Studying Macrophages in the Murine Steatotic Liver Using Flow Cytometry and Confocal Microscopy

Abstract

The study of macrophage functions in the context of metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction associated steatohepatitis (MASH) has been hampered by the fact that until recently all macrophages in the liver were thought to be Kupffer cells, the resident macrophages of the liver. With the advent of single-cell technologies, it is now clear that the steatotic liver harbors many distinct populations of macrophages, likely each with their own unique functions as well as subsets of monocytes and dendritic cells which can be difficult to discriminate from one another. Here, we detail the protocols we utilize to (i) induce MASLD/MASH in mice, (ii) isolate cells from the steatotic liver, and (iii) describe reliable gating strategies, which can be used to identify the different subsets of myeloid cells. Finally, we also discuss the issue of increased autofluorescence in the steatotic liver and the techniques we use to minimize this both for flow cytometry and confocal microscopy analyses.

Keywords: Autofluorescence; Confocal microscopy; Diet-induced obesity; Flow cytometry; Kupffer cells; Lipid-associated macrophages; Liver; MASH; MASLD; Macrophages; NAFLD.