Hieff quick exosome isolation kit for serum plasma

According to the manufacturer’s protocol, plasma EVs were extracted using the Hieff^® Quick exosome isolation kit (for serum/plasma) (cat.41202ES30, Yeasen Biotechnology, Co., Ltd., Shanghai, China) and cryopreserved at − 80 °C until use. Briefly, a 1 ml plasma sample was centrifuged at 4 °C at 3000× g for 10 min. The supernatant was collected and centrifuged at 4 °C at 10,000× g for 20 min. Then, 4 ml PBS was added and thoroughly mixed, 1 ml 41,202-A reagent was added to the sample diluted with PBS, vortexed for 1 min and placed in a refrigerator at 4 °C for 2 h. The mixed liquid was centrifuged at 4 °C at 10,000× g for 60 min, the supernatant was discarded, and the sediment rich in EVs was collected. Then, 400 μl of PBS was added to the sediment to resuspend the EVs. The suspension was centrifuged at 4 °C at 12,000 × g for 2 min, the sediment was discarded, and the supernatant was retained which contained the purified EVs fraction. BCA assays were used to measure the concentration of total protein, then the EVs preparations were stored at − 80 °C.

Blood samples were collected from both groups at 8:00 a.m. after they had fasted for at least 8 hours. Fresh blood samples were subjected to centrifugation at a speed of 3000 × g for 10 minutes at 4°C. The supernatant was subsequently transferred to another tube, and plasma exosomes were isolated using a Hieff™ Quick exosome isolation kit (for Serum/Plasma, Yeasen Biotech Co., Ltd., Shanghai, China) according to the manufacturer's instructions. The morphological characteristics and size distribution of the plasma-derived exosomes were further assessed by transmission electron microscopy and nanoparticle tracking analysis as previously described [20 (link)].