Methods for detection of mitochondrial reactive oxygen species in senescent cells.

Cellular senescence is a pathophysiological process with multifaceted effects. It is involved in wound healing, aging and age-related diseases as well as cancer. On the one hand, senescence is considered as barrier against tumorigenesis by inducing an irreversible/prolonged cell cycle arrest. On the other hand, it may promote tumorigenesis when senescent cells accumulate genomic instability and bypass this cell cycle arrest. Interestingly, the bystander effects mediate the propagation of the genetic instability from senescent cells to their environment through the SASP (Senescence Associated Secretory Phenotype) including proinflammatory cytokines, proteases, growth factors and Reactive Oxygen Species 'ROS.' From several markers explored to detect senescent cells (β-galactosidase, p16, p21, p53, heterochromatin foci, DNA damage,…), ROS arouse particular interest because of their involvement at the chronic supraphysiological level, in the induction and maintain of DNA damage, inflammation, cell cycle disruption and epigenetic instability. In this context, the choice of methods to detect ROS in senescent cells is of particular interest and must take into account relevant parameters as well as the specificity for each species of ROS and the subcellular localization of ROS production. In this chapter, we introduce senescence and ROS, we briefly discuss the advantages and the shortcomings of methods routinely used to detect ROS. In addition, we describe the protocol to detect ROS at mitochondrial level (using the MitoSOX staining) in the BCPAP cell line (from human papillary thyroid carcinomas) expressing BRAF^V600E oncogene known to trigger senescence.