Two-Step Positive Selection Method for the Magnetic Isolation of Lymphatic Endothelial Cells.

With the advent of next-generation sequencing technologies, there is an increased interest in identifying and isolating rare cell populations to understand their roles in homeostatic and pathophysiological states. Lymphatic vessels exist in nearly all vascularized tissues and have become increasingly appreciated as physiological and biological mediators of tissue homeostasis and immune function. Lymphatics, and the lymphatic endothelial cells (LECs) that make up these vessels, are low in number compared to blood endothelial cells and other stromal cell types and share surface markers with these cells. Whole tissue cell isolation therefore often fails to enrich a sufficient population of LECs to study their cellular responses. A combination of molecular markers can help to distinguish these rare LEC populations from other cell types. In this protocol, we describe a dual antibody marker magnet-based isolation method, suitable for isolating viable rare cell populations and isolating bona fide LECs from tissues. The protocol can also be coopted for other cell types best isolated with dual markers for any downstream applications in which cell population purity is a concern such as single-cell RNA sequencing, RNA sequencing, qRT-PCR, western blot, or flow cytometry.