The prostatic fluid sample from each individual was collected in a 1.5-mL sterile Eppendorf tube as described in the literature (25 (link)), followed by ultracentrifugation at 3,000 g and 4 °C for 10 min to collect the supernatant and a dilution with an equal volume of sterile phosphate-buffered saline (PBS, pH 7.4). The diluted supernatant was further centrifuged at 10,000 g and 4 °C for 30 min, followed by collecting and filtering the supernatant with a 0.22-mm filter (Millipore, Burlington, MA). The filtered supernatant was further centrifuged at 150,000 g and 4 °C for 2 h in a CP70ME ultracentrifuge (Hitachi, Japan). To further purify the exosomes, the pellet was washed with a large amount of sterile PBS, centrifuged at 150,000 g and 4 °C for 1 h to collect the precipitate, and resuspended in 100 µL sterile PBS for subsequent experiments.
Cp70me ultracentrifuge
Manufactured by Hitachi
Sourced in Japan
The CP70ME ultracentrifuge is a high-speed centrifuge designed for laboratory applications. It is capable of generating centrifugal forces up to 500,000 x g, enabling the separation and purification of a wide range of biological samples, such as proteins, nucleic acids, and cellular organelles. The CP70ME features a compact design and advanced safety mechanisms to ensure reliable and efficient operation in the laboratory environment.
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Lab products found in correlation
2 protocols using cp70me ultracentrifuge
1
Exosome Isolation from Prostatic Fluid
2
Isolation and Characterization of Small Extracellular Vesicles
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Cell culture medium was collected and centrifuged at 3,000×g for 15 min to remove cellular debris, and the supernatants were transferred to an appropriate vessel for the CP70ME ultracentrifuge (Hitachi, Ltd. Japan) according to the method described by Théry C [46 ]. Successive centrifugations at increasing speeds were performed to throw the pellet away (300×g for 10 min-2000×g for10 min–10,000×g for 30 min). In the last step, the supernatant was collected and centrifuged one more time at 100,000×g for 70 min and only the pellets were kept. The pellet was washed in a large volume of PBS three times to eliminate contaminating proteins and centrifuged at the same high speed. The final sEVs pellets were resuspended in 100 mL PBS and filtered through a syringe filter (0.2 mm, Sartorius). The morphology of sEVs was observed by Transmission Electronic Microscopy (FEI co., CZ). The number and size of sEVs particles were measured by nanoparticle-tracking analysis with Nanosight NS300 (Malvern Instruments).
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