To purify the exosomes, cells were cultured following the procedure described in previous studies [65 (link), 66 (link)]. After filtering with a 0.22-µm filter, the culture supernatants were centrifuged at 3,000 g at 4 °C for 30 min (Thermo Fisher) first, and then further centrifuged at 10,000 g at 4 °C for 1 h (Beckman Coulter, Optima XPN-100) to remove cell debris, dead cells and large vesicles. Subsequently, after centrifugation at 100,000 g at 4 °C for 2 h (Beckman Coulter, Optima XPN-100), the exosomes were pelleted and stored at 80 °C for further use.
Optima xpn 100
Manufactured by Beckman Coulter
Sourced in United States
The Optima XPN-100 is a high-performance centrifuge designed for a wide range of laboratory applications. It features a maximum speed of 100,000 rpm and a maximum RCF of 802,000 x g, making it capable of separating small particles and macromolecules with high efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Allegra x 14r, by Beckman Coulter (8 mentions)
J2 hs, by Beckman Coulter (6 mentions)
70ti rotor, by Beckman Coulter (4 mentions)
Zetaview pmx 110, by Particle Metrix (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Nanosight ns300, by Malvern Panalytical (3 mentions)
Sw41ti, by Beckman Coulter (3 mentions)
0.22 μm filter, by Merck Group (3 mentions)
Stericup 0.45 μm and 0 22 μm pore size polyethersulfone pes top filter, by Merck Group (2 mentions)
Sw28 rotor, by Beckman Coulter (2 mentions)
Dnase, by Applied Biological Materials (2 mentions)
H 7650, by Hitachi (2 mentions)
Optima max xp, by Beckman Coulter (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Shnr1d1, by Addgene (2 mentions)
Pspax2, by Addgene (2 mentions)
Pmd2 g, by Addgene (2 mentions)
Ab236630, by Abcam (2 mentions)
Zetaview, by Particle Metrix (2 mentions)
Ultracentrifuge 25 89 mm polypropylene tubes, by Beckman Coulter (2 mentions)
129 protocols using optima xpn 100
1
Exosome Purification from Cell Cultures
2
Isolation of Bovine Milk Exosomes
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The isolation of exosomes was performed from raw bovine milk that was collected from the dairy farm located inside the BHU campus, followed by a sequential centrifugation process as described previously [34 (link)], with slight modifications. Briefly, the raw milk was subjected to centrifugation at 13,000× g for 30 min using a Remi C-24 BL centrifuge (R-244 rotor), after which the supernatant obtained was ultracentrifuged for 1 h at 90,000× g (type 70 Ti rotor, Optima XPN-100, Beckman, Brea, CA, USA) to remove the microvesicles. The resultant supernatant was further separated and ultra-centrifuged for 2 h at 180,000× g (type 70 Ti rotor, Optima XPN-100, Beckman, USA), followed by the collection of pellets and their redispersion in PBS by using a hand homogenizer. The exosomal solution was then passed through 0.22 syringe filters to obtain uniform exosomes and until further use, they were stored at −80 °C. The protein content was estimated via the Bradford method.
3
Isolation of Outer Membrane Vesicles from E. coli
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E. coli was grown in LB broth (500 mL, 37 °C, 180 rpm) until reaching an OD600 nm value of 1. Bacterial cells were removed through centrifugation at 3000× g for 20 min at 4 °C. Residual bacteria and cellular debris were eliminated by sequential 0.45 and 0.22 µm filtration (VWR Scientific, Bridgeport, NJ, USA). OMVs were collected by centrifugation at 100,000× g for 2 h at 4 °C (Optima XPN-100 Beckman Coulter and rotor 70Ti, Palo Alto, CA, USA). The vesicles were washed in sterile PBS1X through ultracentrifugation and re-suspended in 150 μL of PBS1X. LB agar plates were used to check the sterility of the OMVs. The purified vesicles were stored at −20 °C until use [28 (link)].
