Isolation and Characterization of Hypoxic EVs

Extracellular Vesicles were harvested from primary CDCs at passage 5 or older passages from transduced cells using a hypoxic cycling method used previously by our group^{3 (link)}. Briefly, cells were grown to confluence at 20% O₂/5% CO₂ at 37°C, and then cells were serum-free at 2% O₂/5% CO₂ at 37°C overnight after one wash. Conditioned media was collected and filtered through 0.45 μm filter to remove apoptotic bodies and cellular debris and frozen for later use at −80°C. EVs were purified using centrifugal ultrafiltration with a 1000 KDa molecular weight cutoff filter (Sartorius). EV preparations were analyzed through Malvern Nanosight NS300 Instrument (Malvern Instruments) with the following acquisition parameters: camera levels of 15, detection level less than or equal to 5, number of videos taken 4, and video length of 30 s.