EVs in mouse serum were isolated with an EV isolation kit (SmartSEC Mini EV Isolation System) and detected with an ELISA kit detecting CD81 exosome (EXOEL-CD81A-1) from System Biosciences.
L02 and primary hepatocytes (PMH) were cultured 48 hours at 37°C in serum-free DMEM. The supernatant was collected and centrifuged at 2,000 g for 10 min at 4°C to remove the cell debris [20 (link)]. Then, after filtering through 0.22 μm filters, the filtrate was ultracentrifuged at 100,000 g for 120 minutes in a Beckman SW28Ti rotor. After the resuspension by PBS, the pellets were ultracentrifuged again at 100,000 g for 120 min. The final EV pellets were dissolved in PBS for further experiments [20 (link)].
To isolate CD47+ EVs, a total of 200 μg PMH-EVs were mixed with nonblocking anti-CD47 antibody (REA170)-FITC for 30 min at 4°C. Then, incubated with anti-FITC magnetic beads (Miltenyi Biotec; 1 μL/μg EVs) overnight at 4°C. CD47− EVs and CD47+ EVs were separated by magnetic beads, and both supernatants were washed with PBS and pelleted by ultracentrifugation.
EVs were further analyzed by a Micro BCA Protein Assay Kit to determine protein concentrations (Thermo, #23235). Particle diameters and amounts were observed by the NanoSight system (NS300, Malvern, Ranch Cucamonga, CA, USA). Further details of methods were described in supplementary materials (availablehere ).
L02 and primary hepatocytes (PMH) were cultured 48 hours at 37°C in serum-free DMEM. The supernatant was collected and centrifuged at 2,000 g for 10 min at 4°C to remove the cell debris [20 (link)]. Then, after filtering through 0.22 μm filters, the filtrate was ultracentrifuged at 100,000 g for 120 minutes in a Beckman SW28Ti rotor. After the resuspension by PBS, the pellets were ultracentrifuged again at 100,000 g for 120 min. The final EV pellets were dissolved in PBS for further experiments [20 (link)].
To isolate CD47+ EVs, a total of 200 μg PMH-EVs were mixed with nonblocking anti-CD47 antibody (REA170)-FITC for 30 min at 4°C. Then, incubated with anti-FITC magnetic beads (Miltenyi Biotec; 1 μL/μg EVs) overnight at 4°C. CD47− EVs and CD47+ EVs were separated by magnetic beads, and both supernatants were washed with PBS and pelleted by ultracentrifugation.
EVs were further analyzed by a Micro BCA Protein Assay Kit to determine protein concentrations (Thermo, #23235). Particle diameters and amounts were observed by the NanoSight system (NS300, Malvern, Ranch Cucamonga, CA, USA). Further details of methods were described in supplementary materials (available
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