Methods for Establishing and Using a Stable Cell Line Expressing Both Gaussia Luciferase and Firefly Luciferase to Screen for Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress is one of the major mechanisms underlying the etiology of multiple diseases and drug-induced toxicity. Gaussia luciferase (Gluc) is a naturally secreted protein that has been used as a reporter for the secretory pathway of ER to enable efficient and real-time monitoring of the ER function. The Gluc assay has been widely used and optimized in various labs. In this chapter, we provide an example of the application of the Gluc assay by establishing a stable cell line expressing both Gluc and firefly luciferase (Fluc) to study ER stress in liver cells. We describe the detailed procedures used in our laboratory for Gluc- and Fluc-containing lentivirus production and titration, for establishing a HepG2-based stable cell line through lentivirus transduction and the validation process. In addition, we provide an example of using the established stable cell line to investigate ER stress.