Abstract
Lymphatic vessel endothelial hyaluronan receptor 1, or LYVE-1, is a type 1 integral membrane glycoprotein expressed by lymphatic endothelial cells (LECs). LYVE-1 is commonly used as a biological marker to visually distinguish developing lymphatic vessels from blood endothelial cells (arteries or veins). As our understanding of lymphatic biology is still lacking today, the need to isolate LECs apart from other endothelial cells has taken on greater importance. The following procedure describes a magnetic bead separation procedure for isolating LEC-rich populations of cells from developing mouse embryos.
Keywords: LYVE-1, Endothelial cells, Lymphatic, Liver, Mouse embryo
Background
The lymphatic vasculature forms a secondary circulatory system that functions in draining extracellular fluid from tissue, allows for the transport of lipids, and provides immune cell trafficking and transport function. While our understanding of the lymphatic system has rapidly expanded in the last couple of decades, it is still lacking compared to our knowledge of arterial and venous biology. The identification of LYVE-1 receptor expression by LECs provided a useful tool to distinguish lymphatic tissue but LYVE-1 is known to be expressed in other tissues including liver and spleen sinusoidal cells and pancreatic exocrine and islet of Langerhans cells (Banerji et al., 1999). Encoded by the LYVE1 gene, the biological function of the LYVE-1 receptor has yet to be determined but it has been suggested to participate in tumor metastasis in addition to HA transport across endothelial cells (Jackson, 2003). Despite it being expressed in a variety of tissues, LYVE-1 is still a commonly used marker today to distinguish LECs from other endothelial cells. The goal of this procedure was to isolate LECs from developing mouse embryos with a positive selection approach using dynabeads conjugated to LYVE-1 antibody.
Materials and Reagents
Equipment
Procedure
Data analysis
After isolating RNA using the isolation kit, RNA purity and quality should be checked prior to running any downstream experiments. For the purpose of this experiment, only samples with a 260/280 ratio of 1.9-2.1 were selected for RT-qPCR experiments. Purity was assessed using a Thermo Fisher Scientific Nanodrop 2000 device. However, standard spectrophotometers can also be used to assess quality.
Notes
The physiological role of the LYVE-1 receptor is still under debate, as well as exactly what tissues express the receptor at which time-point during mouse embryo development. LYVE-1 is highly expressed by sinusoidal liver cells (a specialized type of blood vessel) so removal of the liver prior to digesting the embryo would improve the purity of isolating lymphatic endothelial cells. While this protocol describes a method for isolating cells and directly obtaining RNA for quantitative real-time PCR analysis, it can be adapted for a variety of downstream purposes. For example, after step 14 of Part B, cells could be eluted from the Dynabeads (according to manufacturer’s instructions), which would allow for alternative downstream methods to be employed (cell culture or fluorescence-activated cell sorting [FACS]) depending on the needs of the individual.
Recipes
Acknowledgments
This procedure was developed in the laboratory of Dr. Courtney Griffin at the Oklahoma Medical Research Foundation in Oklahoma City, OK. There are no conflicts of interest.
References
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