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Updated: Aug 9, 2026
Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting
Published on: May 1, 2015
Isolation, purification and flow cytometric analysis of human intrahepatic lymphocytes using an improved technique
Mohamed A Morsy1, Paul J Norman, Ragai Mitry
1Institute of Liver Study, King's College Hospital, London, UK.
Insights
Researchers developed improved methods for isolating intrahepatic lymphocytes (IHL), yielding higher viability. These techniques reveal the liver harbors diverse immune cells, including CD8+, NK, and gammadelta T cells, highlighting its immunological importance.
Area of Science:
- Immunology
- Cell Biology
Background:
- The liver is a significant site of immunological activity, hosting diverse intrahepatic lymphocytes (IHL).
- Efficient isolation and characterization of IHL are crucial for understanding liver immunity but present technical challenges.
Purpose of the Study:
- To develop and optimize protocols for the isolation, purification, and flow cytometric analysis of intrahepatic lymphocytes (IHL).
- To characterize the immunophenotypes of IHL populations in histologically normal liver tissue.
Main Methods:
- Developed novel protocols for IHL isolation, reducing enzyme exposure time.
- Utilized monoclonal antibody (mAb)-coated magnetic microbeads for purification of specific IHL subsets.
- Optimized flow cytometric analysis by combining CD45 staining with light scatter properties.
Main Results:
- Achieved higher IHL viability (92-95%) compared to existing methods (39-86%).
- Identified a heterogeneous IHL population in normal liver, with CD8+ T cells being predominant (CD4/CD8 ratio of 1:1.7).
- Found significant proportions of IHL expressing gammadelta T cell receptor (up to 40%) and CD56 (NK marker, one-third).
Conclusions:
- The optimized techniques enable high-viability isolation and detailed characterization of IHL subsets.
- The normal liver contains a diverse array of immune cells, including CD8+, NK, NKT, and gammadelta T cells, underscoring its role in immune surveillance.
- Further research can now determine the physiological functions of these distinct IHL populations.
Abstract:
Intrahepatic lymphocytes (IHL) with their diverse and distinctive subsets emphasise the importance of the liver as a site of immunological activity, but special care is required for their isolation and characterisation. Protocols for IHL isolation, purification and FACS analysis were devised and compared with published extraction protocols. We have reduced the time that IHL are exposed to potentially damaging enzymes during extraction and purified specific subsets using monoclonal antibody (mAb)-coated magnetic microbeads. This has yielded IHL populations with higher viability than previously described protocols (92-95%, compared with 39-86%). Flow cytometric characterisation of IHL subset immunophenotypes was optimised by combining CD45 staining (fluorescence gating) with traditional light scatter properties. Using a panel of mAb and liver biopsies obtained from 23 cadaveric liver transplant donors, we show that the normal liver contains a heterogeneous IHL population with distinctive phenotypes. CD8+ IHL was the predominant population with a mean CD4/CD8 ratio of 1:1.7. Up to 40% of IHL expressed gammadeltaTCR and a third expressed CD56 NK marker; indicating a site of intense immunological activity. The techniques described will allow these cell types to be isolated, fully characterised and their physiological functions to be determined. The histologically normal liver contains heterogeneous and diverse IHL with large numbers of CD8+, NK, NKT and gammadelta+ cells.
