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Isolation of Mononucleated and Binucleated Hepatocytes by Flow Cytometry
Yusuke V Watanabe1, Masaki Nishikawa2, Yasuyuki Sakai1,2
1Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.
Bio-Protocol
|July 28, 2026
Summary
This study introduces a method to separate liver cells (hepatocytes) based on their nucleus count, distinguishing between single and double nuclei within the same ploidy. This allows for a deeper understanding of how different hepatocyte structures impact liver health and disease.
Area of Science:
- Hepatology
- Cell Biology
- Flow Cytometry
Background:
- Polyploid hepatocytes are crucial in liver function, exhibiting diverse nuclear configurations.
- Conventional methods cannot differentiate hepatocytes with identical cellular ploidy but varying nuclear structures (e.g., 1x4n vs. 2x2n).
- This ambiguity limits understanding of functional heterogeneity and roles in tumorigenesis and senescence.
Purpose of the Study:
- To develop and detail a protocol for fractionating mononucleated and binucleated hepatocytes.
- To enable discrimination between hepatocyte subpopulations with the same cellular ploidy but different nuclear configurations.
- To provide a tool for investigating the functional heterogeneity of polyploid hepatocytes.
Main Methods:
- Isolation of primary mouse hepatocytes.
- Staining hepatocytes with the DNA dye Hoechst 33342.
- Utilizing flow cytometry to detect fluorescence differences between mononucleated and binucleated cells.
Main Results:
- Successful isolation of primary mouse hepatocytes.
- Effective staining enabling fluorescence detection.
- Discrimination of mononucleated and binucleated hepatocyte subpopulations achieved.
Conclusions:
- The described protocol effectively separates mononucleated and binucleated hepatocytes.
- This method allows for the investigation of phenotypical differences between hepatocytes with identical cellular ploidy.
- This technique is valuable for studying the functional heterogeneity of polyploid hepatocytes in various liver conditions.
