High speed refrigerated centrifuge

Loratadine tablets [Bayer Healthcare (Shanghai) Co., Ltd., National Drug ApprOVAl Number H10970410]. OVA (Sigma, United States, A5503-5G). Aluminum Hydroxide (Thermo Fisher Scientific, United States, 77161). Mouse OVAsIgE enzyme-linked immunosorbent assay (ELISA) Kit (Shanghai Jianglai Biotechnology Co., Ltd., JL20466). Mouse IL4 ELISA Kit (Shanghai Jianglai Biotechnology Co., Ltd., JL20266). Mouse IL5 ELISA Kit (Shanghai Jianglai Biotechnology Co., Ltd., JL20267). Mouse IL13 ELISA Kit (Shanghai Jianglai Biotechnology Co., Ltd., JL20247). DNA extraction kit (Omega Soil DNA Kit, D5625-01). High-fidelity DNA polymerase (NEB, M0491L). Gel recovery kit (Axygen, AP-GX-250). Fluorescence quantitative polymerase chain reaction (PCR) reagent kit (BioTek, United States, FLx800). High-fidelity DNA polymerase (NEB, M0491L). Gel recovery kit (Axygen, AP-GX-250). Fluorescence quantitative PCR reagent kit (BioTek, United States, FLx800).
Rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, RE-2000A); Circulating water vacuum pump (Zhengzhou Changcheng Science and Technology Trade Co., Ltd., SHB-III); Fc-type ELISA reader (Thermo, United States); High-speed refrigerated centrifuge (Beckman Coulter, Inc., United States); PCR instrument (Bio-Rad, Germany, C1000).

hADMSC-sEVs were collected and purified according to the following processes. We selected the fourth-passage hADMSCs to extract sEVs. After cells’ confluency reaching 70–80%, hADMSCs’ culture medium was replaced with serum-free low glucose DMEM for 48 h to collect cells’ supernatant. To isolate and remove cell particles, dead cells, and cell debris of the obtained supernatant, we performed a series of differential centrifugal precipitation (300×g for 10 min, 2000×g for 10 min, and 10,000×g for 30 min). The supernatant removed the sediment was then filtered through a 0.22-μm filter (Millipore, USA) to remove the large extracellular vesicles further and ultracentrifuged at 100,000×g for 70 min by using the High-Speed Refrigerated Centrifuge (Beckman Coulter, USA). The supernatant was discarded, and the precipitation was resuspended with PBS. Finally, the suspension was ultracentrifuged at 100,000×g for 70 min again, and sEVs were obtained after precipitation collection. The obtained sEVs concentration was measured with bicinchoninic acid (BCA) protein detection kit (Beyotime, Shanghai, China) and stored at − 80 °C for further use. All centrifugations are operated at 4 °C.