Cd73 clone ty 11.8

Mice were perfused with 10 ml PBS, the lungs removed, washed in PBS, and minced into small pieces. The lungs were then digested for 1 h with RPMI 1640 supplemented with 10% FBS, 1 mg/ml type II collagenase (Worthington, Lakewood, NJ, USA), and 50 U/ml deoxyribonuclease I (Worthington, Lakewood, NJ, USA) at 37°C/ 5% CO₂. Single-cell suspensions were obtained by mashing the digested lungs, and the red blood cells were removed by treatment with a hypotonic lysis buffer (Lonza, Morristown, NJ, USA). Cells were analyzed using flow cytometry. Intracellular cytokine staining (ICS) was performed using the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA, USA). GolgiPlug was added to the digestion media and following red blood cell lysis, the cells were incubated with RPMI 1640 supplemented with 10% FBS and Golgi Plug for 3 more hours at 37°C/ 5% CO₂. Cells were surface stained with anti-mouse CD45 (clone 30-F11, eBioscience, Waltham, MA, USA), Ly6G (clone 1A8, BD Biosciences, San Jose, CA, USA), and CD73 (Clone TY/11.8, eBioscience, Waltham, MA, USA). For intracellular staining, cells were permeabilized and stained with IL-10 (clone JES5–16E3, eBioscience) or isotype control (Rat IgG2b K Isotype Control, Biolegend, San Diego, CA, USA). Fluorescence intensities were measured on a FACSCalibur and at least 25,000 events for lung tissue were analyzed using FlowJo.