fig1

Figure 1. Separation of the three main components of conditioned media (EVs, LPPs, and protein) by optimized SEC. (A) Representative dot blots of EV markers (CD9, CD63, and CD81) and the LPP marker (ApoB) across SEC fractions. Graphing software: LAS 4000 mini system and Fiji ImageJ; (B) Elution profiles of EVs, LPPs, and soluble proteins. Densitometry analysis of dot blots for EV markers (CD9, CD63, and CD81) and the LPP marker (ApoB), along with absorbance at 280 nm for total protein, are shown as the % of the total signal for each marker (normalized to 100% as the sum of signals across all eluted fractions). Squares indicate the fractions pooled for further analysis of EVs, LPPs, and protein. Data represent mean ± SEM from at least three independent experiments. Graphing software: GraphPad Prism; (C) Size distribution of particles in EV-enriched fractions, analyzed by NTA. Graphing software: NTA 3.0 software (stated under methods); (D) Representative TEM images of negatively stained EV-enriched fractions. Scale bars = 1 μm. Graphing software: JEM1400 Flash transmission electron microscope (Jeol) and Image J. EVs: Extracellular vesicle; Fr.: fraction; LPP: lipoprotein particle; SEC: size-exclusion chromatography; NTA: nanoparticle tracking analysis; TEM: transmission electron microscopy; SEM: standard error of the mean.