Pmx 110

Exosome pellets were resuspended in 1 ml of PBS and examined with a ZetaView PMX 110 instrument (Particle Metrix, Meerbusch, Germany). We used NTA software (ZetaView) to analyze the particle size and quantity (Min et al., 2019 (link)).

RAW264.7 cells were cultured in DMEM containing EXO-free FBS (ultracentrifugation at 120,000 ×g for 12 h) before LPS stimulation. RAW264.7 cells were cultured to 80% confluence, and then the control group was not given any treatment. The LPS group was stimulated with 1 µg/mL LPS (#L2880; Sigma, St Loui, MO, USA) for 24 h, and the supernatant was collected and subjected to a series of gradient centrifugation to extract the EXOs.
For the extraction of renal EXOs, 20 mg of renal cortex was digested with collagenase at 37 °C for 120 min. The samples were then subjected to EXOs extraction. The gradient centrifugation included 300 ×g for 10 min and 2000 ×g for 10 min, followed by 10000×g for 30 min. The supernatants were then centrifuged at 120000 ×g for 70 min. The pellets were washed once with PBS, centrifuged again at 120000×g for 70 min and resuspended in PBS (Type 70 Ti rotor; Beckman Coulter Optima, USA) .
The morphology of RAW264.7-EXO was examined using a transmission electron microscope (Hitachi HT770, Tokyo, Japan). Nanoparticle tracking analysis (NTA) was performed by Zetaview, PMX 110 (Particle Metrix, Meerbusch, Germany), and the protein levels was quantified using the BCA Protein Assay Kit (Aspen, Wuhan, China) following the manufacturer’s instructions.

The culture medium was replaced with a serum-free culture medium when SCCs had reached 80–90% confluency. Exosomes were isolated from cell culture supernatants from L-SCCs and H-SCCs after 24–48 h of culture by differential ultracentrifugation. Briefly, the cell culture medium was centrifuged at 300 × g for 10 min, the supernatant was collected, centrifuged at 2,000 × g for 10 min, the supernatant collected again, and centrifuged at 16,000 × g for 30 min. The resulting supernatant went through two rounds of centrifugation at 100,000 × g for 70 min. The exosome-containing pellet was resuspended in PBS for the identification of functional assays. For identification, the morphology and size of the exosomes were assessed by the transmission electron microscopy (TEM) (HITACHI, H-7650, Tokyo, Japan) and ZetaView analyzer (Particle Metrix, Pmx110, Meerbusch, Germany). Exosomes were quantified using a BCA assay and 20 μg/ml exosomes were used for the functional assays. For osteogenic differentiation assay, osteogenic inducing fluid containing 20 μg/ml exosomes were used for the duration of both RT-qPCR and Alizarin red, with the medium being changed every 3 days.