Protocol for the assessment of mTOR activity in mouse primary hepatocytes

Ana Belén Plata-Gómez; María Crespo; Celia de la Calle Arregui; Lucía de Prado-Rivas; Guadalupe Sabio; Alejo Efeyan

doi:10.1016/j.xpro.2021.100918

Protocol for the assessment of mTOR activity in mouse primary hepatocytes

STAR Protoc. 2021 Oct 29;2(4):100918. doi: 10.1016/j.xpro.2021.100918. eCollection 2021 Dec 17.

Authors

Ana Belén Plata-Gómez¹, María Crespo², Celia de la Calle Arregui¹, Lucía de Prado-Rivas¹, Guadalupe Sabio², Alejo Efeyan¹

Affiliations

¹ Metabolism and Cell Signaling Laboratory, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain.
² Myocardial Pathophysiology, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029 Madrid, Spain.

Abstract

We present a protocol for measuring the activity of the mechanistic target of rapamycin (mTOR) pathway in ex vivo isolated mouse primary hepatocytes. It can be used as a tool for genetic, pharmacological, metabolomic, and signal transduction procedures. We discuss critical aspects for improving yield, viability, and modulation of the mTOR pathway. This protocol can be adapted to other signaling cascades and is compatible with multiple readouts. For complete details on the use and execution of this protocol, please refer to Ortega-Molina et al. (2021).

Keywords: Cell culture; Cell isolation; Cell-based Assays; Metabolism; Metabolomics; Signal Transduction.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Culture Techniques / methods*
Cells, Cultured
Hepatocytes / metabolism*
Liver / cytology*
Mice
Signal Transduction / physiology
TOR Serine-Threonine Kinases* / analysis
TOR Serine-Threonine Kinases* / metabolism

Substances

mTOR protein, mouse
TOR Serine-Threonine Kinases