Ap5333b ev

Total exosomes were isolated by Total Exosome Isolation Reagent for Serum (Life Technologies, Austin, USA) according to the manufacturer's instructions. Briefly, 600 μl of serum, removed from cells and debris by a centrifugation step at 2,000 g for 30 min., were incubated with 120 μl of the exosome isolation reagent at 4°C, for 30 min., Following centrifugation at 10,000 g for 10 min., the total exosomes were precipitated, and exosomal proteins were extracted from the pellet with RIPA buffer (150 mM NaCl, 1% NP40, 0.5% Na-deoxycholate, 0.1% SDS, 50 mM Tris pH 8). The quality of the extracted exosomes was verified on a Western blot (as described in our previous work [23 (link)]) 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). The purity of the extracted exosomes (not lysed) was checked on a Western blot using the antibody specific for AGO2 protein (ab32381, abcam, San Francisco, USA). AGO2 protein isolated from HeLa cells served as positive control.

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).