Isolation of Primary Alveolar Macrophages

Primary alveolar macrophages were isolated from rodents following established protocols with minor modifications [58 (link)]. Briefly, mice and rats were killed and the lungs were carefully removed. The lung was cannulated via the trachea and BAL was performed using 10×5 ml ice-cold PBS. The lavage fluid was centrifuged at 400 g for 5 min and the pelleted cells were resuspended in PBS. Cell viability was determined by trypan blue exclusion. The cells were cultured in Ham’s F-12K medium supplemented with 15% FBS for 45 min at 37 °C and the non-adherent cells was washed away with PBS. The adherent cells were recovered and diluted as needed. Alveolar macrophages were identified as F4/80⁺ and CD11c⁺ cells by flow cytometry using anti-F4/80 (Abcam) and anti-CD11c antibody (Abcam) [59 (link)].