(a) Schematic overview of a Proteinase K protection assay analyzing the integrity of EVs after fractionation with SEC, using four different conditions: a No treatment, two-hour incubation, lyse with Triton X, one-hour incubation b Proteinase K application, two-hour incubation, lyse with Triton X, one-hour incubation. c No treatment, one-hour delay, add PMSF for one hour, lyse with Triton X, one-hour incubation. d Proteinase K application, one-hour incubation, add PMSF to inhibit Proteinase K for one hour, lyse with Triton X, one-hour incubation. (b) Alix and Albumin concentrations after the Proteinase K protection assay, as measured with Simoa. Data represent the average of two technical replicates of the Simoa assay measurements from a single experiment with one sample. This experiment was conducted 3 times with similar results.