scientific, 15070-063). Then BMDMs were seeded into 12-well plates at a density of 1 million cells/well and incubated overnight before use.