Evsecond l70

EVs were purified from human serum samples using size exclusion chromatography on a drip with extracellular vesicle isolation by size exclusion chromatography on a EVSecond L70^® drip column (GL Sciences, Tokyo, Japan). The column was initially equilibrated with 700 μL of FBS twice, followed by three washing steps using 1500 μL of PBS. After washing, 100 μL of the collected human serum sample was loaded onto the column, followed by the collection of 12 consecutive fractions in 100 μL of PBS. The CD9 expression in these fractions was analyzed using western blotting, and CD9-positive fractions were recognized as the exosome-rich portion. The obtained EVs using this system were previously authenticated with TEM and western blotting, and the diameters of the particles in the EV fractions were analyzed with the Zetasizer nano ZSP [24 (link)].

AML12 cells were differentiated by culturing in DMEM/Ham’s F-12 without FBS for 2–4 days and then treated with or without PA for 48 h. The cells were then further cultured in serum-free medium in 100 mm dishes for four days. The conditioned medium (approximately 130 mL) was filtered through 0.22 μm membrane filters after removing the cells by centrifugation (2000× g, 10 min). The supernatant was concentrated to 10 mL using Amicon Ultra-15 Centrifugal 30 K filter units (Merck, Darmstadt, Germany). EVs were collected using size-exclusion chromatography (SEC). After removing the storage solution, 700 μL of exosome-depleted FBS (Gibco A2720801, Lot No. 2243287p) was added to a size-exclusion column (EVSecond L70, GL Sciences Inc., Tokyo, Japan), and then 4.5 mL of PBS filtered with 0.22 μm membrane filters was run through the column. Concentrated culture medium samples were diluted to 1.5 mL with filtered PBS, and the sample was overlaid on the size-exclusion column followed by elution with 100 μL of filtered PBS per fraction. The flow-through was collected in 100 µL fractions, and fractions 1–9 were combined for characterization and intracellular uptake analysis.