Protocol for the assessment of mTOR activity in mouse primary hepatocytes
| Autor: | María Crespo, Lucía de Prado-Rivas, Alejo Efeyan, Celia de la Calle Arregui, Guadalupe Sabio, Ana Belén Plata-Gómez |
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| Rok vydání: | 2021 |
| Předmět: |
Science (General)
Cell Culture Techniques General Biochemistry Genetics and Molecular Biology Q1-390 Mice Metabolomics Protocol Animals Cell isolation Mechanistic target of rapamycin PI3K/AKT/mTOR pathway Cells Cultured Primary (chemistry) General Immunology and Microbiology biology General Neuroscience TOR Serine-Threonine Kinases Cell biology Metabolism Liver Cell culture biology.protein Hepatocytes Cell-based Assays Signal transduction Ex vivo Signal Transduction |
| Zdroj: | STAR Protocols STAR Protocols, Vol 2, Iss 4, Pp 100918-(2021) |
| ISSN: | 2666-1667 |
| DOI: | 10.1016/j.xpro.2021.100918 |
| Popis: | Summary 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). Graphical abstract Highlights • Describes the purification and culture of primary hepatocytes from mice • Describes the assessment of mTORC1 activity manipulating nutrient and growth factor signals • Protocol adaptable to other signaling cascades and compatible with multiple readouts 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. |
| Databáze: | OpenAIRE |
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