Rodent eCSF nanovesicle purification and protein expression.

Abstract

<p>(<b>A</b>) Flow chart of the experimental design after CSF labeling with a tracer dye (fast green) and eCSF collection. (<b>B</b>) Histogram of size distribution of eCSF nanoparticles (per ml) determined by nanoparticle tracking analysis (NanoSight). The nanoparticles were obtained from e14 CSF from three rat litters (N = 3). The mean nanoparticle diameter was 77 nm and was obtained by Gaussian curve fitting. (<b>C</b>) Electron micrographs of rat embryonic purified nanovesicles. Scale bar: 30 nm. (<b>D</b>) Immunoblots for rat exosomal marker proteins CD63 and HSP70, and additional proteins known to be in exosomes. PTEN and PKM2. (<b>E</b>) Quantification of phosphoenol pyruvate kinase enzymatic activity determined from rat nanovesicles. (<b>F</b>) Quantification of IGF pathway-related proteins in nanovesicles isolated from e15 rat CSF using the phospho (p)-pathscan assay. Error bars: SEM. Experiments were reproduced with nanovesicles isolated from three litters (N = 3).</p