Measuring Endocytosis During Proliferative Cell Quiescence

Quiescence (also called “G0”) is the state in which cells have exited the cell cycle but are capable to reenter as required. Though poorly understood, it represents one of the most prevalent cell states across all life. Many biologically important cell types reside in quiescence including mature hepatocytes, endothelial cells, and dormant adult stem cells. Furthermore, the quiescence program occurs in both short- and long-term varieties, depending on the physiological environments. A barrier slowing our understanding of quiescence has been a scarcity of available in vitro model systems to allow for the exploration of key regulatory pathways, such as endocytosis. Endocytosis, the internalization of extracellular material into the cell, is a fundamental and highly regulated process that impacts many cell biological functions. Accordingly, we have developed an in vitro model of deep quiescence in hTERT-immortalized RPE1 cells, combining both long-term contact inhibition and mitogen removal, to measure endocytosis. In addition, we present an analytical approach employing automated high-throughput microscopy and image analysis that yields high-content data allowing for meaningful and statistically robust interpretation. Importantly, the methods presented herein provide a suitable platform that can be easily adapted to investigate other regulatory processes across the cell cycle.