Nanovesicles extraction
Veerle Baekelandt
Published: 2021-08-05 DOI: 10.17504/protocols.io.bw5wpg7e
Abstract
Nanovesicles extraction (Exosomal isolation, SHSY-5Y)
Steps
1.
seed cells for 24h 0m 0s in 3 x 15 cm dishes at a density of 20 x 106 cells per plate
2.
wash cells of all the plates with 2 times 10mL of 1x PBS
3.
Add 10mL DMEM with 1% exosome depleted FBS to each plate for 24h 0m 0s
Preparation of exosome depleted FBS:
Commercial FBS was filtrated through 0.22 µm filter and 140000x g,0h 0m 0s
4.
collect medium.
purify nanovesicles with differential centrifugation (see substeps)
4.1.
300x g (remove cells)
4.2.
15000x g (remove cell debris)
4.3.
filter through 0.22 µm filter (filtration of apoptotic bodies)
4.4.
140000x g,4°C
5.
decant supernatant and collect nanovesicles in 50µL cell lysis buffer (for western blotting) or 50 µl PBS+ PI (for intact cell cross-linking)
