Cf633 α bungarotoxin

After preincubating for 15 minutes with anti-mouse CD16/32 antibodies (eBioscience), Fixable Viability Stain 780 (BD Bioscience) was used to exclude dead cells. Subsequently, lung cells were labeled with antibodies. For staining indirectly labeled antibodies, the primary antibodies were stained for 30 minutes and washed out followed by the secondary antibodies staining. The antibodies used were as follows: APC/Fire 750 anti-mouse F4/80, BV650 anti-mouse/human CD11b, BV421 anti-mouse Ly6G, PE/Cyanine7 anti-mouse Ly6C, BV421 anti-mouse Ki67, PE anti-mouse PDPN, APC anti-mouse CD31, APC anti-mouse CD45, FITC anti-mouse CD326/EPCAM, BV510 donkey anti-rabbit IgG, AF488 donkey anti-rabbit IgG, rabbit anti-pro-Sftpc (Millipore), rabbit anti-mouse CCSP (Proteintech), CF633-α-Bungarotoxin (Biotium) (all others from Biolegend). Debris and aggregates were≠ excluded and live cells were detected on a LSRFortessa (BD Biosciences) and then analyzed by FlowJo X 10.0.7 software (Tree Star Inc.). BD FACS Aria II appliances were used for AT2 lineage–labeled cell sorting; the sorting strategies are shown in the figures. For isolation of lung mesenchymal cells, cells negatively isolated by a CD31 via MagCellect Magnet column (R&D Systems) were further negatively sorted by EPCAM and CD45 via a MagCellect Magnet column.

DMAB-anabasine dihydrochloride and PHA568487 were obtained from Tocris Biosciences (Minneapolis, MN, USA); (−)-nicotine hemisulfate salt, and Escherichia coli 0111:B4 lipopolysaccharide was purchased from Sigma (St. Louis, MO). Anti-mouse CD16/CD32 monoclonal antibody (IM7) was purchased from eBioscience (San Diego, CA, USA). PE rat anti-mouse Ly-6A/E (clone D7) was purchased from BD Biosciences (San Jose, CA, USA). Phospho-Akt1 (Ser473) (D7F10) XP® rabbit mAb (Akt1 Specific) and PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit were obtained from Cell Signaling (Danvers, MA, USA). CF633 α-Bungarotoxin was purchased from Biotium (Fremont, CA, USA). α7nAChR antibody (H-302) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). BV421 rat anti-mouse CD144 was purchased from BD Horizon. Alexa Fluor 488 anti-mouse CD309 (VEGFR2, Flk-1) antibody was purchased from BioLegend. The E. coli K1 (serotype) strain, isolated from patients with biliary infection, was kindly provided by Dr. Thomas Martin (University of Washington, USA) [33 (link)].