Detection of CD81.GFP in the AChE+ fractions derived the crude exosomes treated with RIPA buffer.
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293T (2 x 106) were plated in a 10 cm plate and transfected with CD81.GFP. Transfected cells were cultured in exosome-free medium for 3 days. Culture medium was collected, pooled (about 30 ml) and used to isolate crude exosomes as described above. The crude exosome pellet was lysed in 100 μl RIPA buffer, subjected to 3 rounds of freezing and thawing on dry ice, and diluted in 400 μl PBS, followed by the same 6%-18% OptiPrep gradient centrifugation. Aliquots of each fraction were used for AChE activity assay (24 μl, A). The remaining fractions (426 μl) were diluted in 4 ml PBS and spun by 100,000 g, 70 min. The pellets were lysed in the RIPA buffer, followed by Western blotting using an anti-TSG101, or CD81.GFP antibody. The AChE activities were mean ± SD of triplicates; the data were representative of three independent experiments.