20 ml plastic column

EVs were recovered by size-exclusion chromatography as described by [10 (link)]. Briefly, the 500 µL of concentrated supernatant was mixed with the lipophilic dye FM5-95 (1.75 µM, Thermo Fisher, Waltham, MA, USA) and incubated for 15 min at room temperature protected from light. Simultaneously, a 20 mL plastic column (Takara, Kusatsu, Shiga, Japan) was loaded with 10 mL of Sepharose CL 2B (Sigma) and equilibrated with Dulbecco’s phosphate-buffered saline (DPBS, Billings, MT, USA). The supernatant was placed onto the Sepharose, and 45 fractions (approx. 300 µL each) were eluted in DPBS and collected in a black microtiter plate with black bottom (Greiner Bio-One, Kremsmünster, Austria). The fluorescence of the fractions was measured immediately in a SpectraMax M2 plate reader (Molecular Devices, San Jose, CA, USA). Adjacent fractions with consistent positive relative fluorescence units (RFUs) above 1.5 were pooled and labeled “EV sample”. The protein concentration of the EV sample was measured with a Qubit4 (Thermo Fisher). The protein content of the unpooled fractions was quantified by microBCA (Thermo Fisher). All samples were snap-frozen and preserved at −80 °C until further use.

EVs were separated by size-exclusion chromatography (SEC) as described previously [15 (link)]. Briefly, the concentrated supernatant was mixed with the fluorescent lipophilic dye FM5-95 (Thermo Fisher, Waltham, MA, USA) at a concentration of 1.75 µM (5 µL, 0.1 mg/mL), on a rotary incubator for 15 m at room temperature with protection from light. The sample was loaded onto a 20 mL plastic column (Takara) containing 10 mL of Sepharose CL 2B (Sigma) equilibrated with Dulbecco’s phosphate buffered saline (DPBS, Thermo Fisher). Forty-five fractions (approx. 300 µL each) were eluted with DPBS and collected in black microtiter plates with black bottom (Bunzl, London, UK). The fluorescence of the fractions was measured immediately in a SpectraMax M2 plate reader (Molecular Devices, San Jose, CA, USA). Adjacent fractions with consistent positive relative fluorescence units (RFU) above the baseline were pooled and named “EV sample”. The protein concentration of the EV sample was determined with a Qubit4 (Thermo Fisher). The protein content of the unpooled fractions was quantified by microBCA (Thermo Fisher). All samples were frozen in liquid nitrogen and preserved at −80 °C until further use.