Encyclopedia of Experiments: Cancer Research
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Transcript
Microglial cells release immune mediators in the form of extracellular vesicles or EVs, including larger apoptotic bodies and smaller micro-sized vesicles, into growth media in vitro.
To isolate EVs, begin by taking microglia conditioned media. This culture media contains microglial cells along with the released EVs. Centrifuge the media to pellet the cells.
Collect the supernatant containing the EVs, debris, and protein aggregates in a fresh tube. Centrifuge again to remove cellular debris and large apoptotic bodies.
Transfer the supernatant into an ultracentrifuge tube. Centrifuge at high speed.
Discard the supernatant containing spent media and resuspend the EV and protein aggregate pellet in the desired buffer.
Concomitantly, take a size-exclusion chromatography column with an appropriate filter at its base.
Tightly pack the column with a gel-filtration matrix consisting of spherical gel beads that form a porous sieve of defined pore size.
Load the EV and protein aggregate suspension on top of the stationary phase.
As the suspension passes through the column, larger-sized EVs move through interparticle spaces between the gel beads, traveling a shorter path. In contrast, the smaller-sized protein aggregates move into the pores, following a longer path.
Consequently, EVs elute out first from the column.
Collect the fractions containing EVs and use them for further analysis.
