Tricorn 5 100 glass columns

100 µL liposome sample was incubated with 900 µL completed DMEM medium (containing 10% FBS) for four hours at 37 °C, 5% CO₂ modeling cell culture conditions. 10 µL of incubated sample was injected into a Jasco HPLC system (Jasco, Tokyo, Japan) consisting of a PU-2089 pump with an UV-2075 UV/Vis detector and using a Tricorn 5/100 glass columns (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), filled with Sepharose CL-2B (GE Healthcare Bio-Sciences AB) were used, and the eluent was PBS with a flow rate of 0.5 mL/min. The UV-Vis chromatograms were collected at 450 nm wavelength corresponding to the absorption maximum of Cu(I)-neocuproine complex.

Flu-SEC was used to study the release of soluble proteins from EVs upon a freeze–thaw cycle. 10 µL of REV sample was injected into a Jasco HPLC system (Jasco, Tokyo, Japan) consisting of a PU-2089 pump with a UV-2075 UV/Vis detector and a FP-2020 fluorescence detector controlled by the Chromnav software v. 1.17.02. Tricorn 5/100 glass columns (GE Healthcare Bio-Sciences AB) were packed with Sepharose CL-2B (GE Healthcare Bio-Sciences AB), and the eluent was PBS with a flow rate of 0.5 mL/min. The fluorescence chromatograms were collected at excitation and emission wavelength corresponding to the intrinsic fluorescence of proteins (280/340 nm), and the area under the curve of the EV peak and the free protein peak was used to quantify the amount of unbound proteins.