Exoquick exosome precipitation solution

Serum amounts of total exosomes were quantified by the Exosome Antibodies & ELISA Kit (System Biosciences, Mountain View, California, USA), which is specific for the exosomal protein CD63. At first, exosomes were precipitated from 250 μl serum, removed from cells and debris by a centrifugation step at 2,000 g for 30 min., with 63 μl ExoQuick exosome precipitation solution (BioCat, Heidelberg, Germany) and then resuspended in 200 μl exosome binding buffer according to the manufacturer's instructions. Fifty μl of these exosomal protein samples and CD63 protein standards (undiluted, diluted 1:2, 1:4, 1:8, 1:16, 1:32 and 1:64) were added to the micro-titer plate. ELISA assay was carried out following the manufacturer's instruction. The absorbance at 450 nm was measured on a spectrophotometric plate reader (Tecan, Männerdorf, Switzerland), and the amounts of CD63 protein were calculated according to the exosome protein standard curve. The quality of the extracted exosomes was verified on a Western blot using 3 different antibodies specific for the exosomal markers Mucin1 (CD227, BD Biosciences, Franklin Lakes, New Jersey, USA), CD63 (AP5333b-ev, ABGENT, San Diego, California, USA) and CD9 (AP1482d-ev, ABGENT, San Diego, California, USA).

Exosomes were isolated by the ExoQuick exosome precipitation solution (BioCat, Heidelberg, Germany) according to the manufacturer's instructions. Four hundred μL of serum were incubated overnight, on ice, with 100 μl of the ExoQuick solution. Following precipitation of exosomes, exosomal RNA was extracted using the mirVana PARIS kit (Life Technologies) and exosomal protein was extracted using RIPA (150mM NaCl, 1% NP-40, 0.5% sodium-deoxycholate, 0.1% SDS, 50mM Tris/HCl, pH 8) buffer. The extraction of exosomes was verified by Western Blot using an antibody specific for the exosomal marker proteins CD63 (BioCat), Mucin1 (CD227, BD Biosciences) and GAPDH (Sigma, Supplementary figure 4).