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. 2023 May 16;31(7):2220–2239. doi: 10.1016/j.ymthe.2023.05.012

Figure 5.

Figure 5

Cre mRNA is detected in brain-derived EVs extracted from the brain

(A) Schematic illustration of the protocol used to isolate brain-derived EVs (bdEVs). (B) Density distribution of 10 fractions as result of iodixanol gradient centrifugation at 100,000 × g for 18 h. EV-enriched fractions were isolated in densities ranging from 1.105 to 1.165 g/mL (middle region) (n = 10). (C) Quantification of protein amount per fraction (in percentage) before and after 100,000 × g purification step. Before 100,000 × g purification step (blue bars), protein is highly enriched in the first fractions decreasing until fraction 10. After 100,000 × g purification step (yellow bars), the majority of free protein was washed out and the highest percentage of protein was located in EV fractions 6, 7, and 8 (n = 4). (D) Particle size distribution of each fraction (represented by mode) was evaluated by nanoparticle tracking analysis (NTA) (red bars). Fraction 1 showed the higher mode with 140 nm and decreasing in each fraction until fraction 10, which showed the mode of 90 nm (n = 3). (E) Particle concentration in each fraction was evaluated by NTA (green bars), with fractions 6, 7, and 8 accounting for more than 50% of total particles, while fractions 1 and 2 and 9 and 10 showed a lower concentration (n = 3). (F) Representative western blotting of 10 fractions obtained after ODG and ultracentrifugation of each fraction in PBS (loaded per volume) show the presence of positive EV markers HSC70 and Flotilin-1. The endoplasmic reticulum protein calnexin was detected in low levels in EV-enriched fractions. (G) Distribution of Cre exRNA in bdEV fractions was evaluated by qRT-PCR (Ct value). Fractions 6, 7, and 8 showed higher levels of Cre exRNA when compared with the other fractions (n = 4) (same volume was used as starting point). (H) Transmission electron microscopy of pool 1 (fractions 1–5) showed lipoproteins (red arrow) and few canonical bdEVs (blue arrow), pool 2 (fractions 6–8) was highly enriched in bdEVs (blue arrow) with cup-shaped format, and pool 3 (fractions 9–10) presented very low number of particles and some protein aggregates (orange arrows). Scale bars, 500 nm (big pictures) and 200 nm (pool 2, crop). Values are presented as mean ± SEM.