Hieff quick exosome isolation kit

Based on exosome purification protocols, cells were cultured in Exo-Clear cell growth medium (SBI, USA) for 36 h before collection. The supernatant was filtered using a 0.22 μm Millipore Express PES Membrane (Millex, USA) to remove dead cells. The filtrate was then centrifuged at 10,000 × g for 30 min to eliminate cell debris (Optima XPN-100 Ultracentrifuge, Beckman, USA). After ultracentrifugation at 100,000 × g for 70 min, the pellet containing exosomes was washed in PBS and then centrifuged at 100,000 × g for 70 min to remove contaminant proteins. Exosomes in the co-culture system were enriched using the Hieff Quick exosome isolation kit (for Cell Culture Media) (Yeasen, China) according to the instructions.

Small EVs release inhibitor GW4869 was dissolved in DMSO at 5 mg/mL and then diluted to 0.3 mg/mL in PBS. Each mouse received 100 μl local injection solution (30 mg/mouse) in both hind leg muscles. Control mice were injected with a 6% DMSO solution in PBS. After 6 h of injection, muscle tissues were cultured in 6 well plate for 12 h. Cell culture medium was collected into a 15 mL centrifuge tube, 3000 × g, centrifuged for 10 min. Carefully collect the supernatant and transfer it to a new centrifuge tube and was thoroughly mixed with exosome extraction reagent (Hieff® Quick exosome isolation kit, 41201ES25, Yeason). The centrifuge tube containing the mixture was taken out at 4 °C and centrifuged at 10000 × g for 60 min. The supernatant was discarded and precipitation was collected and prepared for immunoblot analysis.

HPVECs were plated in a 10 cm dish at a density of 1 × 10⁶ cells per dish with DMEM. The culture medium was discarded after 72 h, the cells were washed 3 times in serum-free medium, and 10 ml serum-free medium was added to each dish. After 48 h, HPVECs were centrifuged twice at 2000 rpm per minute, and the HIEFF™ Quick exosome isolation kit (41201ES50, Yeasen, Shanghai, China) was used to extract exosomes. Then, the cell supernatant and isolated exosomes (2:1) were added into the centrifuge tube for incubation overnight at 4 °C. On the following day, the mixture was centrifuged at 10,000 rpm per minute at 4 °C for 1–2 h. The supernatant was removed, whereas the precipitate (exosomes) was collected. Based on the volume ratio of the initial medium and the resuspension (10:1), the precipitate was resuspended in phosphate-buffered saline (PBS). Then, 30 μL of the resuspension (exosomes) was placed in an Eppendorf tube and mixed with the radio-Immunoprecipitation Assay (RIPA) lysis buffer of equal volume and maintained on ice. Microwave methods were employed to lyse the mixture twice, and the bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Jiangsu, China) was used to determine the protein concentration in exosomes.

The FLS exosomes were isolated using Hieff^® Quick exosome isolation kit (Yeasen, China) according to the kit’s protocol. The exosomes were quantified by a bicinchoninic Acid (BCA) kit (Sangon, China). Nanoparticle tracking analysis (NTA) was carried out by the Nanosight NS300 (Malvern, UK). The marker proteins of exosomes were analyzed by Western blot, and the medium without culturing with FLS served as negative control (NC). The miR-19b-3p expression in the exosomes of OA patients-FLS and Controls-FLS was assessed by RT-qPCR.

Blood was collected from neonatal mice (postnatal day 7; P7) and centrifuged at 3000× g for 10 min at 4 °C to isolate plasma, then 10,000× g for 20 min at 4 °C to remove residual blood cells. A Hieff ^® Quick exosome isolation kit (for serum/plasma) was used for the isolation of plasma exosomes based on the manufacturer’s instructions (Yeasen Biotech, Shanghai, China). Each exosome sample was purified from 200 μL plasma (collected from 5 neonatal mice and EDTA-Na2 was used as an anticoagulant at the working concentration of 1.5 mg/mL).The isolated exosomes used for subsequent experiments and proteomic sequencing were further purified by 100 kD ultrafiltration tubes (Millipore, Boston, MA, USA) to centrifuge at 14,000× g for 20 min at 4 °C, then sterilized by 0.22 μM filters and diluted in sterile PBS to the final concentration of 400 μg/mL after being quantified by a BCA protein quantitative kit (TransGen Biotech, Beijing, China).

ESCC cells were placed in Roswell Park Memorial Institute (RPMI) 1640 medium containing EVs-free 10% fetal bovine serum (FBS) and cultured for 3 d. Then, the cell supernatant was collected and centrifuged to remove cell debris. The EVs were extracted using Hieff™ Quick exosome isolation kit (41201ES50, YEASEN, Shanghai, China). The supernatant and exosome separation reagent were added into the Eppendorf (EP) tube with a proportion of 2:1 overnight, and then centrifuged at 100,00 g for 1–2 h and the precipitate was resuspended in phosphate buffered saline (PBS). The EV suspension (30 μl) together with an equal volume of radioimmunoprecipitation assay (RIPA) lysis buffer was continuously dissolved in microwave (2 times, 10 s/time). Then, the EVs were purified at 4°C at 12,000 × g for 2 min, and the supernatant was retained and stored at −80°C. Protein quantification of EV was performed by bicinchoninic acid (BCA) kit (Beyotime, Nantong, China).
EVs identification: Transmission electron microscope (TEM; JEM-1010, JEOL, Tokyo, Japan) was adopted to observe EV morphology, tunable resistive pulse sensing (TRPS) to examine concentration and diameter of EVs, and Western blot analysis to test EV markers (CD81 and CD9) and non-marker protein (Calnexin and GM130) (Li Z et al., 2019 (link)).