Hieff quick exosome isolation kit plus

Fetal Bovine Serum (Bio-Channel, BC-SE-FBS07), DMEM/F-12 (BiologicalIndustries, 01-170-1A), EDTA-Trypsin (Biosharp, BL512A), EndoFectin™Max Reagent (GeneCopoeia, EF013), Cell Value Added-Toxicity Assay Kit (Biosharp, BS350B), PA (Aladdin, S161420), OA (Maclean, S817542), ORO Staining Kit (Solarbio, G1262), BlazeTaq™ SYBR®Green Mix (GeneCopoeia, QP031), Dnase QP031), BCA Protein Reagent (P0009-1, P0009-1), Hematoxylin (Solarbio, H8070), Eosin Staining Reagent (Beyotime, C0105-2), Masson Trichrome Staining Reagent (Solarbio, G1340), TRIzol®Reagent (Life, Cat. no. 15596-018), SureScript™ First-Strand cDNA Synthesis Kit (GeneCopoeia, QP056), Immobilon Western HRP Substrate Luminol Reagent (Affinity, KF001), 30% Acrylamide/Bis solution (29:1) (Solarbio, A1010), 24-well plate (VIRYA, 3512409), FBS(Bio-Channel, BC-SE-FBS07), Hieff® Quick exosome isolation kit Plus (YEASON, China).
Tissue embedding kit (Jiangsu Shitai, 20084), Real-timePCR (Thermo Fisher Scientific, TCR0096, USA), UV spectrophotometer (ALLSHENG, USA), benchtop low-speed centrifuge (Weil Ltd., China), inverted fluorescence microscope (Zeiss, Observer.A1, Germany), gel imager (Monad, USA), ice maker (FM150KE, China), 4 °C refrigerator (Haier, China), baking machine (Thermo Scientific, DB-B2, USA), TEM(ZCIBIO, China), NTA(Particle Metrix, China).

The hUC-MSCs were provided by the Central Laboratory of Yan'an Hospital, Kunming Medical University. hUC-MSCs were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA), and the adherent cells were incubated in 10% fetal bovine serum (FBS)(Bio-Channel, BC-SE-FBS07) under cell incubation conditions (37 °C and 5% CO2) for at least 24 h. Then, the hUC-MSCs culture supernatant was collected, centrifuged(Eppendorf, Centrifuger5418), filtered and concentrated to remove dead cells, cell debris and decontamination. The hUC-MSCs exosomes were extracted from the cell supernatants using the Hieff® Quick exosome isolation kit Plus (YEASON, China) according to the kit instructions, resuspended in sterile PBS(Solarbio, NO. P1003), and stored at − 80 °C (Haier, DW-86L626). The protein concentration of exosomes was determined using the BCA protein assay kit (beyotime, P0009-1). The extracted hUC-MSCs exosomes were characterized by transmission electron microscopy (TEN; ZCIBIO, ZC1099). Particle size was analyzed by nanoparticle tracking analysis (NTA; Particle Metrix, ZetaView), and Western blot was used to detect exosome surface markers TSG101, CD9 and CD63.