Accuri6 flow cytometer

Flow cytometry was performed in single-cell suspensions from mouse bronchial alveoli lavage fluid (BALF) as reported previously (31 (link)). The pellet was then re-suspended in 10 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 resuspended 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). Neutrophils were gated as 7AAD⁻Ly6G⁺CD11b⁺ and analyzed according to the manufacturer’s guidance on a BD Accuri6 flow cytometer. All antibodies used are listed in Table. 1.

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)).

Flow cytometry was performed in single cell suspensions from mouse lung as reported^{(12 (link))} before and in brief as follows: freshly harvested sections of mouse lung were minced and incubated in 50 μg/ml Liberase solution (Roche) for 30 min at 37°C and agitated at 750rpm. The cells were then disassociated with an 18-gauge needle. The tissue digest was passed through a 40 μm cell strainer into a tube with wash buffer and centrifuged at 400 × 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 × 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). IL-17A⁺ and Foxp3⁺ cells were analyzed according to manufacturer’s guidance on a BD Accuri6 flow cytometer.