Sucrose Gradient Density Profiling of sEVs

For determination of sEV density on sucrose gradients, sEVs were labeled with the fluorescent red carbocyanine DiI (Invitrogen, Carlsbad, CA, USA) at a concentration of 1.0 mmol/L in 50 mM trehalose PBS and re-isolated at 110,000× g for 1.5 h according to established methods [16 (link)]. Flotation of DiI labeled sEVs on a continuous sucrose gradient (2.0–0.25 M sucrose, 20 mM HEPES/NaOH, pH 7.4) was performed similarly as previously described using a Beckman Coulter SW 41 rotor [16 (link),18 (link)]. For each gradient, 400 μg of sEV protein was loaded except for the primary melanocyte sEVs in which case 80 μg of protein was loaded. The primary melanocytes grow very slowly, requiring multiple months of culture to accumulate small amounts of sEV material. The gradient was produced using a Gradient Master (Biocomp Instruments, Fredericton, NB, Canada) and was spun, after loading sEVs, for >15 h at 100,000× g. Post centrifugation, 1 mL fractions were collected from the bottom up. The density of each fraction was calculated using a refractometer [18 (link)]. Two hundred microliters of each fraction were added to a black 96-well plate, and DiI sEV fluorescence detected using a Tecan M200 infinite pro microplate reader according to established methods [16 (link),18 (link)].