This study isolated EVs of plasma samples of 111 PDAC patients, CP patients and normal controls. 3 mL plasma sample was collected from each subject. We performed ultracentrifugation (UC) method for EVs isolation. Firstly, we centrifugated plasma samples at 3000 × g for 15min after thawing at 37°C in order to remove cell debris. Then, we diluted supernatant of each sample by 7-fold volume of phosphate-buffered saline (PBS). Next we centrifuged the mix again at 13,000 × g for 30min and a 0.22μm filter was applied in order to filter large vesicles. Then, a P50AT2-986 rotor (CP100NX; Hitachi, Brea, CA, USA) was used at 150,000 × g, 4°C for 4h to pellet EVs. Pellet was resuspended in PBS and centrifuged again at 150,000 × g 4°C for 2h. Finally, the EVs enriched fraction pellet was re-suspended in 100µL PBS.
P50at2 986 rotor cp100nx
Manufactured by Hitachi
Sourced in United States
The P50AT2-986 rotor is a laboratory equipment component designed for use with the CP100NX centrifuge. It is a high-speed rotor capable of generating the necessary centrifugal force for various sample separation and processing applications in a research or clinical laboratory setting.
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3 protocols using p50at2 986 rotor cp100nx
1
Isolation of Extracellular Vesicles from Plasma
2
Optimized Plasma sEV Isolation by Ultracentrifugation
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A total of 3mL plasma was collected from each patient, and each plasma sample was divided into two aliquots (2mL for sEV isolation, 1mL for direct RNA isolation). The ultracentrifugation (UC) method was optimized according to the method previously described [31 ,32 ]. After thawing at 37°C, plasma samples were centrifugated at 3,000 × g for 15min to remove cell debris. Then, the supernatant was diluted by seven-fold volume of PBS, centrifuged at 13,000 × g for 30min, and processed through a 0.22μm filter to remove large particles. The supernatant was ultracentrifuged using a P50AT2-986 -rotor (CP100NX; Hitachi, Brea, CA, USA) at 150,000 × g, 4°C for 4h to pellet the sEVs. The pellet was resuspended in phosphate-buffered saline (PBS) and centrifuged again at 150,000 × g 4°C for 2h. After PBS washing, the sEVs enriched fraction pellet was re-suspended in 100µL PBS. Full description of methodologies was also submitted to EV-TRACK [33 ] (ID: EV190033), summarized in an EV-RNA specific checklist [34 ] (Supplementary file 1), and checked by a MISEV2018 checklist [34 ] (Supplementary file 2).
3
Plasma-derived small extracellular vesicle isolation
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A total of 4 mL of plasma was collected from each participant, and sEVs were extracted by ultracentrifugation. After being thawed at 37°C, the plasma samples were centrifuged at 3000× g for 15 min, the supernatants were diluted with seven times the volume of PBS. After centrifugation at 13000× g for 30 min. The supernatants were filtered into the overspeed centrifugal tube using the P50AT2‐986 rotor (CP100NX; Hitachi, Brea, CA, USA), and were then centrifuged at high speed at 150,000× g at 4°C for 4 h. The precipitates were resuspended in PBS and centrifuged again at 150,000× g 4°C for 2 h. The sEV‐enriched precipitates were then resuspended in 200‐μL PBS.
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