Analytical Centrifugation in Density Gradients

Addressing challenges in the removal of unbound dye from passively labelled extracellular vesicles

“…The gradient was centrifuged at +4 °C with 200 000× g (40 291 rpm, k -factor 129) for 3.5 h (13.2 ml, Open-Top Thinwall Ultra-Clear Tube, Optima L-80 XP ultracentrifuge with SW 41 Ti Rotor, Beckman Coulter, USA). Upon the centrifugation the EVs move from their original bottom layer (density of >1.215 g ml −1 ) to the top of the layer with the density of ∼1.195 g ml −1 with a velocity dependent on the cube of their size, 37 i.e. , the EVs concentrate on the 0–35% iodixanol interface according to their buoyant density.…”