Exosome/sEV Isolation (Figure 1): SP (200 µL) was first passed through a 0.22 μm filter to enrich for sEVs . The resulting filtrate was processed by either ultracentrifugation, three commercial exosome-EV isolation reagents [miRCURY® Exosome Cell/Urine/CSF kit (Qiagen, NV; Germany), miRCURY® Exosome Serum/Plasma kit (Qiagen, NV; Germany), ExoQuick® ULTRA EV Isolation kit for Serum and Plasma (System Biosciences, Palo Alto, CA, USA)] or a non-commercial exosome-EV isolation reagent [ExoGAG (NasasBiotech, Santiago de Compostela, Spain)] previously tested for isolating plasma EV from endometrial cancer patients . They all were precipitation-based methods, although ExoQuick® ULTRA EV Isolation kit for Serum and Plasma can also include a final solid extraction phase (precipitation + column-based method). Different experimental conditions were tested for each isolation reagent (Figure 1). Ultracentrifugation (UC).
The filtered SP fluid plus 9 mL of PBS was ultra-centrifuged at 100,000× g in a SW40 rotor for 2 h at 4 °C to sediment the sEVs, which mainly contain exosomes, as described elsewhere . The pellet was resuspended in 100 µL PBS and frozen at −80 °C.
-miRCURY ® Exosome Cell/Urine/CSF kit (miRCURY Cell/Urine/CSF)
The exosomal fraction from SP filtered fluid was isolated according to the manufacturer’s recommendations. Briefly, filtered fluid was supplemented with PBS till 1mL and subsequently, 400 µL of Precipitation buffer B was added. Samples were incubated at 4 °C for 1 h. The mixture was centrifuged at either 10,000× g (manufacturer’s protocol) or 1500× g for 30 min at room temperature. Pellet fraction was resuspended in 100 µL of Resuspension buffer and frozen at −80 °C. Full paper
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