4
Isolation of Outer Membrane Vesicles from Klebsiella pneumoniae
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OMVs were isolated from liquid cultures of K. pneumoniae-pGR and K. pneumoniae-PRM-GFP as previously described with modifications [46 (link)]. First, 10 mL of overnight (O/N) bacterial culture was inoculated in 600 mL of LB containing 100 µg mL−1 ampicillin. The bacterial inoculum was cultured at 37 °C under orbital shaking (180 rpm) for 8–12 h, up to the OD600 nm value of 1. The cultures were centrifuged at 4000× g at 4 °C for 20 min, to remove bacterial cells. Supernatants were decanted and filtered using vacuum Stericup™ 0.45 μm and 0.22 μm pore size polyethersulfone (PES) top filter (Millipore, Burlington, MA, USA), to deflect remaining bacteria and cell debris. Vesicles were collected from cell-free supernatant culture by ultracentrifugation at 100,000× g (centrifuge Optima XPN-100 Beckman Coulter and rotor SW28) at 4 °C for 1.5 h. Pellets were washed in sterile phosphate buffered saline 1X (PBS) by ultracentrifugation (100,000× g at 4 °C for 1.5 h). Vesicular pellets were suspended in 250 μL of PBS 1X and OMVs sterility was checked by inoculating 10 μL of vesicles on LB-agar plates. OMV samples were treated with DNase (Applied Biological Materials (abm), Richmond, BC, Canada) according to the manufacturer’s protocol and stored at −20 °C until use.
5
Isolation and Purification of Outer Membrane Vesicles from Klebsiella pneumoniae
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OMVs were isolated from liquid cultures of K. pneumoniae-pGR and K. pneumoniae-PRM-GFP as previously described with modifications [42] (link). Ten milliliters of overnight (O/N) bacterial culture were inoculated in 600 mL of LB containing 100 µg mL -1 ampicillin. The bacterial inoculum was cultured at 37 °C under orbital shaking (180 rpm) for 8-12 hours, up to the OD 600 nm value of 1. The cultures were centrifuged at 4000 × g at 4 °C for 20 min, to remove bacterial cells. Supernatants were decanted and filtered using vacuum Stericup™ 0.45 μm and 0.22 μm pore size polyethersulfone (PES) top filter (Millipore, Massachusetts, USA), to deflect remaining bacteria and cell debris. Vesicles were collected from cell-free supernatant culture by ultracentrifugation at 100000 × g (centrifuge Optima XPN-100 Beckman Coulter and rotor SW28) at 4 °C for 1.5 hours. Pellets were washed in sterile phosphate buffered saline 1X (PBS) by ultracentrifugation (100.000 × g at 4 °C for 1.5 hours). Vesicular pellets were suspended in 250 μL of PBS 1X and OMVs sterility was checked by inoculating 10 μL of vesicles on LB agar plates. OMV samples were treated with DNase (Applied Biological Materials -abm, British Columbia, Canada) according to the manufacturer's protocol and stored at -20 °C until use.
6
Exosome Isolation and Characterization
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The cell culture solution was centrifuged at 120,000 g for 90 minutes using an OptimaTM XPN-100 ultracentrifuge (Beckman Coulter) to obtain exosomes derived from the HCT8 and HCT8FU cells. The exosomes were resuspended in PBS and placed on a copper grid, fixed with 1% glutaraldehyde, and treated with uranyl acetate. The images of the exosomal samples were observed under a transmission electron microscope (FEI Tecnai G2 Spirit TEM, USA) at 80 kV. ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) was used to measure the size distribution and concentration of the exosomes.
7
Exosome Isolation from Lung Cancer Cells
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Exosomes were obtained from conditioned medium culturing A549/SEN, A549/CR and hypoxic cisplatin-resistant A549 cells (hA549/CR). In short, supernatants were filtered using a 0.22 μm syringe filter and isolated by centrifugation at 120,000 g for 90 min (4 °C). Next, exosome pellets were collected by centrifugation at 120,000 g (4 °C) for 90 min again, followed by a wash with PBS. In the final step, exosome pellets were resuspended in PBS and stored at -80 °C. The process of exosome purification was completed using OptimaTM XPN-100 (Beckman Coulter) and the concentration of exosomes was measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific).
8
Isolation and Characterization of Small Extracellular Vesicles from Adipose-Derived Stem Cells
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When the confluency of ADSCs reached 80–90%, the medium was changed to DMEM/F12 medium containing 10% FBS without sEVs and continued to culture for 72 h. The supernatant was then centrifuged at a low speed, and large vesicles were removed by filtration using a 0.22 μm pore size filter. The culture medium was subjected to ultracentrifugation at 120,000 × g for 90 min (OptimaTM XPN-100, Beckman Coulter, USA). The resulting sEV precipitates were resuspended in pre-cooled PBS, followed by another round of centrifugation. Subsequently, the sEV precipitates were resuspended in an appropriate volume of phosphate buffered saline (PBS) and characterized using nanoparticle tracking analysis (NTA) with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany). The sEV solution was placed on copper grids (Zhongjingkeyi Technology, Beijing, China), stained with 50 μL of uranium acetate, and visualized using transmission electron microscopy (FEI Tecnai G2 Spirit TEM, USA) for sEV visualization.
9
Isolation and Characterization of Small Extracellular Vesicles
10
Isolation of Tumor-Derived Small Extracellular Vesicles
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