Isolation and Analysis of Liver Macrophages

Liver macrophages were isolated from mice as described (36 (link)). Briefly, mouse livers were cut into small pieces and digested for 1 hour at 37°C in RPMI 1640 medium containing 0.05% collagenase/dispase (Roche) and 0.01% trypsin inhibitor (Gibco, Thermo Fisher Scientific). The liver suspension was pressed through a 40 μm cell strainer, then centrifuged at 800g for 10 minutes at 4°C. The supernatant was removed and erythrocytes were lysed with red blood cell lysis buffer for 2 minutes at 4°C, then centrifuged at 800g for 10 minutes at 4°C. The cell pellet was resuspended in 40% Percoll gradient (MilliporeSigma, P1644). Cell suspensions were loaded on the top of the 80% Percoll gradient and centrifuged at 376g for 25 minutes at room temperature without brake. The middle layer was suspended with 10 mL of RPMI 1640 medium. Cell suspensions were spun at 376g for 5 minutes at room temperature, and supernatant was removed. Cells were resuspended in 2% FCS and 1 × 10⁶ cells were enumerated. Subsequently, cells were stained with F4/80 (BioLegend, catalog 123107) and CD11b antibody (BioLegend, catalog 101211) for 30 minutes in the dark (0.5 μL/1 × 10⁶ cells). Finally, the cells were washed and suspended in 0.5 mL PBS. The stained cells were analyzed using flow cytometry (BD LSRFortessa). Final calculations were performed by FlowJo software (FlowJo LLC).