Fig. 2. Overview of high-resolution density gradient fractionation of EVs.

a, Schematic of the generation of lEVs or sEVs and non-vesicular (NV) fractions by high-resolution iodixanol density-gradient fractionation (12–36%). Crude pellets of lEV-Ps or sEV-Ps were resuspended in ice-cold PBS and mixed with ice-cold iodixanol (OptiPrep)/PBS for a final 36% iodixanol solution. The suspension was added to the bottom of a centrifugation tube and solutions of descending concentrations of iodixanol (30%, 24%, 18%, 12%) in PBS were carefully layered on top yielding the complete gradient. The bottom-loaded 12%–36% gradient was subjected to ultracentrifugation at 120,000g for 15 h. Twelve individual fractions of 1 ml were collected from the top of the gradient. The first six fractions are pooled in a tube, and the last five fractions are pooled in a second tube. The tubes are filled with PBS and mixed. Following ultracentrifugation at 120,000g for 4 h, the two pellets represent purified EVs and NVs, respectively. The images in panel a are modified from Zhang et al.¹⁵ and Jeppesen et al.⁹. b, Representative photographs of the most important steps during the high-resolution gradient fractionation procedure from a.