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Updated: Jul 17, 2025
An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes
Published on: April 26, 2019
Studying Macrophages in the Murine Steatotic Liver Using Flow Cytometry and Confocal Microscopy
Zhuangzhuang Liu1,2, Pieter A Louwe1,2,3, Charlotte L Scott4,5,6
1Laboratory of Myeloid Cell Biology in Tissue Damage and Inflammation, VIB-UGent Center for Inflammation Research, Ghent, Belgium.
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.
Insights
Researchers developed new methods to identify diverse liver macrophage populations in metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatitis (MASH). This advancement clarifies cellular roles in these liver conditions.
Area of Science:
- Immunology
- Hepatology
- Cell Biology
Background:
- Liver macrophages, previously considered solely Kupffer cells, are now recognized as diverse populations in metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH).
- Understanding the distinct functions of these myeloid cell subsets is crucial for deciphering disease pathogenesis.
Purpose of the Study:
- To establish robust protocols for isolating and identifying distinct myeloid cell subsets within the steatotic liver.
- To provide reliable gating strategies for flow cytometry analysis of liver myeloid cells in MASLD/MASH models.
- To address and mitigate challenges associated with increased autofluorescence in steatotic liver samples.
Main Methods:
- Induction of MASLD/MASH in mouse models.
- Isolation of cells from steatotic livers.
- Development of specific gating strategies for myeloid cell subset identification using flow cytometry and confocal microscopy.
- Implementation of techniques to minimize autofluorescence.
Main Results:
- Successful induction of MASLD/MASH in mice.
- Establishment of protocols for isolating diverse myeloid cell populations from the steatotic liver.
- Defined gating strategies enabling discrimination between distinct macrophage, monocyte, and dendritic cell subsets.
- Methods to overcome autofluorescence interference in flow cytometry and microscopy.
Conclusions:
- Single-cell technologies reveal significant heterogeneity within liver myeloid cells in MASLD/MASH.
- The presented protocols and gating strategies facilitate accurate identification and characterization of these subsets.
- These advancements are essential for future research into the functional roles of liver myeloid cells in metabolic liver diseases.
