2.23. Isolation of mouse alveolar macrophages

Alveolar macrophages (AMs) were harvested by bronchoalveolar lavage (BAL). Here, we described an adapted protocol as following (Busch et al., 2019). Mice were euthanized. Lungs were lavaged with 0.5 ml of warm BAL buffer (2 mM EDTA and 0.5% FBS in PBS) for nine times. Then the collected BAL fluid (BALF) was cooled on ice and filtered through a 70 μm cell strainer into the 15‐ml tube with 1.5 ml complete medium (RPMI 1640 supplemented with 10% FBS with penicillin and streptomycin). Filtered BALF was centrifuged at 300 × g, 5 min at 4°C. Remove supernatant. A 1 ml haemolysis buffer was added for 2 min incubation at room temperature to lyse residual erythrocytes. Complete medium was added to stop lysis and cells were collected by centrifugation as before. Remove supernatant. Resuspend the pelleted cells and plate with complete medium. Then we collected the adherent cells after 2 h to 4 h of cultivation. For PLD2i (PLD2 inhibition) administration, mice were intraperitoneally injected CAY10594 (2 mg/kg). After 4 h, mice were sacrificed for AM isolation.