Isolation and Characterization of EVs Containing Mitochondria from OIS Cells

Oncogene activation triggers oncogene-induced senescence (OIS), a tumor-suppression mechanism characterized by mitochondrial dysfunction and the secretion of various factors collectively known as the senescence-associated secretory phenotype (SASP). Recent evidence highlights that extracellular vesicles (EVs), nanosized membrane-bound particles, are part of the SASP and act as a novel intercellular communication pathway. Additionally, EVs containing mitochondrial compartments are hypothesized to function as a mitochondrial quality control mechanism, eliminating damaged mitochondria in senescent cells. However, the exact role of mitochondria-enriched vesicles in OIS remains elucidated. The diversity of protocols for isolating and characterizing EVs complicates the unification of research findings. Here, we provide a concise overview of current protocols for investigating vesicles-containing mitochondria in oncogene-induced senescent cells including size exclusion chromatography, NTA analysis, flow cytometry, confocal microscopy, and Western blot.