Isolation of Liver Monocytes from V. vulnificus-Infected Mice

We intraperitoneally injected the mice with 1 mg BrdU (BD Bioscience) in 200 μl PBS, then with1×10⁸ CFU of V. vulnificus two hours later. We harvested the liver from each infected mouse six hours after infection, isolating liver monocytes by following the previous protocol [42 (link)]. Briefly, livers were mashed in IMDM with 10% FBS. The upper cell suspension was filtered by nylon mesh and spun at 4°C, 2000 rpm for 5 min. The cell pellet was resuspended in 12 ml 35% Percoll (GE Healthcare), and carefully underlaid with an equal volume of 75% Percoll, and centrifuged at 1000 × g for 20 min at room temperature without break. Cells at the interface were collected, washed and resupended in IMDM (10% FBS) for further staining. We stained the cell surface with CD11b and F4/80 (Biolegend) and used a BrdU Flow Kit (BD Bioscience) for BrdU intracellular staining, following the manufacturer’s protocol.