0.1 m glycine hcl

tEVs were isolated from serum using ExoQuick solution (System Bioscience, Palo Alto, CA, USA) with some modifications to the manufacturer’s instructions. After centrifugation to remove cell debris, the serum of six animals in each group were pooled. A total of 2 mL of serum and 500 μL of solution were mixed. After the mixture was centrifuged, the pellet was resuspended in 200 μL phosphate-buffered saline (PBS).
nEVs were isolated as described by Mustapic et al. [52 (link)] with some modifications. Exosomes isolated from serum were incubated with anti-CD171 (L1CAM; Bioss Antibodies, Beijing, China) antibody for 1 h at 4 °C on a rotating mixer. After adding Pierce Streptavidin Plus Ultralink Resin (Thermo Fisher Scientific, Waltham, USA) and PBS, the samples were again incubated for 1 h at 4 °C on a rotating mixer. The samples were then pelleted by centrifugation at 200× g for 10 min at 4 °C. The supernatants were removed from the samples and the pellets were resuspended in 200 μL 0.1-M glycine-HCl (Biosesang, Seongnam, Korea). After mixing for 10 s and vortexing for 30 s, the samples were pelleted by centrifugation at 4500× g for 10 min at 4 °C. Finally, the supernatants were transferred to new tubes before Tris-HCl (Biosesang) and PBS were added.

The nEVs were isolated as described by Mustapic et al., with some modifications in this study [32 (link)]. Briefly, tEV samples containing PBS were incubated with anti L1 cell adhesion molecule (L1CAM; CD171) antibody (Bioss Antibodies, Beijing, China) for 1 h at 4 °C on a rotating mixer. Following the addition of Pierce^TM Streptavidin Plus Ultralink™ Resin (Thermo Fisher Scientific, Waltham, MA, USA) and PBS, the mixture was incubated for 1 h at 4 °C on a rotating mixer and then centrifuged at 500× g for 10 min at 4 °C. The supernatants were removed, and the pellets were resuspended in 0.1 M glycine-HCl (Biosesang, Seongnam, Korea). Following the mixing and vortexing, the samples were pelleted by centrifugation at 4500× g for 10 min at 4 °C. The supernatants were transferred to fresh tubes, and Tris-HCl (Biosesang, Seongnam, Korea) and PBS were added.