Extracellular Vesicle Isolation Protocol

EVs were harvested from the cells 48 h after transfection with LMP1. The collected EVs were enriched using the ExtraPEG method, as previously described (74 (link)). Briefly, the medium was centrifuged at 500 × g for 5 min and at 2,000 × g for 10 min in an Eppendorf 5804R centrifuge using an S-4-104 rotor, followed by 10,000 × g for 30 min in an Eppendorf 5804R centrifuge using an FA-45-630 rotor to remove cells and cellular debris. Subsequently, a 1:1 volume of 16% (2×) polyethylene glycol (average M_n, 6,000) (catalog number 25322-68-3; Alfa Aesar) and 1 M sodium chloride was added to the samples, and the samples were incubated overnight at 4°C. The incubated samples were centrifuged at 3,214 × g for 1 h in an S-4-104 rotor. The pellet was then washed with 1× phosphate-buffered saline (PBS) and centrifuged at 100,000 × g for 70 min in a Beckman Max-E centrifuge using a TLA120.2 rotor. The collected EV samples were resuspended in particle-free PBS for nitrilotriacetic acid (NTA) or resuspended in 2× Laemmli sample buffer (4% SDS, 100 mM Tris [pH 6.8], 0.4 mg/ml bromophenol blue, 0.2 M dithiothreitol [DTT], 20% glycerol, 2% β-mercaptoethanol [BME]) for immunoblot analysis.