Isolation, characterization, and experimental applications of apoptotic bodies in vitro.

Apoptotic bodies (ApoBDs) are a type of extracellular vesicle (EV) generated during the final stages of apoptosis. These vesicles were traditionally regarded as apoptotic cellular waste. More recently, they have begun to be recognized as mediators of intercellular communication via the delivery of diverse factors, with emerging roles in various physiological processes including immune regulation, cancer progression, and tissue repair. However, the lack of standardized methods for isolating and characterizing ApoBDs has limited both their study and potential application. Here, we present simple, efficient, and reproducible protocols for ApoBD isolation, characterization, and application to downstream experimental approaches. This protocol includes a two-step differential centrifugation to isolate ApoBDs and flow cytometry-based characterization, as well as optional steps such as caspase inhibitor-based modulation of ApoBD release and fluorescent labeling of ApoBDs for their tracking. Furthermore, we provide methods to evaluate ApoBD uptake by recipient cells, enabling subsequent downstream functional studies. These approaches promote the reproducibility and standardization of ApoBD research, providing a foundation to deepen our understanding of their functional roles and advance future clinical applications.