Membrane topological changes through fusion and fission processes can lead to the generation of new internal lipid compartments inside GUVs: intraluminal vesicles (ILVs). To investigate ILV formation, where extra-vesicular media is encapsulated within these new compartments, a membrane-impermeable 10 kDa dextran labelled with Alexa Fluor 488 (AF488) or Cascade Blue (CBlue) was added to the bulk medium. Formation of ILVs is characterised by the inclusion of AF488 or CBlue in the lumen of these internal vesicles.