Isolation and Flow Cytometry of Mouse Lung Cells

Single cell suspensions from bronchoalveolar lavage fluid or from mouse lung isolates were subjected to flow cytometry. To isolate single-cell suspensions from mouse lung, the lungs were minced and incubated in 50 μg/ml Liberase solution (Roche) for 30 min at 37C and agitated at 750rpm. The cells were then disassociated with an 18-gauge needle. The tissue digest was passed through a 40μm cell strainer into a tube with wash buffer and centrifuged at 400 x g, 4°C for 5 min. The pellet was then re-suspended in 50 mL 1% BSA (VWR Life Sciences) in PBS and centrifuged at 400 x g, 4°C for 5 min and the supernatant was discarded. The cell pellet was re-suspended at 2.5 × 10⁷ cells/mL in FACS buffer. Single cell suspensions were then incubated with anti-CD16/CD32 (BD Bioscience) to block Fc receptor binding (20 min, 4 °C). Cells were pelleted by centrifugation and re-suspended in primary fluorochrome-conjugated antibodies (see above in ice-cold FACS buffer. After washing with 1% BSA in PBS, at least 100,000 live cells per sample were collected for analysis on a BD Accuri6 flow cytometer, in which neutrophils were counted as Ly6G and CD11b double positive after excluding dead cells which were 7-AAD positive. Data analysis was performed using FlowJo software (FlowJo, LLC) as in (31 (link)).