Exoquick tctm

Culture medium was replaced with fresh Essential 8 medium daily in human iPSCs culture. The spent medium was collected daily from day 2 to the end of culture and filtered through 0.45 μm syringe filter. Exosomes were isolated from the conditioned medium, using ExoQuick-TC^TM (System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s instructions. Briefly, the conditioned medium was incubated with the exosome precipitation solution at 4 °C for 12 h and subsequently centrifuged at 1500× g for 30 min. After discarding the supernatant, the pellet was resuspended in phosphate buffered saline (PBS) and the collected iPSC-Exo was stored at −80 °C until use.

Culture medium was collected and exosomes were isolated using ExoQuick-TC^TM (System Biosciences, Palo Alto, CA, USA, EXOTC50A-1) according to the manufacturer’s instructions. Briefly, the medium was centrifuged at 3000 g for 15 min and the supernatant was incubated with the exosome precipitation solution at 4 °C overnight. After subsequent centrifugation at 1500 g for 30 min, the pellet was resuspended in phosphate buffered saline (PBS). Size distribution of the isolated exosomes was analyzed by nanoparticle tracking analysis (NTA) using NanoSight NS300 (Malvern Panalytical, Malvern, UK). BCA protein assay kit (Thermo Fisher Scientific, 23227, Waltham, MA, USA) was used for quantification of exosomes.

GC cells were cultured in RPMI-1640 supplemented with 10% exosome-free FBS for 48 h. The conditioned medium was collected and exosomes were isolated by using ExoQuick-TC^TM (System Biosciences, USA) according to the manufacturer’s instructions. The final exosome pellets were resuspended in PBS and stored at −80 °C for further experimental needs. According to the published literatures, the concentration of exosomes was determined by using bicinchoninic acid assay (BCA, Thermo Scientific, USA). Purified exosomes were labeled with the PKH67 green fluorescent linker Mini Kit (Sigma, USA) according to the manufacturer’s instructions.

Once CPC-N and CPC-P were 80–90% confluent, complete medium was replaced with a serum-free medium, which was collected after 48 h and processed to isolate EVs. The isolation was carried out by ExoQuick-TCTM (System Biosciences, Mountain View, CA, United States), following the protocol supplied with the reagent without introducing any modification. Briefly, culture medium from each cell population was pooled and centrifuged at 3000 g for 15 min at 4°C to eliminate cells and cellular debris. The supernatant was transferred to sterile tubes adding 1 ml of ExoQuick-TCTM Exosome Isolation Reagent to each 5 ml of cell culture medium. Tubes were mildly agitated until the separation between the two phases was no longer visible, then incubated at 4°C overnight. The next day tubes with ExoQuick-TC/culture medium mixture were centrifuged at 1500 g for 30 min at 4°C to allow the precipitation of the white/beige pellet. Then the supernatant was discarded, and the residual ExoQuick-TC solution was spun down by centrifugation at 1500 g for 5 min at 4°C. The EVs were quantified using Bradford Assay (Gupta et al., 2021 (link)), then pooled and stored at −80°C until their use for specific assays (Belviso et al., 2016 ).

Exosomes were collected by density gradient ultracentrifugation according to previously published protocol^{46 (link)}. In brief, the polarized macrophages were incubated for 48 h in complete PRMI1640 medium with 10% FBS that was previously depleted of contaminating vesicles by overnight centrifugation at 100,000×g. The conditioned medium was collected and centrifuged at 800×g for 10 min, followed by a centrifugation step of 3000×g for 30 min to remove cell debris. Next, the supernatant was filtered using a 0.22-µm filter (Millipore). The exosomes were pelleted by ultracentrifugation at 100,000×g for 90 min, washed in PBS, pelleted again and re-suspended in PBS. Measurement of the exosome particle number was performed using a CD63 ExoELISA Complete Kit (System Biosciences, USA) following the manufacturers’ instructions. For Nano-LC–MS/MS analysis, exosome pellets were also isolated using ExoQuick-TC TM (System Bioscience) according to the manufacturer’s protocol. For exosome uptake experiments, exosome preparations were labeled with PKH67 Fluorescent Cell Linker Kits (Sigma-Aldrich) according to the manufacturer’s instructions, followed by washing through Exosome Spin Columns (MW3000) (Invitrogen, USA) to remove excess dye. Next, exosomes were incubated with GC cells, which were examined under a confocal microscope or analyzed using flow cytometry at the indicated time points.