OptiPrep density gradient UC

Density gradient UC using OptiPrep^TM [60% (w/v) solution of iodixanol in water; Axis-Shield PoC AS, Oslo, Norway] was performed according to the manufacturer’s protocol with specific modifications. A discontinuous gradient of 50%, 35% and 20% (w/v) iodixanol was made by mixing 50% (w/v) iodixanol working solution with appropriate amounts of a homogenization medium (HM; 0.25 M sucrose, 150 mM NaCl, 20 mM HEPES pH 7.4). Iodixanol working solution was prepared by mixing 5 volumes of OptiPrep with 1 volume of dilution medium (DM; 0.25 M Sucrose, 0.9 M NaCl, 120 mM HEPES pH 7.4). The LEV pellets after UC were dissolved in 0.417 mL of DM and mixed with 5 volumes (2.085 mL) of OptiPrep solution. Next, 2.5 mL of control (50% iodixanol alone), or sample in 50% iodixanol was overlaid with 2.5 mL each of 35%, 20% and 0% solutions and centrifuged at 200,000× g at 4°C for 2 h (SW 41 Ti rotor, Beckman Coulter). The 1 mL fractions were then collected from the top of the gradient in the control tube, and the density of each fraction was determined via a standard curve created with absorbance values at 340 nm. The EV fractions at densities between 1.17~1.24 (between fractions 5 and 6; F5-6) were collected using a 1 mL-syringe from the sample tube. To remove iodixanol, the fraction was diluted in 60 mL of HBS, ultracentrifuged at 150,000× g at 4°C for 3 h, and resuspended with 200 μL of HBS. LEVs purified by OptiPrep density gradient UC (density-purified LEVs) were analysed for total protein concentration using a Bradford assay, and their distribution, average diameter and particle numbers were measured using DLS with Zetasizer Nano ZS or TRPS with qNano Gold. Density-purified LEVs were then used for validating the biological effects of UC-purified LEVs and for high resolution image analysis using bio-transmission electron microscopy (TEM) and cryo-electron microscopy (EM).