title: Isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting.
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 CD34(Low)CD31(Pos)VEGFR-3(Pos)PODOPLANIN(Pos) 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.
authors.affiliation: Murdoch Childrens Research Institute, The Royal Childrens Hospital; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne; zerina.lokmic@mcri.edu.au.
authors.forename: Zerina
authors.initials: Z
authors.lastname: Lokmic
authors.affiliation: Murdoch Childrens Research Institute, The Royal Childrens Hospital; Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton.
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Ng
authors.affiliation: Murdoch Childrens Research Institute, The Royal Childrens Hospital.
authors.forename: Matthew
authors.initials: M
authors.lastname: Burton
authors.affiliation: Murdoch Childrens Research Institute, The Royal Childrens Hospital; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne; Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton.
authors.forename: Edouard G
authors.initials: EG
authors.lastname: Stanley
authors.affiliation: Murdoch Childrens Research Institute, The Royal Childrens Hospital; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Penington
authors.affiliation: Murdoch Childrens Research Institute, The Royal Childrens Hospital; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne; Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Elefanty
mesh_terms: D042783:Endothelial Cells / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008199:Lymphadenitis / Q000473:pathology; D008209:Lymphedema / Q000473:pathology; D020784:Matrix Metalloproteinase 8 / Q000737:chemistry
chemical_list: D020784:Matrix Metalloproteinase 8