Ab92573

U87MG cells were cultured in exosome-depleted DMEM (A2720801, GIBCO BRL, Green Island, NY) containing 10% FBS under normoxia (21% O₂) or hypoxia (1% O₂). After 48–72 h of incubation, the culture medium (30 mL) was collected and centrifuged at 300 × g for 10 min, 2000 × g for 15 min and 12,000 × g for 30 min to remove the floating cells and cell debris, followed by filtering through 0.22-μm filter. The supernatant was subjected to centrifugation at 100,000 × g for 2 h. After PBS wash, another round of ultracentrifugation was performed at 100,000 × g for 2 h. The precipitate was re-suspended in 100 μL PBS and stored at −80 °C for further or immediate use.
Observation of morphological characteristics of EVs was performed under a TEM (JEM-1010; JEOL, Tokyo, Japan). Then, the size was measured using Zetasizer Nano ZS90 instrument (Malvern, UK) with five videos of typically 60 s’ duration taken, and the data was analyzed by Zetasizer software v7.11, Malvern Instruments. The EV surface marker proteins rabbit anti-CD63 (ab134045, 1: 1000, Abcam, Cambridge, UK), rabbit anti-TSG101 (ab125011, 1: 1000, Abcam) and rabbit anti-Calnexin (ab92573, 1: 20000, Abcam) were determined using Western blot analysis.

RIPA lysate, including a protease inhibitor, was applied to extract the total proteins from treated HepG2 cells or exosomes. After centrifugation at 4°C, the supernatant was taken. After the protein concentration was determined by the BCA method, the total protein (40 μg) in each group was separated by 12% SDS-PAGE electrophoresis and transferred into PVDF membranes. Then the target protein in membrane was sealed with 5% skim milk, and addressed with primary antibodies at 4°C overnight and the corresponding secondary antibodies for 2 h. After the reaction of the ECL reagent (Bio-Rad) for 5 min, the results were obtained by developing imaging in a dark room. And the primary antibodies contained CD63 (1 : 5,000, ab134045, Abcam), CD9 (1 : 2000, ab92726, Abcam), TSG101 (1 : 5,000, ab125011, Abcam), Calnexin-CNX (1 : 20,000, ab92573, Abcam), cleaved caspase-3 (1 : 500, ab32042, Abcam), Cyclin D1 (1 : 10000, ab134175, Abcam), Alix (1 : 1000, ab275377, Abcam), Hrs (1 : 1000, ab155539, Abcam), Rab27A (1 : 3000, ab55667, Abcam), Vps4A (1 : 3000, ab229806, Abcam), BAG5 (1 : 2000, ab97660, Abcam), TRPV6 (1 : 400, 39563, SAB), and GAPDH (1 : 10000, ab8245, Abcam).

Sections were incubated overnight at 4°C with primary antibodies; mouse anti-Calnexin (1/1000; EPR3632; ab92573; Abcam), mouse anti-CD31/PECAM (1/500; JC/70A; ab9498; Abcam), rabbit anti-FAM20A (1/250; OACD03385; Aviva), and rabbit anti-Fam20C (1/250; OAAB01003; Aviva). Secondary antibodies used were Cy3-conjugated donkey anti-mouse (Thermo Fisher Scientific; 1:500) and Alexa 488-conjugated donkey anti-rabbit (Jackson Immunoresearch Laboratories, West Grove, PA; 1:500). Helix pomatia agglutinin Alexa 647 (1/1000; Thermo Fisher Scientific) staining was achieved by overnight incubation. Nuclear staining was achieved by 20 min incubation at room temperature in Hoechst 33342 (Thermo Fisher Scientific). No cellular autofluorescence and no nonspecific labeling were detected in these conditions. Images were collected by confocal microscopy (Zeiss LSM8) and processed using ZEN (Zeiss) and ImageJ softwares.

Alpha-MEM (Gibco, Green Island) containing 20% FBS (Gibco) was centrifuged for 18 h at 200,000 g to deplete exosomes. BMSCs were cultured in exosome-free medium containing 10% FBS, and BMSCs at passages 4-6 were selected for exosome collection. A total of 2 × 10⁶ MSCs were cultured in 100 mm culture dishes under normal/hypoxic conditions for 72 h, and 10 ml of the supernatant was taken. Exosomes were then isolated from the supernatant after centrifugation^{16 (link)}. Then, exosomes were resuspended in PBS.
For identification of exosomes, samples were evaluated under a JEM-2100 transmission electron microscope (TEM; JEOL, Tokyo, Japan). Images were acquired using the PARTICLEMEIRIX system. Nanoparticle tracking analysis (NTA) was performed using the NanoSight NS300 system (Malvern Instruments, Malvern, UK). The Brownian motion of exosomes in PBS was recorded and tracked, and the size distribution was analyzed using the Stokes-Einstein equation. The exosome characteristics were identified by detecting the expression of the exosome-specific surface markers with rabbit anti-CD63 (1:2000, ab216130, Abcam, UK), rabbit anti-TSG101 (1:10,000, ab125011, Abcam), rabbit anti-CD81 (1:10,000, ab109201, Abcam) and rabbit anti-Calnexin (1:100,000, ab92573, Abcam) antibodies by Western blot analysis.

EV pellet obtained by ultracentrifugation was fixed in 2% glutaraldehyde overnight at 4℃, washed with PBS, fixed with 1% OsO4 for 1 h, dehydrated in ethanol and embedded using epoxy resin. The embedded material was sectioned using a microtome and saturated with sodium periodate and 0.1N hydrochloric acid. After 10 minutes, the size and morphological characteristics of EV were examined with the use of a TEM (JEM‐1010, JEOL, Tokyo, Japan). The EV suspension was mixed with an equal volume of 4% paraformaldehyde and deposited on a Formvar carbon‐coated EM grid. Images were acquired using a TEM (Hitachi, Tokyo, Japan). Western blot analysis was applied to identify the EV surface marker proteins of rabbit anti‐CD63 (ab134045, 1:1000, Abcam, Cambridge, UK), rabbit anti‐CD81 (ab109201, 1:5000, Abcam), and rabbit anti‐Calnexin (ab92573, 1:20 000, Abcam).