Total exosome isolation from serum kit

MSCs or MRC5 was cultured with respective medium supplemented with 10% EVs-removed FBS for 24–48 h. Before the next sub-cultivation, the cellular supernatant was collected and centrifuged at 500 g for 10 min to discard any cell contaminations. The supernatant was next centrifuged at 10,000 g for 30 min to remove cell debris and apoptotic bodies. Then, raw MSCs-EVs or MRC5-EVs was isolated by ultracentrifugation at 100,000 g for 70 min and washed by Dulbecco’s phosphate-buffered saline (DPBS). Finally, purified MSCs-EVs or MRC5-EVs was ultracentrifuged at 100,000 g for 2 h and the pellet was resuspended in PBS. All the procedures were performed at 4 °C.
Peripheral blood-derived EVs were isolated by Total Exosome Isolation (from serum) Kit (Invitrogen, 4,478,360) according to the manufacturer’s instructions. Briefly, 200 μL serum and 40 μL exosome isolation reagent was mixed by a pipette and vortexed until there is a homogenous solution, followed by the incubation for 30 min at 4 °C. Then, the pellet was centrifuged at 10,000 g for 10 min at RT and the supernatant was discarded. Finally, peripheral blood-derived EVs were collected and resuspended in a convenient volume of PBS. The total protein concentration of EVs was detected using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, 23,225, Madison, WI, USA) following the manufacturer’s instructions.

The sera derived from middle-aged females that underwent tMCAO followed by WBV or No-WBV were used to isolate EV. Serum EV were isolated using the Total exosome isolation from serum kit (Invitrogen) as described in a previous publication (Kerr et al., 2018 (link)). Briefly, 100 μl of each sample was centrifuged at 2,000 × g for 30 min and the supernatant was then incubated with 20 μl of Total exosome isolation reagent for 30 min at 4°C followed by centrifugation at 10,000 × g for 10 min at room temperature. Supernatants were then discarded, and the pellet was re-suspended in 50 μl of PBS and 100 μl of lysis buffer. The concentration and size distribution of the isolated exosomes were analyzed by a NanoSight NS300 system (Malvern Instruments Company, Nanosight, and Malvern, United Kingdom).

Exosomes from 1 mL serum sample were isolated using the “Total Exosome Isolation (from serum)” kit (Invitrogen) according to manufacturer’s instructions. The reagent contained in the kit forces less-soluble components, such as vesicles, out of solution, allowing them to be collected by a short, low-speed centrifugation (10,000 × g for 10 min). Pellet was re-suspended as described in each experiment and analyzed as described in the text.

Exosomes were isolated from the stored supernatant using a Total Exosome Isolation (from serum) Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The purified exosome solution (10 µl) was crossed with a hydrophilic collodion membrane for 15 min and treated with 1% uranyl acetate for 30 s. The grid was then placed on a TEM sample table for observation and photographed using iTEM. The purified exosome solution was also quantified and sized using the NanoSight LM10 system (Malvern Panalytical, UK) following the manufacturer's instructions.