Cd11b pe cyanine7

To obtain single-cell suspensions from lung tissue, lungs were perfused with sterile PBS and removed en bloc, and perfused lungs were digested in RPMI medium containing collagenase XI (0.7 mg/mL; Sigma-Aldrich) and type IV bovine pancreatic DNase (30 μg/mL; Sigma-Aldrich). RBCs were lysed with RBC Lysis Buffer (BioLegend) as described elsewhere (62 (link)). Single-cell suspensions were incubated with a Fc receptor block (553141, BD Bioscience) to reduce nonspecific antibody binding. The flow cytometry panel used to identify immune cell subtypes is shown in Supplemental Figure 3; in short, antibodies used in these experiments included CD45-Brilliant Violet 650 (catalog 103151), Ly6G-APC/Cyanine7 (catalog 127623), F4/80-PE/Cy5 (catalog 123111), MerTK-Brilliant Violet 605 (catalog 151517), CD11c-AF-700 (catalog 117320), CD11b- PE/Cyanine7 (Cat.101216), MHCII-FITC (catalog 107605), and CD64-APC (catalog 139306) from BioLegend as well as SiglecF-PE (catalog 552126) from BD Biosciences. Dead cells were excluded using DAPI (catalog MBD0015, MilliporeSigma). Flow cytometry was performed using BD LSR II and BD FACS Aria III flow cytometers (BD Biosciences), and data were analyzed with FlowJo software.

To quantify the number of neutrophils, samples were collected in pools containing 50 Tg(lysC:DsRed2)^nz50 transgenic larvae, where lysozyme is marked with red fluorescent protein labeling. The larvae were euthanized as previously described and maintained in a 2% fetal bovine serum (FBS)/1X PBS (phosphate-buffered saline) (Thermo Fisher, Waltham, MA, USA) solution at 4 °C until processing. Subsequently, the samples were macerated in a 40 µm cell strainer (Corning Incorporated, New York, NY, USA) and resuspended in 2% FBS/1X PBS. Cells were incubated with CD11b-PE/Cyanine7 (BioLegend, San Diego, CA, USA) and Ly6G-APC (BD Biosciences, San Jose, CA, USA) antibodies. The samples were examined on a BD FACSCanto II and analyzed using FlowJo v10 (Tree Star) software.