MVB–PM fusion is distinct from other forms of vesicle-mediated exocytosis. (a) Schematic model showing the markers used in this study for the different types of cargo delivery of vesicles fusing with the PM. (b) Time-lapse imaging (heat maps) of a fusion event of the exosomal protein CD63-pHluorin. (c) Time-lapse images of soluble (NPY-pHluorin) and membrane protein (VAMP2-pHluorin) fusion events. (d) Fluorescent signal duration of NPY (mean = 0.85 s), VAMP2 (mean = 2.12 s), and CD63 (mean = 106.55 s) fusion events. n ≥ 13 events per reporter. (e) 3D heat maps of three consecutive CD63-pHluorin fusion event frames. (f) Western blot for exosomal markers (CD63 and Alix) on EVs purified from the supernatant (soluble) and EVs attached to the cell surface (PM attached) isolated after short trypsinization of the cells. (g) Direct comparison between signal duration of fusion events of CD81- and CD9-pHluorin relative to CD63-pHluorin. n ≥ 20 events per reporter. ***, P < 0.001; ****, P < 0.0001 using Student’s two-tailed two-sample t test.