FIGURE 3.

Optimization on the resin bed volume of SEC columns. (a,b) Fraction distribution of particle (blue) and protein (red) concentrations of CL‐4B (a) and CL‐6B (b) columns with the bed volume of 20 ml. Optimization using FBS (n = 3) and verification with human serum (HS) from Donor 1 are shown. A total of 52 fractions were collected in each experiment, 500 μl of PBS was used for eluting each fraction. The dashed lines indicate the cumulative percentage. The 10%, 50%, 90% and 99% data points were labelled with triangles on each cumulative curve. The transparent shades illustrate the SD of each fraction. (c) Statistical comparison of the resolution (Rs) in EV isolated from FBS. The dashed red line indicates the threshold of baseline separation (Rs = 1.5). *Significant difference (P < 0.05), n = 3, Tukey's multiple comparisons. (d,e) Immunoblotting of protein markers of EVs and contaminants for all SEC fractions using CL‐4B (d) and CL‐6B (e) columns in the HS experiment. The sample loading volumes or sample loading protein amounts are labelled