Time-lapse confocal laser scanning microscopy analysis of FOOD formation.

Abstract

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.