Exoquick exosome isolation reagent

Exosomes derived from SGC-7901 cells were labelled with PKH26 kit (PKH26 Red Fluorescent Cell Linker Kit, Sigma, USA). 50 μL ultracentrifugation exosomes suspended in PBS were added with 100 μL Solution C. 0.5 μL PKH26 was dissolved in another 100 μL solution C. The diluted exosomes were added to the diluted PKH26 rapidly, mixed and incubated for 5 min, and 250 μL sterile FBS was added to stop staining. The exosomes labelled with PKH26 were isolated using ExoQuick™ Exosome Isolation Reagent (SBI, USA). 200 μg exosomes labelled with PKH26 were added to SGC-7901/ADR cells. After 24 h, the SGC-7901/ADR cells were fixed by 4% paraformaldehyde, stained with DAPI, and washed with PBS for three times. Fluorescence signal of PKH26 was observed under a Carl Zeiss LSM710 laser scanning confocal microscope (Oberkochen, Germany).

Extracellular vesicles (EVs) were isolated from 200 µL of plasma sample using 60 µL of ExoQuick^® Exosome Isolation Reagent (SBI, System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s instructions. EV pellets were suspended in sterile PBS. For FACS analysis and uptake experiments, isolated EVs were stained with 5 uM CFSE dye (ThermoFisher Scientific, Waltham, MA, USA) for 30 min at 37 °C. For EV treatment of cells (uptake and functional experiments), EVs from each group were pooled and added in a volume of 3% of the cell culture medium volume. We chose to fix the volume, rather than the number, of each EV pool to recapitulate the different amounts of circulating EVs found in patients’ samples. Given the paucity of plasma, in terms of volume, in each patient, it was not possible to perform any further analyses or characterization of circulating extracellular vesicles.

Venous blood samples (5 mL) were collected from an antecubital vein into chilled BD vacutainer™ serum separation tubes (Vacutainer, Becton, Dickinson and Company, Franklin Lakes, New Jersey) at four points of time (preoperative (pre-OP), 24 h postoperative (post-OP), 7 d post-OP, and 3 mo post-OP). After 30 min clotting time, separation of serum was performed immediately by centrifuging at 1,700 x g for 15 minutes in a refrigerated centrifuge. Platelets were removed by centrifuging the serum samples at 3,000 x g at 4°C for 15 min. Circulating sEVs were precipitated from 250 μL platelet poor serum using the Exoquick™ exosome isolation reagent (SBI, Palo Alto, CA, USA) according to manufacturer's instruction and resuspended in 30 μL phosphate-buffered saline (PBS). Subsequently, samples were diluted 2.5∗10⁵-fold with ultrapure water and analyzed by nanoparticle tracking analysis (NTA, Zeta View, Particle Metrix, Meerbusch, Germany) as described previously [22 (link), 23 (link)]. In preparation for this study, we validated the optimum parameters for NTA, so that the analysis of all samples could be conducted with identical acquisition parameters (supplementary table S1, S1 Fig).