Extracellular Vesicle Isolation from NPCE Cells

EV samples were purified from NPCE cells under both normal (EVs-N) and oxidative stress conditions (EVS-OS) according to a PEG (Cat# 89510, Sigma, St. Louis, MO, USA)-based isolation method [40 (link),41 (link)] as previously described, with minor modifications. NPCE cells were plated at 5 million cells per 75 cm² and after reaching 90% confluence, the cells were exposed for 1.5 h 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH)compound (Cat# 440914, Sigma, St. Louis, MO, USA). NPCE cell-conditioned medium was aspirated and centrifuged at 1500× g for 10 min to remove cells, followed by filtration through PVDF filter (0.22 µm, Millipore, Billerica, MA, USA) to remove large cellular debris. Precipitation solution (50% PEG8000, 0.5 M NaCl in PBS) was added to the cleared conditioned medium (1:5 v/v, respectively), mixed by flicking the tube and incubated overnight at 4 °C. After incubation, the tubes were centrifuged at 1500× g for 30 min at 4 °C to acquire the pellet of EVs. The supernatant was discarded and pelleted EVs were dissolved in 500 µL PBS for further analysis. The EVs-N were isolated from non-treated (NT) NPCE cells following the same procedure as described above.