miRCURY Exosome Serum/Plasma Kit

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	Follow manufacturer’s instructions

Protocol tips
Follow manufacturer’s instructions

Publication protocol

Exosome/sEV Isolation (Figure 1): SP (200 µL) was first passed through a 0.22 μm filter to enrich for sEVs [6]. 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 [22]. 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 [17]. 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.
-miRCURY ® Exosome Serum/Plasma kit (miRCURY S/P)
Both protocols (for serum and for plasma) were independently followed. SP filtered fluid was supplemented with PBS till 0.5 mL. Then the corresponding amounts of reagents were added according to the manufacturer’s instructions. Samples were incubated at 4 °C for 1 h and centrifuged at 1500× g 30 min (protocol serum) or 500× g 5 min (protocol plasma) at room temperature. Pellet fraction was resuspended in 270 µL of Resuspension buffer and frozen at −80 °C.


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