Cd64 apc

Heart single-cell suspension and flow cytometry was prepared as previously described [30 ]. Briefly, hearts were perfused, extracted, finely minced, and then incubated with digestive enzymes at 37 °C on a rocking shaker at 50 rpm for 45–60 min. Samples were homogenized with a 40 μm cell strainer. Red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis buffer. Next, samples were centrifuged at 400× g for 5 min at 4 °C, and the pellet was then suspended with FACS buffer. Cells were immune-stained with the following anti-mouse antibodies: CD45-Alexa Fluor^®® 700 (BioLegend, 103128, San Diego, CA, USA), CD11b-PerCP (BioLegend, 101228, CA, USA), F480-PE (BioLegend, 123110, CA, USA), CD206-BV421 (BioLegend, 141717, CA, USA), CD64-APC (BioLegend, 161006, CA, USA), Ly-6G- Brilliant Violet 510™ (BioLegend, 127633, CA, USA), Ly-6C-PE/Cyanine7 (BioLegend, 128018, CA, USA), Propidium iodide (Sigma, 25535-16-4, Darmstadt, Germany). Cells were incubated (30 min, 4 °C) with the antibody mixture in a staining buffer (PBS containing 1% bovine serum albumin and 0.05% sodium azide) and then washed twice with a staining buffer. Cells were acquired using a LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo V.10 software (FlowJo, Ashland, OR, USA).

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.