Total exosome isolation kit

Human umbilical mesenchymal stem cells (UMSCs; Jiangsu Heze Biotechnology Co., Ltd., China) were cultured in minimum essential medium (MEM) with 10% fetal bovine serum (FBS). The FBS had been centrifuged at 100,000 g to eliminate preexisting bovine-derived exosomes. After 48 h in culture, exosomes were isolated from UMSC culture supernatant using a total exosome isolation kit (Life Technology, Grand Island, NY, USA). The culture medium collected from UMSCs was centrifuged at 2,000 g for 30 min to remove dead cells and debris, and then transferred to a new tube containing 0.5 volumes of the Total Exosome Isolation reagent. The mixture was incubated at 4 °C overnight and centrifuged at 10,000 g for 1 h at 4 °C. The pellets (exosomes) were resuspended in phosphate buffer saline. The concentration of exosomes was determined using a bicinchoninic acid (BCA) protein assay kit (Takara, Japan). The exosomes were attached to aldehyde/sulphate latex beads (4 μm, Molecular Probes, Invitrogen), then incubated with a FITC‐conjugated antibody against CD63 (Abcam), and the expression of CD63 was analyzed by flow cytometry. In separate experiments, the expression of exosome marker CD9 was also analyzed by Western blot.

Islets exosomes were isolated with ExoQuick-TC Exosome Precipitation Solution (SBI, Palo Alto, USA). After islets were incubated in RPMI-1640 medium with cytokines cocktail or STZ for 24 hours, the media was collected and centrifuged at 3000g for 15 min at 4°C. Then, the supernatant was mixed with appropriate volume of ExoQuick-TC and refrigerated overnight at 4°C followed by a centrifugation step of 1500g for 30 min. Discarded the supernatant and obtained the pellets of the exosomes.
Isolation of exosomes from serum was performed using Total Exosome Isolation Kit (Life Technologies, Carlsbad, USA) according to the manufacturer’s instruction. The serum volume was fixed at 1000μl for testing exosomal miRNAs by qRT-PCR of samples both from mice and human. Because the blood that can be collected from one mouse is scant, sera of three or four mice were mixed together to achieve the fixed volume of one sample, and each group included 3samples. Firstly, the serum sample was centrifuged at 2000 × g for 30 minutes to remove cells and debris. Then, the clarified serum was transferred to a new tube and add 0.2 volumes of the Total Exosome Isolation reagent, following vortexing and incubating at 4°C for 30 minutes. After incubation, the sample was centrifuged at 10000 × g for 10 minutes at room temperature. Discarded the supernatant, exosomes were contained in the pellet at the bottom.

The exosome-producing medium was prepared to remove residual exosomes from FBS as referred [35 ] with modification. Generally, 50% (v/v) FBS in DMEM medium was centrifuged at 2,000 × g for 10 min, and then centrifuged at 100,000 × g (Beckman Optima L90-K with 90Ti rotor; Beckman Coulter Taiwan Inc., Taipei, Taiwan) for 16 hr at 4° C. The supernatant were collected and diluted into 10% (v/v) FBS by serum-free DMEM, and were sterile through 0.22 μm filter. For production of hepatoma-derived exosomes, 1 × 10⁶ Hep3B or Huh7 cells were plated in culture medium overnight, and were replaced into exosome-producing medium for successive culture for 2 days. Exosome-containing medium (100 mL) were collected and exosomes were isolated by Total Exosome Isolation kit (Life Technologies, Grand Island, NY, USA) according to suggested protocol. The pellets containing secreted exosomes were further washed by DEPC-treated PBS, centrifuged at 100,000 × g for 60 min (Beckman Optima MAX-E with TLA-120.2 rotor), and repeated twice to remove residual serum protein. The protein content in the exosome solution was determined by protein assay reagent (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA).

To isolate exosomes derived from TAMs associated with epithelial ovarian cancer and human EOCSKOV3 cells, which were obtained from FuHeng BIO (Shanghai, China), cells were first cultured in RPMI-1640 for 48 h. We centrifuged the cell supernatants twice (2000g for 10 min, then 2500g for 30 min to deplete the cells or fragments), added the total exosome isolation kit (Life technology) overnight, and then centrifuged at 10,000g for 1 h. Exosomes were resuspended in PBS (Gibco) and stored at −80 °C. The exosome concentration was detected by the BCA Protein Assay.

Human umbilical mesenchymal stem cells (UMSCs; Jiangsu Heze Biotechnology Co., Ltd., China) were cultured in Dulbecco's modified Eagle's medium:F12 containing 20% foetal bovine serum (FBS). The FBS was centrifuged at 110 000 g to remove bovine‐derived exosomes. Exosomes were isolated from UMSC culture medium (48 hours) using a total exosome isolation kit (Life Technology). The culture medium was first centrifuged at 2000 g for 30 minutes to get rid of debris and dead cells, and then transferred to a new tube containing 0.5 volumes of the Total Exosome Isolation reagent. The mixture was incubated at 4°C overnight and centrifuged at 10 000 g for 1 hour at 4°C. The pellet was re‐suspended in PBS, and the protein concentration was determined using a BCA protein assay kit (Takara).
The morphology of the exosomes was revealed by transmission electron microscopy. The exosomes were attached to aldehyde/sulphate latex beads (4 μm; Molecular Probes; Invitrogen), then incubated with an FITC‐conjugated antibody against CD63 (Abcam), and the expression of exosome marker CD63 was analysed by flow cytometry and Western blot.

Exosomes from sera was extracted using total exosome isolation kit (Life technologies) [45 (link)]. Briefly, 15 ml serum was centrifuged at 3,000 g for 30 min and thereafter at 10,000 g for 30 min to remove cells and debris. Sera were further diluted with PBS and centrifuged at 10,000g for 10 min at room temperature. The pelleted exosomes was solubilized with PBS and utilized for characterization.