Tx 400 rotor

Exosomes were isolated from the fresh or frozen cell culture supernatants as previously described 30 . Briefly, cells were removed by centrifugations at 300g for 10 min (TX-400 rotor, ST16R, Thermo Fisher, Massachusetts, USA). Subsequently, supernatants were centrifuged at 2,000g for 10 min to remove smaller cellular debris and at 10,000g for 30 min to remove apoptotic bodies (F15-6x100y rotor, ST16R, Thermo Fisher, Massachusetts, USA). Exosomes were then harvested by ultracentrifugation at 100,000g for 70 min in a swing rotor (SW32Ti, XPN-100, Beckman, California, USA). All centrifugation steps were performed at 4℃, and isolated exosomes were suspended in phosphate-buffered saline (PBS) and stored at -80℃. The protein content of exosome suspension was quantified using the bicinchoninic acid (BCA) assay (Beyotime, P0010, Beijing, China). The representative markers CD63 (1:1000, Abcam, California, USA) and CD81 (1:1000, Abcam, California, USA) of exosomes were assayed by Western blotting. Furthermore, the morphology and size distribution of exosomes were detected by transmission electron microscope (TEM) (JEM-1400, JEOL Ltd., Japan) and nanoparticle tracking analysis (Nanosight LM10, Malvern, Worchestershire, UK), respectively. The protein content of the MSC-Exo preparations was normalized to total protein content as quantified by BCA assay.

Fixed cells were pelleted using a Sorvall Legend X1R centrifuge mounted with a TX-400 rotor (Thermo Scientific) at 3000×g for 5 min. Supernatants were decanted and algal pellets resuspended in 1 mL of 10% neutral buffered formalin (Epredia™ HiPur™). Algal pellets were gross processed and embedded by HistoWiz Inc. using a Standard Operating Procedure and fully automated workflow. Paraffin blocks were sectioned in-house using a Leica microtome at a width of 4 μm followed by adhesion to charged slides (HistoBond®). Slides were stored at room temperature in the dark until further processing.