SEC-based Isolation of Microglia-derived Extracellular Vesicles: A Technique to Isolate Extracellular Vesicles from Microglia Conditioned Culture Media

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.

Transfer the conditioned culture medium from microglia or macrophage cultures into a conical tube and centrifuge it at 1,200 x g for 10 minutes to pellet the cells. Transfer the supernatant into a new conical tube and centrifuge for another 20 minutes to eliminate apoptotic bodies.

Then, transfer the supernatant into a 10.4-milliliter polycarbonate tube. Place the tube into a 70.1 Ti rotor and ultracentrifuge it at 100,000 x g for 90 minutes at 4 degrees Celsius to pellet the EVs. After centrifugation, discard the supernatant and resuspend the pellet in 200 microliters of 0.2-micrometer filtered PBS.

To isolate the EVs, prepare a home-made size exclusion chromatography column by washing and sterilizing a glass chromatography column and placing a 60-micrometer filter at the bottom. Stack the column with cross-linked agarose gel filtration base matrix to create a stationary phase of 0.6 centimeters in diameter and 20 centimeters in height.

Then, rinse the phase with 50 milliliters of 0.2-micrometer filtered PBS. If necessary, store the column at 4 degrees Celsius for later use. Place the resuspended EV pellet on top of the stationary phase and collect 20 sequential fractions of 250 microliters while continuing to add PBS to the top of the stationary phase. The fractions can be stored at minus 20 degrees Celsius.