Back to Search
Start Over
Induction and Validation of Cellular Senescence in Primary Human Cells.
- Source :
-
Journal of visualized experiments : JoVE [J Vis Exp] 2018 Jun 20 (136). Date of Electronic Publication: 2018 Jun 20. - Publication Year :
- 2018
-
Abstract
- Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated β-galactosidase (SA-β-gal), the rate of DNA synthesis using 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.
- Subjects :
- Cell Line cytology
Humans
Cell Line metabolism
Cellular Senescence physiology
Subjects
Details
- Language :
- English
- ISSN :
- 1940-087X
- Issue :
- 136
- Database :
- MEDLINE
- Journal :
- Journal of visualized experiments : JoVE
- Publication Type :
- Academic Journal
- Accession number :
- 29985363
- Full Text :
- https://doi.org/10.3791/57782