Cellulose acetate membrane

For exosome isolation, FBS without small extracellular vesicles was prepared by ultracentrifugation for 24 h at 100,000 g and 4 °C. U251 cells were seeded on T150 cell culture flask in cell medium composed of EMEM Eagle's minimum essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with small extracellular vesicles-depleted 5% fetal bovine serum (FBS) (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 1% non-essential amino-acids solution (Gibco) and 1% antibiotic–antimycotic solution (penicillin/streptomycin/amphotericin B; Gibco). After 72 h incubation, medium was collected and centrifuged for 10 min at 300 g, 4 °C and then 10 min at 2000 g, 4 °C. Afterwards, medium was passed through 0.22 μm-pore-size filter with cellulose acetate membrane (Corning) and concentrated by centrifugation at 2100g, 4 °C to final volume of 3 mL using Amicon® Ultra concentrators with Ultracel-100 regenerated cellulose membrane (Merck). Concentrated media was diluted with PBS to 8 mL, loaded on 2 mL of 20% sucrose cushion and ultracentrifuged for 135 min at 100,000 g. The pellet was thoroughly resuspended in PBS and stored at −80 °C.