We first identified priority cytokine candidates based on known liver fibrosis pathophysiology and previously published literature, then selected from the assays available at Myriad-RBM, which were primarily multiplexes (Luminex, Myriad-RBM Inc., Austin TX). Then purified EVs from 20 MM and 20 ZZ plasma samples (Table ), were suspended in a mixture of water and PBS (1:2). EVs in solution were lysed using an equal volume of IP lysis buffer. Lysed EV samples to which Triton X was added at final concentration of 1% were run on the MilliPLEX Human High Sensitivity T Cell Magnetic Bead Panel Luminex kit for measurement of 21 unique cytokines per plex according to manufacturer’s instructions (EMD Millipore, St. Louis, MO).
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