Extracellular Vesicle Isolation Protocol

EVs were isolated with a differential centrifugation method as previously described [24 (link)] with slight modifications. Briefly, ascitic fluids and peritoneal lavages were centrifuged at 300× g for 10 min, followed by centrifugation at 2500× g for 20 min at the moment that the sample was collected, and frozen at −80 °C. Then, samples were centrifuged at 10,000 g for 30 min (Thermo Scientific Heraeus MultifugeX3R Centrifuge (FiberLite rotor F15-8x-50c)). The supernatant was filtered through 0.22 µm filters (Merck Millipore), and the obtained sample was transferred to ultracentrifuge tubes (Beckman Coulter), which were filled with phosphate-buffered saline (PBS), to perform two consecutive ultracentrifugation steps at 100,000 g for 2 hours each on a Thermo Scientific Sorvall WX UltraSeries Centrifuge with an AH-629 rotor. The pellet containing the EVs was resuspended in 50 µL of PBS. From those, 5 µL were isolated for nanoparticle tracking analysis (NTA) and quantification, and the rest was frozen at −80 °C with 500 µL of Qiazol for RNA extraction.