FLOW CYTOMETRY-BASED ISOLATION OF DERMAL LYMPHATIC ENDOTHELIAL CELLS FROM NEWBORN RATS

Abstract

The lymphatic system plays a key role intissue homeostasis, fatty acid transport, andimmune surveillance. Pathologically, dysfunctionof the lymphatic system results in edema,and increased lymphangiogenesis cancontribute to tumor metastasis. Lymphaticvessels are composed of lymphatic endothelialcells (LECs) that can be identified by distinctmarker molecules such as Prox-1, podoplanin,VEGFR-3 and LYVE-1. Primary LECsrepresent a valuable tool for the study of basicfunctions of the lymphatic system. However,their isolation remains a challenge, particularlyif rodent tissues are used as a source.We developed a method for the isolation ofrat dermal LECs from the skin of newbornrats based on sequential enzymatic digestionwith trypsin and Liberase followed by flowcytometric sorting using LYVE-1 specificantibodies. Cells isolated according to thisprotocol expressed the lymphatic markersProx-1, podoplanin, LYVE-1 and VEGFR-3,and displayed an endothelial-like morphologywhen taken into culture. These primary cellscan be used for studying lymphatic biology inrat models, and the protocol we describe heretherefore represents an important extensionof the experimental repertoire available forrats and for modeling the human lymphaticsystem.