Ly6g apc 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.

Mice under indicated conditions were anesthetized, and kidneys were harvested and cut into pieces. Then kidney tissues were digested in a buffer (HBSS supplemented with 0.05% collagenase I and 2 mM CaCl₂) under 37 °C for 25 min. After digestion, the kidney tissues were filtered through a 70-μm nylon mesh. The cell suspension was centrifuged at 500g for 5 min, and the resulting cell suspension was treated with Fcγ receptor blocker (101320, BioLegend) for 10 min followed by incubating with the following fluorescent antibodies (all from BioLegend): CD45 BV421 (103134), CD11b FITC (101206), Ly6G APC/Cyanine7 (127624), Ly6C PE (128008), F4/80 APC (123116), 7AAD (420404), and CD38 PE/Cyanine7 (102717). Flow cytometry was performed on a FACSCanto II (BD Biosciences), and data were analyzed by FlowJo software 10.4.