Time-lapse confocal laser scanning microscopy analysis of FOOD formation

Apoptotic cells undergo various morphological changes as they die, including membrane blebbing, apoptopodia formation, and the generation of apoptotic bodies. Recently, a novel step in the disassembly of apoptotic adherent cells was described, wherein cells retract from the substrate leaving behind phosphatidylserine and F-actin rich membrane remnants that are tightly anchored to the substrate. Over time, this membrane, coined the 'FOotprint Of Death' or 'FOOD' rounds into extracellular vesicles known as 'FOOD-derived Apoptotic cell derived extracellular vesicles' or 'F-ApoEVs'. Here, we detail an imaging approach to monitor the morphological changes of apoptotic cells as they retract from the extracellular substrate and generate FOOD and F-ApoEVs, using fluorescent conjugated annexin V and actin cytoskeletal stains. With some variations, we expect this approach to be compatible with a range of fluorescent dyes, stains, cell types, and methods of apoptosis induction to detect the formation of FOOD/ F-ApoEV during apoptosis.