Flow cytometry analysis and confocal images of GMVs derived from HeLa cells. (a) Two-dimensional (2D) dot plots of side light scattered area (SSC-A) versus forward light scattered area (FSC-A) from flow cytometry exhibit two distinct populations for HeLa cells (blue dots) and GMVs (red dots). The inset image is the bright field image of a single GMV. (b) Confocal images of GMVs stained with FM 4-64 dye. The confocal image (c), 3D image (d), and Z-stack image (e) of a single vesicle all exhibit spherical structure with good symmetry. (f)-(h) Images of GMVs incubated with fluorescein demonstrate good integrity. Scale bar is 20 μm for (b) and 10 μm for (c)-(h).