Extracellular Vesicle Isolation Protocol

30 mL of conditioned medium (CM) was collected from approximately 2 × 10⁷ sub‐confluent HCT cells cultured in serum‐free medium for 24 h. CM was centrifuged at 3000 g at 4°C for 20 min. Supernatants were collected and concentrated 20‐fold using Amicon Ultra‐15 30 kDa MWCO centrifugal filters (Merck Millipore, Darmstadt, Germany). EVs were isolated by size exclusion chromatography (SEC) according to Brahmer et al. (2019 (link)). 10 mL syringes were packed with Sepharose CL‐2B (Thermo Fisher Scientific). Columns were washed with one column volume PBS before applying 2 mL of concentrated and precleared CM. Fractions of 1 mL were collected and analyzed for particle count by nanoparticle tracking analysis. Fractions 4–6 were collected as particle‐rich fractions and concentrated 30‐fold using Amicon Ultra‐2 30 kDa MWCO centrifugal filters (Merck Millipore, Darmstadt, Germany). Protein content was measured using Pierce 660 nm Protein Assay Reagent (Thermo Fisher Scientific). Absorption was measured at 660 nm with a M1000 plate reader (Tecan, Männedorf, Switzerland).