FIGURE 1.

Characterization of mouse hepatocyte-derived EVs. Mouse AML12 hepatocytes were grown under serum-free conditions for 36 h after which EV^Norm were isolated by ultra-centrifugation of conditioned medium and characterized by (A) NTA [mean ± SEM for particle diameter (nm) is indicated]; (B) comparative Western blot with AML12 cell lysates for common EV- or cell-associated proteins (25 μg EV or cellular protein per lane; uncropped data are shown in Supplementary Figure 1); or (C) TEM (×56,000; scale bar = 100 nm). (D) EV concentration in conditioned medium from AML12 cells that were maintained under serum-free conditions for 16 h, 24 h (with a medium exchange at 16 h), 40 h (with medium exchanges at 16 and 24 h), or 48 h (with medium exchanges at 16, 24, and 40 h). The mean EV output per cell per hour over 0–16, 16–24, 24–40, or 40–48 h was computed by expressing particle number at each collection (from NTA analysis) as a function of the cell number (from cell count at each collection time) and the duration of the incubation for EV collection (8 or 16 h).