Cp100nx

Extracellular vesicles were isolated according to recommendations of the International society of extracellular vesicles⁸¹ (link) and as in Pastuzyn et al.⁶⁶ (link) with minor modifications (Figure 4I). Control and AS patient derived neurons were cultured in T175 flasks for 6 weeks. 25 mL of conditioned media from two flasks were pooled to obtain one replicate of extracellular vesicles. The corresponding cell pellets were pooled and aliquoted for protein and mRNA analysis. Post media harvest, cell debris was removed by centrifuging at 200 x g for 10 min. Clarified media then centrifuged at 10,000 x g for 30 mins to remove microvesicles. The supernatant was carefully removed and ultracentrifuged at 100,000 x g for 2 hr (Hitachi CP 100NX). The supernatant was discarded and the pellet suspended thoroughly in 2 mL cold PBS followed by another round of ultracentrifugation at 100,000 x g for 1 hr. The EV pellets were then suspended in PBS and stored at −80°C for immunoblotting and mass spectrometry analysis.

The supernatant of E7 plasmid–transfected HuBM-MSCs subjected to 48 hours of culture was collected and centrifuged at 3000g for 10 min at 4°C. The supernatant was collected and filtered using a 0.22-μm filter (Biosharp) to remove the remaining cellular debris. Then, the supernatant was ultracentrifuged at 110,000g for 120 min at 4°C using an ultrahigh-speed centrifuge (CP100NX, HITACHI, Japan). The exosome pellets were resuspended in PBS, and the amount of exosomal protein was measured by the BCA Protein Assay kit (Beyotime). The exosomes were stored at 80°C.

EV proteins were extracted from concentrated EV‐rich SEC qEV fractions using a RIPA buffer (Thermo Scientific) with a protease inhibitor cocktail (Roche), or extracted using differential centrifugation method (CP100NX, Hitachi). Human embryonic kidney cell line HEK293 was obtained from ATCC (Manassas, VA), authenticated by short tandem repeat profiling, then cultured with DMEM/RPMI‐1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10 % foetal calf serum (Gibco BRL) and 1% penicillin plus streptomycin (HyClone, Logan, UT) in a humidified incubator (5% CO₂) under 37 °C. The protein content of EVs/cells were examined by Pierce BCA Protein Assay Kit (Thermo Scientific) for quantification and lysed in 1× SDS‐PAGE loading buffer (1% SDS, 11% glycerol, 10% β‐mercaptoethanol, 0.1 M Tris, pH 6.8) for assay. The expression of proteins was assessed by Western Blotting in 10 μg of HEK293T/MC38/CT26 cell lysate/mixed plasma EV samples/extracted cellular EV samples with the appropriate antibodies (for human proteins, ARG1/PD‐L1/CD81/CD9, identical to immunogold labelling assay; Calnexin, GTX629976, GeneTex; for mouse proteins, CD63, ab217345; CD9, ab92726; Calnexin, ab133615, Abcam), illuminated with the Clarity Western ECL substrate (Bio‐Rad, Hercules, CA), and visualized with the Amersham Imager 600 (GE Healthcare, Chicago, IL). Blotted bands were quantified with Image J software.

TFENs were isolated and purified following previous studies¹⁶ (link)^,22 (link). Tea flowers and phosphate-buffered saline (PBS) solution (1:2, g/mL) were placed in a blender and chopped at a high speed for 5 min. The obtained juices were centrifuged at 3000×g for 20 min (Hitachi, CP100NX, Tokyo, Japan) and 10,000×g for 1 h to remove the large plant tissues and cell debris. Subsequently, TFENs were collected by ultracentrifugation at 150,000×g for 2 h, and re-suspended in PBS. To further purify TFENs, their suspension was transferred to a discontinuous sucrose gradient (8%, 15%, 30%, 45%, and 60%, w/v), and ultracentrifuged at 150,000×g for 2 h. Finally, TFENs in the 15%/30% layer were harvested and washed 3 times. The amounts of TFENs were quantified on the basis of their protein amounts, which were determined using a BCA protein assay kit. The resultant TFENs were stored at −80 °C.

M-vesicles were derived in accordance with previously described protocols with some modification³² (link). RAW 264.7 cells at full confluence were isolated from the culture dish and washed with PBS three times. Next, the cells were resuspended in ice-cold 0.1 × TM buffer (Sangon Biotech, Shanghai, China) containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The cell suspended solution was repeatedly grinded by a Dounce homogenizer for 20 passes to disrupt the cells, followed by centrifugation at 3200×g for 5 min (ST 16, Thermo Fisher Scientific). The pellet was removed and the supernatant was centrifuged at 20,000×g for 25 min (Thermo Fisher Scientific). The upper supernatant was collected again and centrifuged at 100,000×g for 60 min (CP100NX, Hitachi, Tokyo, Japan). The eventual pellet was collected, dispersed in water and extruded through 400 and 200 nm polycarbonate porous membranes by an Avanti mini extruder (LF-1, Avestin, Ottawa, Canada) to generate M-vesicles. M-vesicle material was stored at −80 °C for future study. To prepare MRaNPs, the M-vesicles were mixed with the as-prepared RaNPs at a membrane protein-to-PLGA weight ratio of 1:1. The mixture was sonicated for 3 min using a bath sonicator. The obtained MRaNPs were stored at 4 °C until use.