Exosome isolation kit

Exosomes were extracted using an exosome isolation kit (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s protocol. Briefly, after ADSCs reached 60–65% confluence, the culture medium was replaced with DMEM supplemented with 10% exosome-free FBS (Cell Max, Beijing, China), and the cells were cultured for another 48 h. The supernatants were collected and centrifuged at 2000×g for 30 min and then passed through a 0.22-μm filter to remove dead cells and cellular debris. The cell-free culture medium was collected and exosome extraction reagent was added to the medium at a ratio of 1:2. The culture medium/reagent mixture was mixed well and incubated at 4 °C overnight. After incubation, the samples were centrifuged at 10,000×g for 1 h at 4 °C, and the pelleted exosomes were resuspended with PBS. The protein content of the exosome suspension was determined using a BCA quantitation kit (Solarbio, Beijing, China).
The morphology, size, and marker (CD81 and CD9) expression of ADSC-Exos were analyzed by transmission electron microscopy (TEM, JEM-1400, JEOL Ltd., Tokyo, Japan), nanoparticle tracking analysis (NTA) using a NanoSight NS300 (Malvern Panalytical, Malvern, UK), and western blotting, respectively. ADSC-exosomes were used for experiments or stored at − 80 °C.

The BMMSCs were cultured in DMEM with exosome-free FBS (Hyclone, South Logan, UT, USA). The culture medium was collected, filtered using a 0.22-μM cell filter, and then concentrated using ultrafiltration (Millipore Corp., Bedford, MA, USA). Exosomes were obtained following the manufacturer’s protocol of exosome isolation kit (Invitrogen, Carlsbad, CA, USA). A total of 10 μL extracted exosomes were diluted with equal volume of PBS and then negatively stained using 3% sodium phosphotungstate solution for 1 min. After washing with distilled deionized water and drying at room temperature, the exosomes were observed and photographed under a transmission electron microscope (Hitachi, Tokyo, Japan).
Dynamic light scattering was performed to measure exosome particle diameter. Particle size distribution was analyzed using Zetasizer Nano ZS90 (Malvern Panalytical, UK). The diluted samples in PBS in the ratio of 1:20 were manually loaded into the sample chamber. Three videos (60 s) were recorded of each sample. Data was analyzed using DTS v5.10 software (Malvern Panalytical, UK).

In the present study, we used 10- to 12-week old C57BL/6 female mice. The animal study protocol was approved by the Institutional Animal Care and Use Committee (IACUC; approval number 18–086)) of the University of Tennessee Health Sciences Center (Memphis, TN, USA). All experiments were performed in accordance with relevant guidelines and regulations. Mice were divided into two groups, a control (n = 6) and a treatment group (n = 6). The control mice received Lieber-DeCarli control liquid diet and the treatment group mice received a single dose of ethanol (5 g kg⁻¹). The animals were sacrificed at the end of the study and blood was collected to isolate plasma. Further, we isolated exosomes from plasma using a validated exosome isolation kit (Invitrogen, Life Technologies, NY) and characterized as described in the preliminary study and previous reports^{28 (link)}.

All cell culture media (DMEM, RPMI 1640) and cell culture supplements were purchased from Gibco Inc. (Billings, MT, USA), and cell culture plasticware was obtained from ThermoFisher, unless otherwise mentioned. Millipore deionized water of 18.0 Ω/cm² was used for reagent dilution when required. MTT assay reagents (Cell Proliferation Kit I (MTT) Cat# 11465007001) were purchased from Roche. Exosome surface marker antibodies were obtained from Abcam (Cat# ab275018: Exosome Panel; CD9, CD63, CD81, TSG101, Hsp70, Calnexin). Annexin-V was purchased from BD Bioscience (Cat# 556419), and propidium iodide (PI) was obtained from Invitrogen, ThermoFisher Scientific (Waltham, MA, USA). The exosome isolation kit (total exosome isolation (cell culture), Cat# 4478359) was purchased from Invitrogen^TM by Thermo Fisher Scientific.

Serum total exosomes were isolated by exosome isolation kit (Invitrogen, Thermo Scientific, Vilnius Lithuania, cat# 4478360) following manufacturer's instructions. Briefly, 1 ml of serum sample was centrifuged (relative centrifugal force, 2,000 g) for 30 min. The supernatant was transferred into a new tube on ice. 200 μl of total exosome isolation reagent was added into the serum supernatant. The mixture was incubated at 4°C for 30 min. After incubation, the sample was centrifuged (relative centrifugal force, 10,000 g) for 10 min. The supernatant was discarded. The pellet was completely resuspended in 200 μl phosphate-buffered saline (PBS, Jiangsu KeyGen Biotech Co., Ltd, cat# KGB5001). The exosomes were stored at −80°C.