Flavoperidol

Mice were euthanized with carbon dioxide. Lungs were perfused with PBS trough the right ventricle until no blood was visible in the tissue. Lungs were removed, minced and placed in a digestion buffer containing collagenase type 4 (1.6 mg/ml; Roche), DNase type 1 (50 units/ml; Roche) and Flavoperidol (1 μM, Sigma-Aldrich) in RPMI 1640 medium. Lungs were incubated at 37 °C for 15 min while on a rotator. Red blood cells were lysed using RBC lysis buffer (eBioscience) at 4 °C for a 5 min incubation. The obtained cell suspension was filtered using a 70-micron filter, washed and cells were resuspended in PBS. Cells were then incubated with anti-Fc blocking mAb (anti CD16/32, clone 93, BioLegend) and viability dye Zombie Aqua (BioLegend) for 10 min. After this, cells where stained with an antibody cocktail containing APC Cy7-conjugated CD45, PE-conjugated F4/80, BV605-conjugated CD11c, v450-conjugated CD11b, PE Cy7-conjugated CD64, AF700-conjufgated Ly6c, FITC-conjugated Ly6g, AF647-conjugated SiglecF mAb for 30 min. All labeled cells were passed through a FACS Aria II flow cytometer (BD Biosciences) and after proper gating 4 subsets of lung MPs were sorted^{29 (link)}. Data were analyzed with FlowJo 8.3.3 software (TreeStar Inc.). The antibodies used for FACS are listed in Supplementary Table 1.