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Figure 2. Isolation and characterization of lEV and sEV from MEG-01 cells. (A) Schematic of the differential centrifugation protocol used to isolate lEVs and sEVs from MEG-01 cell conditioned medium; (B) lEV and sEV were isolated from MEG-01 cell culture medium using differential centrifugation, and resuspended in the same volume of PBS. Quantification of total protein concentration in lEV and sEV preparations, as measured by BCA assay; (C) Same amount of protein as measured by BCA was loaded on the gel, and analyzed using SDS-PAGE and western blotting. SDS-PAGE gel (top) showing protein composition of EV fractions and western blot analysis (bottom) confirming the presence of canonical EV markers (CD41, CD63, CD81, HSP70, and Annexin A1) in MEG-01 EVs; (D) NTA profiles showing particle size distribution of large (left) and small (middle) EVs. Same settings were used when the particle size was measured using NTA. Bar graph (right) quantifies the modal size of lEV and sEV, revealing a significantly smaller size for sEVs; (E) TEM images of negatively stained lEVs (left) and sEVs (right). Scale bars = 100 nm; (F and G) MACSPlex analysis of surface molecules on the isolated MEG-01 sEVs. PBS was used as a vehicle control to assess non-specific binding of the detection antibodies. (F) Representative flow cytometry plots show clear staining for sEV surface molecules compared to vehicle control. The left panel shows successful detection of 37 EV-related antibody-coated beads, and the right panel shows successful detection of surface molecules on the EVs captured by each antibody-coated bead; (G) Quantification of the surface molecules as shown in (F). Representative immunoblots (C), NTA profiles (D), TEM images (E), flow cytometry plots (F), and MACSPlex fluorescence profiles (G) from one of three independent biological experiments with similar results are shown. Quantitative data in (B and D) are presented as mean ± SD from three independent biological experiments. Statistical significance was determined using an unpaired two-tailed Student’s t-test; ^**P < 0.01. APC: Allophycocyanin; BCA: bicinchoninic acid; EV: extracellular vesicle; HSP70: heat shock protein 70; lEV: large extracellular vesicle; MACSPlex: multiplex bead-based assay for extracellular vesicle surface protein analysis; NTA: nanoparticle tracking analysis; PBS: phosphate-buffered saline; PE: phycoerythrin; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SD: standard deviation; sEV: small extracellular vesicle; TEM: transmission electron microscopy.