Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting
Identifieur interne : 001C27 ( Main/Exploration ); précédent : 001C26; suivant : 001C28Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting
Auteurs : Zerina Lokmic ; Elizabeth S. Ng ; Matthew Burton ; Edouard G. Stanley ; Anthony J. Penington ; Andrew G. ElefantySource :
- Journal of Visualized Experiments : JoVE [ 1940-087X ] ; 2015.
Descripteurs français
- KwdFr :
- MESH :
- anatomopathologie : Lymphadénite, Lymphoedème.
- cytologie : Cellules endothéliales.
- Cytométrie en flux, Humains, Matrix metalloproteinase 8.
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : Matrix Metalloproteinase 8.
- cytology : Endothelial Cells.
- methods : Flow Cytometry.
- pathology : Lymphadenitis, Lymphedema.
- Humans.
Abstract
Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.
Url:
DOI: 10.3791/52691
PubMed: 25992474
PubMed Central: 4652192
Affiliations:
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Ng</name><affiliation><nlm:aff id="ID1">Murdoch Childrens Research Institute, The Royal Children's Hospital</nlm:aff><wicri:noCountry code="subfield">The Royal Children's Hospital</wicri:noCountry></affiliation><affiliation><nlm:aff id="ID3">Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton</nlm:aff><wicri:noCountry code="subfield">Clayton</wicri:noCountry></affiliation></author><author><name sortKey="Burton, Matthew" sort="Burton, Matthew" uniqKey="Burton M" first="Matthew" last="Burton">Matthew Burton</name><affiliation><nlm:aff id="ID1">Murdoch Childrens Research Institute, The Royal Children's Hospital</nlm:aff><wicri:noCountry code="subfield">The Royal Children's Hospital</wicri:noCountry></affiliation></author><author><name sortKey="Stanley, Edouard G" sort="Stanley, Edouard G" uniqKey="Stanley E" first="Edouard G." last="Stanley">Edouard G. Stanley</name><affiliation><nlm:aff id="ID1">Murdoch Childrens Research Institute, The Royal Children's Hospital</nlm:aff><wicri:noCountry code="subfield">The Royal Children's Hospital</wicri:noCountry></affiliation><affiliation><nlm:aff id="ID2">Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne</nlm:aff><wicri:noCountry code="subfield">The University of Melbourne</wicri:noCountry></affiliation><affiliation><nlm:aff id="ID3">Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton</nlm:aff><wicri:noCountry code="subfield">Clayton</wicri:noCountry></affiliation></author><author><name sortKey="Penington, Anthony J" sort="Penington, Anthony J" uniqKey="Penington A" first="Anthony J." last="Penington">Anthony J. Penington</name><affiliation><nlm:aff id="ID1">Murdoch Childrens Research Institute, The Royal Children's Hospital</nlm:aff><wicri:noCountry code="subfield">The Royal Children's Hospital</wicri:noCountry></affiliation><affiliation><nlm:aff id="ID2">Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne</nlm:aff><wicri:noCountry code="subfield">The University of Melbourne</wicri:noCountry></affiliation></author><author><name sortKey="Elefanty, Andrew G" sort="Elefanty, Andrew G" uniqKey="Elefanty A" first="Andrew G." last="Elefanty">Andrew G. Elefanty</name><affiliation><nlm:aff id="ID1">Murdoch Childrens Research Institute, The Royal Children's Hospital</nlm:aff><wicri:noCountry code="subfield">The Royal Children's Hospital</wicri:noCountry></affiliation><affiliation><nlm:aff id="ID2">Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne</nlm:aff><wicri:noCountry code="subfield">The University of Melbourne</wicri:noCountry></affiliation><affiliation><nlm:aff id="ID3">Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton</nlm:aff><wicri:noCountry code="subfield">Clayton</wicri:noCountry></affiliation></author></analytic><series><title level="j">Journal of Visualized Experiments : JoVE</title><idno type="eISSN">1940-087X</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Endothelial Cells (cytology)</term><term>Flow Cytometry (methods)</term><term>Humans</term><term>Lymphadenitis (pathology)</term><term>Lymphedema (pathology)</term><term>Matrix Metalloproteinase 8 (chemistry)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Cellules endothéliales (cytologie)</term><term>Cytométrie en flux ()</term><term>Humains</term><term>Lymphadénite (anatomopathologie)</term><term>Lymphoedème (anatomopathologie)</term><term>Matrix metalloproteinase 8 ()</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Matrix Metalloproteinase 8</term></keywords><keywords scheme="MESH" qualifier="anatomopathologie" xml:lang="fr"><term>Lymphadénite</term><term>Lymphoedème</term></keywords><keywords scheme="MESH" qualifier="cytologie" xml:lang="fr"><term>Cellules endothéliales</term></keywords><keywords scheme="MESH" qualifier="cytology" xml:lang="en"><term>Endothelial Cells</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Flow Cytometry</term></keywords><keywords scheme="MESH" qualifier="pathology" xml:lang="en"><term>Lymphadenitis</term><term>Lymphedema</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Humans</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Cytométrie en flux</term><term>Humains</term><term>Matrix metalloproteinase 8</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34<sup>Low</sup>CD31<sup>Pos</sup>VEGFR-3<sup>Pos</sup>PODOPLANIN<sup>Pos </sup>LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.</p></div></front></TEI><affiliations><list></list><tree><noCountry><name sortKey="Burton, Matthew" sort="Burton, Matthew" uniqKey="Burton M" first="Matthew" last="Burton">Matthew Burton</name><name sortKey="Elefanty, Andrew G" sort="Elefanty, Andrew G" uniqKey="Elefanty A" first="Andrew G." last="Elefanty">Andrew G. Elefanty</name><name sortKey="Lokmic, Zerina" sort="Lokmic, Zerina" uniqKey="Lokmic Z" first="Zerina" last="Lokmic">Zerina Lokmic</name><name sortKey="Ng, Elizabeth S" sort="Ng, Elizabeth S" uniqKey="Ng E" first="Elizabeth S." last="Ng">Elizabeth S. Ng</name><name sortKey="Penington, Anthony J" sort="Penington, Anthony J" uniqKey="Penington A" first="Anthony J." last="Penington">Anthony J. Penington</name><name sortKey="Stanley, Edouard G" sort="Stanley, Edouard G" uniqKey="Stanley E" first="Edouard G." last="Stanley">Edouard G. Stanley</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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