Cell Isolation

Primary cells from untreated WT mice were isolated after anesthesia with ketamine and xylazine and exsanguination by opening the vena cava caudalis. In the case of alveolar epithelial cells, heparin (Rotexmedica) was included in the anesthesia mix.

AMs were isolated as described earlier (Cakarova et al., 2009). Briefly, the lungs were lavaged (2 mM EDTA in PBS, 5 ml), and the cell suspension centrifuged (300 × g, 10 min, 4°C). Afterward, the cells were suspended in RPMI1640 (10% FCS, 1% Glu, 1% P/S) and seeded in cell culture plates. The medium was exchanged 2 h later to remove non-adherent cells. Cell purity and morphology was routinely checked by light microscopy. The next day, the medium was changed to RPMI1640 (2% FCS, 1% Glu) 2 h before infection.

AECs were harvested from perfused and digested lungs (5,000 U dispase) as described previously (Cakarova et al., 2009) with some modifications as follows: after generation of the single cell suspension by passing macerated lungs through different cell strainers (100, 70, and 30 μm), the suspension was centrifuged (200 × g, 10 min) and resuspended in PBS containing 3% FCS and 10 mM EDTA. Unwanted cells were depleted by the addition of biotinylated anti-CD45, anti-CD16/CD32, anti-CD31 antibodies (BD Biosciences) and MagniSort Streptavidin Negative Selection Beads (eBioscience, Frankfurt am Main, Germany). Then, cells were seeded in DMEM (10% FCS, 1% Glu, 1% P/S) in cell culture plates and incubated (37°C, 5% CO₂, overnight). Cell morphology was routinely checked by light microscopy. In addition, FACS staining was performed for cell purity and was always above 90% (see also Cakarova et al., 2009). Two hours before the experiment, the medium was changed to DMEM containing 2% FCS and 1% Glu.

For isolating PMNs, the femurs and tibiae of mice were flushed with PBS, and cells were filtered through a 70 μm cell strainer as described earlier (Shrivastav et al., 2018). PMNs were then positively isolated using the mouse anti-Ly6G MicroBead kit (Milteny Biotec GmbH, Telterow, Germany) according to manufacturer’s instruction, seeded in RPMI1640 containing 2% FCS and 1% Glu and infected immediately. This cell isolation method is considered to provide highly pure and viable cells (Swamydas et al., 2015). Furthermore, the positive selected PMNs can strongly induced proinflammatory cytokines (as assed by ELISA, data not shown), indicating that these cells have not lost their capacity to respond to bacteria.