Dataset Information

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells.

ABSTRACT: Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

SUBMITTER: Finlon JM

PROVIDER: S-EPMC6350926 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells.

Finlon Jeffrey M JM Burchill Matthew A MA Tamburini Beth A Jirón BAJ

Journal of visualized experiments : JoVE 20190107 143

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from t ...[more]

PMID: 30663671

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Project description:Background:Circulating endothelial cells (CECs) and their subpopulations could be potential novel biomarkers for various malignancies. However, reliable enumerable methods are warranted to further improve their clinical utility. This study aimed to optimize a flow cytometric method (FCM) assay for CECs and subpopulations in peripheral blood for patients with solid cancers. Patients and methods:An FCM assay was used to detect and identify CECs. A panel of 60 blood samples, including 44 metastatic cancer patients and 16 healthy controls, were used in this study. Some key issues of CEC enumeration, including sample material and anticoagulant selection, optimal titration of antibodies, lysis/wash procedures of blood sample preparation, conditions of sample storage, sufficient cell events to enhance the signal, fluorescence-minus-one controls instead of isotype controls to reduce background noise, optimal selection of cell surface markers, and evaluating the reproducibility of our method, were integrated and investigated. Wilcoxon and Mann-Whitney U tests were used to determine statistically significant differences. Results:In this validation study, we refined a five-color FCM method to detect CECs and their subpopulations in peripheral blood of patients with solid tumors. Several key technical issues regarding preanalytical elements, FCM data acquisition, and analysis were addressed. Furthermore, we clinically validated the utility of our method. The baseline levels of mature CECs, endothelial progenitor cells, and activated CECs were higher in cancer patients than healthy subjects (P<0.01). However, there was no significant difference in resting CEC levels between healthy subjects and cancer patients (P=0.193). Conclusion:We integrated and comprehensively addressed significant technical issues found in previously published assays and validated the reproducibility and sensitivity of our proposed method. Future work is required to explore the potential of our optimized method in clinical oncologic applications.

Dataset Information

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells.

Publications

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells.

Similar Datasets

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