OptiPrepTM density gradient centrifugation

Purification of exosomes by density gradient was described as previously²². OptiPrep^TM aqueous iodixanol solution (60% w/v) was mixed with a homogenization buffer (containing 0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH7.4) to prepare the solutions of 5, 10, 20, and 40% iodixanol. The gradient was set up by layering 3 ml of 40, 20, and 10% iodixanol solutions and 2.8 ml of 5% solution in a 13.2 ml ultra-clear tube (Beckman Coulter). Exosome pellets obtained by ultracentrifugation from TECs were resuspended and overlaid onto the top of the gradient, with centrifugation for 18 h at 100,000 × g and 4 °C (SW41Ti rotor, Beckman Coulter). Individual fractions of 1 ml were collected from the top of the gradient and diluted with 20 ml in PBS and centrifuged for 2 h at 200,000 × g and 4 °C. The pellets were resuspended in 100 µl PBS and stored at −80 °C for further analysis. A standard curve was made by the absorbance values at 340 nm of 5, 10, 20, and 40% iodixanol solutions to estimate the density of each fraction collected from samples.