EVs were isolated from culture medium of DPCs with or without treatment of LPS (0.1, 1, 10 µg/mL) for 3 days. Collected culture medium was centrifuged as previously published protocols23 (link)
: 300 g for 10 min, 2000 g for 10 min, and 10,000 g for 30 min to eliminate cells and debris, followed by ultracentrifugation at 110,000 g for 70 min (Optima L-90 K; Beckman Coulter, USA). After washed once with PBS and centrifugation at 110,000 g for 70 min, supernatant was carefully removed and the pellet was resuspended in PBS. Besides, fetal bovine serum was ultracentrifuged at 100,000 g for 12 h to prepare EV-free serum, which was utilized for subsequent cell culture. Particle size distribution of collected EVs was identified by nanoparticle tracking analysis (NTA) (NanoSight NS300, Malvern, UK). After loaded onto a copper grid and stained with 2% (w/v) phosphotungstic acid for 5 min, EV morphology was examined by transmission electron microscopy (TEM; Japan). Protein concentration of EVs was quantified with a BCA Protein Assay Kit (Kangwei, Beijin, China), and the EV surface markers CD9 (1:1000, DF6565, Affinity Biosciences, USA), CD63 (1:1000, DF2305, Affinity Biosciences, USA), CD81(1:1000, Zen, Chendu, China), and TSG101 (1:1000, ab125011, Abcam, UK) were analyzed by western blot analysis.
: 300 g for 10 min, 2000 g for 10 min, and 10,000 g for 30 min to eliminate cells and debris, followed by ultracentrifugation at 110,000 g for 70 min (Optima L-90 K; Beckman Coulter, USA). After washed once with PBS and centrifugation at 110,000 g for 70 min, supernatant was carefully removed and the pellet was resuspended in PBS. Besides, fetal bovine serum was ultracentrifuged at 100,000 g for 12 h to prepare EV-free serum, which was utilized for subsequent cell culture. Particle size distribution of collected EVs was identified by nanoparticle tracking analysis (NTA) (NanoSight NS300, Malvern, UK). After loaded onto a copper grid and stained with 2% (w/v) phosphotungstic acid for 5 min, EV morphology was examined by transmission electron microscopy (TEM; Japan). Protein concentration of EVs was quantified with a BCA Protein Assay Kit (Kangwei, Beijin, China), and the EV surface markers CD9 (1:1000, DF6565, Affinity Biosciences, USA), CD63 (1:1000, DF2305, Affinity Biosciences, USA), CD81(1:1000, Zen, Chendu, China), and TSG101 (1:1000, ab125011, Abcam, UK) were analyzed by western blot analysis.