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

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.