Anti siglec f microbeads

Lungs from naïve Ifih1^(−/−), Mavs^(fl/fl) and Mavs^(fl/fl)x Itgax-Cre were perfused with 20–30 ml of PBS. Lungs were then removed, injected with 700 μl of collagenase buffer [10ml RPMI, 1ml FBS, 25 μl 0.5M CaCl₂, 25 μl 0.5M MgCl₂, 50 μl HEPES, 100 μl L-glutamine, 100 μl Penn-Strep, 2 μl gentamycin, 25 μl DNase (VWR, Catalog # 77001–900), and 2 ml Liberase (Sigma-Aldrich)], and finally placed in 15 ml conical tube containing 2 ml of collagenase buffer. Lungs were digested or 30min at 37°C while shaking at 160–180 rpm. After which, 5 ml of ice cold stop buffer [25 ml RPMI, 1.25 ml FBS, 50 μl 0.5M EDTA] was added to each tube. Lungs were pushed through a 70 μm filter to generate a single cell suspension. Single cell lung suspensions were labeled with anti-Siglec-F MicroBeads (Miltenyi Biotec, Cat. No. 130-118-513) and selected for using LS Columns (Miltenyi Biotec, Cat. No. 130-042-401). Siglec F⁺ were eluted from the column, spun down, and resuspended in DMEM at 5×10⁶ cells per ml. Subsequently 5×10⁵ Siglec F⁺ alveolar macrophages were transferred i.t. to Mavs^(fl/fl)x Itgax-Cre. Twenty-four hours later mice were challenge with A. fumigatus as described above.

Lung tissue was digested as described above to obtain a single-cell suspension. SiglecF⁺ cells were isolated using anti-SiglecF microbeads (Miltenyi Biotec) and plated in non-treated 6-well plates in RPMI 1640 media containing 1x GlutaMAX, 1x pyruvate, 1x penicillin/streptomycin, 10% fetal bovine serum (FBS), and 20 ng/ml recombinant GM-CSF (Biolegend), as previously described (31 (link)). Culture medium was replaced after 16 h of incubation at 37°C and again every 2 days in culture. Cells were collected on days 1 or 6 after plating. Where indicated, AMs were treated overnight with 5 μM of the Wnt/β-catenin inhibitor XAV939 (Sigma) or 1 μM of the Stat3 inhibitor Napabucasin (Fisher Scientific) before cells were harvested.