Cd11b and f4 80

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.

Primary bone marrow-derived macrophages (BMDMs) were prepared as previously described [21 (link)]. Male BALB/c mice were used to isolate BMDMs for the efficacy study whereas for the biodistribution study, mice with a mixed background (L2G85 mice bred to wild-type FVB mice) expressing the CAG-luc-eGFP L2G85 transgene were used (FVB-Tg(CAG-luc,-GFP)L2G85Chco/J). Briefly, femurs and tibias of mice (8-10 weeks) were harvested and muscle tissue removed from the bones in a sterile fume hood. Bone marrow was flushed from the bones using a sterile syringe with Dulbecco's modified Eagle's medium (DMEM): F12 cell culture medium (Gibco) supplemented with 10% foetal bovine serum, 2 mM glutamine, and 1x penicillin/streptomycin (Invitrogen). The bone marrow was suspended in the medium before being passed through a cell strainer (40 μm) and then cultured in DMEM: F12 media containing 20 ng/ml murine recombinant macrophage colony-stimulating factor (MCSF-1). Bone marrow suspensions were cultured at 37°C and 5% CO₂, and medium was replaced every other day. On day 7, macrophages were considered fully differentiated as determined by the expression of both CD11b and F4/80 (BioLegend) by flow cytometry. Mature BMDMs were then polarised towards an M2-like phenotype by the overnight addition of recombinant murine interleukin- (IL-) 4 (20 ng/ml).