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Published on: January 22, 2021
Multiparametric Flow Cytometry-Based Immunophenotyping of Mouse Liver Immune Cells
Lenka Vanekova1,2, Marketa Pimkova Polidarova1, Vaclav Veverka1,2
1Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo namesti 542/2, 160 00 Prague, Czech Republic.
Insights
This study presents a rapid protocol for characterizing liver immune cells. The optimized method uses liver perfusion and enzymatic digestion for accurate immune cell analysis in mice.
Area of Science:
- Immunology
- Hepatology
- Cell Biology
Background:
- The liver's immune microenvironment is critical for its functions and disease outcomes.
- Understanding liver immunity aids in predicting therapeutic responses.
- Characterizing liver-associated immune cells is essential for research.
Purpose of the Study:
- To develop an optimized, simple, and rapid protocol for characterizing the liver immune cell milieu.
- To improve the accuracy of immune cell determination and characterization in liver tissue.
Main Methods:
- Mouse liver perfusion using phosphate buffer saline to obtain immune cell suspension.
- Enzymatic digestion and mechanical dissociation of liver tissue.
- Cell purification followed by multiparametric flow cytometry immunophenotyping.
Main Results:
- Demonstration of a protocol for detecting and quantifying various liver immune cells.
- Identification of liver endothelial cells, T cells, B cells, NK cells, NKT cells, neutrophils, monocytes, dendritic cells, macrophages, and Kupffer cells.
- Successful characterization of the liver immune microenvironment.
Conclusions:
- The presented protocol is effective for characterizing the liver immune microenvironment.
- This method facilitates a better understanding of immune signaling and responses in the liver.
- The protocol is a prerequisite for accurate immune cell determination and can aid in predicting therapy outcomes.
Abstract:
The liver is a complex organ that governs many types of metabolisms, including energy metabolism and other cellular processes. The liver also plays a crucial role in important functions in immunity, and the activity of liver tissue-associated immunity affects the outcome of many liver pathologies. A thorough characterization of the liver immune microenvironment may contribute to a better understanding of immune signaling, the mechanisms of specific immune responses, and even to improved predictions about therapy outcomes. In this paper, we present an optimized, simple, and rapid protocol to characterize the liver-associated immune cell milieu. We believe that the most suitable technique for obtaining a complex immune cell suspension and for removing contaminating blood cells is to perform mouse liver perfusion, using only phosphate buffer saline. Combining an enzymatic digestion and a mechanical dissociation of liver tissue, followed by cell purification, improves downstream applications. This combination is an essential prerequisite for immune cell determination and characterization. We then demonstrate a flow cytometry-based multiparametric immunophenotyping along with a gating strategy to detect and quantify liver endothelial cells, T cells (helper and cytotoxic), B cells, NK cells, NKT cells, neutrophils, monocytes (subsets included), dendritic cells (subsets included), macrophages and Kupffer cells.
