Purification of Subpopulations of Extracellular Vesicles: A Technique to Purify Subpopulations of Extracellular Vesicles Using Density Gradient Ultracentrifugation
Transcrição
Extracellular vesicles or EVs are nano-sized, heterogenous, membrane-bound vesicles secreted by various cells, including brain tumor cells. The small EVs are a subpopulation comprising endosomal-derived and membrane-shed EVs.
To purify the small EVs, begin by taking pre-extracted EVs suspended in a sucrose supplemented buffer that maintains a homogenous solution and preserves the structural integrity of EVs.
To this solution, then, add an appropriate volume of iodixanol – an inert and non-toxic density gradient medium – to achieve the desired density.
Iodixanol solubilizes the contaminating proteins to be removed later.
Next, add layers of iodixanol solutions of successively decreasing densities to create a discontinuous density gradient.
Centrifuge the tube at high speed.
Depending upon their floatation rates, the denser EVs migrate to an equally-dense layer of gradient medium while less-dense EVs migrate further to a lighter layer, and the densest contaminants form a pellet at the bottom of the tube.
Now, transfer small fractions of the gradient layers in fresh tubes containing a suitable buffer.
Thereafter, centrifuge the tubes at ultra-high speed in chilled conditions.
During centrifugation, low temperature prevents EVs from high-speed-induced heat damage.
After the pure EVs pellet down, remove the supernatant. Resuspend the EVs in an appropriate buffer and proceed for characterization and proteomics analysis.
