Nanovesicles extraction

seed cells for 24h 0m 0s in 3 x 15 cm dishes at a density of 20 x 10⁶ cells per plate

wash cells of all the plates with 2 times 10mL of 1x PBS

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

collect medium.

purify nanovesicles with differential centrifugation (see substeps)

300x g (remove cells)

15000x g (remove cell debris)

filter through 0.22 µm filter (filtration of apoptotic bodies)

140000x g,4°C

decant supernatant and collect nanovesicles in 50µL cell lysis buffer (for western blotting) or 50 µl PBS+ PI (for intact cell cross-linking)

Intact cell cross-linking