The isolation and characterization of extracellular vesicles were conducted according to the guidance of Minimal information for studies of extracellular vesicles 2018 (MISEV2018)56 (link). Extracellular vesicle isolation EPC-EVs were isolated with Umibio exosome isolation kits (Umibio, cat. no: UR52121, Shanghai, China) according to the manufacturer’s guidelines. Briefly, EPCs were cultured in medium with extracellular vesicle-free FBS. Fifty millilitres of culture media was harvested and centrifuged at 3000 × g and 4 °C for 10 minutes and at 10,000 × g and 4 °C to remove cells and debris. The supernatant was transferred to a new container without disturbing the pellet. Then, 25 ml of total extracellular vesicle isolation reagent was added, and the reaction mixture was mixed by rotation. The mixture was incubated at 4 °C for two hours and was then centrifuged at 10,000 g for 1 hour at 4 °C. The supernatant was discarded, and the pellet was resuspended with 1000 µl of 1x PBS. The isolated extracellular vesicles were stored at −80 °C for long-term storage.
Exosome isolation kit
Manufactured by Umibio
Sourced in China
The Exosome Isolation Kit is a laboratory equipment used for the extraction and purification of exosomes from biological samples, such as cell culture media or bodily fluids. The kit utilizes a proprietary method to selectively isolate exosomes, which are small extracellular vesicles produced by cells. The core function of the kit is to provide a reliable and efficient way to obtain purified exosome samples for further analysis and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Zetaview pmx 110, by Particle Metrix (4 mentions)
Exosome concentration solution, by Umibio (2 mentions)
Tsg101, by Abcam (1 mentions)
Optima xpn 100 ultracentrifuge, by Beckman Coulter (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
Ht7800, by Hitachi (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab236630, by Abcam (1 mentions)
H 7800, by Hitachi (1 mentions)
220 twin, by Particle Metrix (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
Exosome rna isolation kit, by Thermo Fisher Scientific (1 mentions)
0.2 m syringe filter, by Thermo Fisher Scientific (1 mentions)
Hibernate e solution, by Transnetyx (1 mentions)
Papain, by Worthington (1 mentions)
16 protocols using exosome isolation kit
1
Isolation and Characterization of Extracellular Vesicles
2
Exosome Isolation from Cell Culture
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Exosome isolation kits (Umibio, China) are for exosome isolation. Cells were cultured in a complementary medium with 10% exosome-depleted FBS and 1% penicillin–streptomycin. After 3 days of culture, the cells were collected and transferred to centrifuge tube. Cells were spun at 3000 g for 10 min. The supernatants were collected and treated with exosome concentration solution for 2 hrs at 2°C. Then, the precipitate was collected to isolate exosomes by ultracentrifugation at 1000×g for 60 min. The exosomes were harvested from resuspended precipitate at 12000×g for 2 min and purified by exosome purification filter. Exosomes were identified with transmission electron microscopy as described previously [34 (link)].
3
Serum Exosome Isolation and Characterization
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Serum samples were used for exosome isolation with exosome isolation kits (Umibio®, Cat. No: UR52136, China) according to the manufacturer's instructions. In brief, an initial spin was performed at 3000g 4 ℃ for 10 min and 10,000g 4℃ for 20 min for each sample (0.6 mL serum sample) to remove cellular debris and other impurities. The supernatant was diluted with pre-cooled 1× PBS (HyClone, SH30256.01, pH = 7.0), then 0.6 ml Blood PureExo Solution (BPS) was added. Mixtures were vortexed and incubated at 4℃ for up to two hours and then centrifuged at 10,000xg 4℃ for 60 min to precipitate exosome pellets. Pellets were resuspended with 1 ×PBS and purified with Exosome Purification Filter at 3000g 4℃for 10 min. The resuspension volume for exosome pellets was 6 μl for 0.6 ml starting volumes in this study. All exosomes were stored at −80℃ immediately after isolation until further analysis. Transmission electron microscopy (TEM) (JEOL, 1230) and nanoparticle tracking analysis (NTA) (Particle Metrix, ZetaView PMX 110) were performed to validate the exosome isolation and purification [13 (link), 14 (link)].
4
Exosome Isolation from Serum Samples
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Serum samples were used for exosome isolation with Umibio® exosome isolation kits (Umibio, Cat. No: UR52136, China) according to the manufacturer's instructions.In brief, an initial spin was performed at 3000×g 4 °C for 10 min and 10,000×g 4 °C for 20 min for each sample to remove cells and debris, then the corresponding amounts of reagents were added proportional to the starting sample volume, according to the manufacturer's instructions. Mixtures were vortexed and incubated at 4 °C for up to 2 h and then centrifuged at 10,000×g 4 °C for 60 min to precipitate exosome pellets. Pellets were resuspensed with 1 × PBS and purified with Exosome Purification Filter at 3000×g 4 °C for 10 min. The resuspension volume for exosome pellets was 200 μL for 20 mL starting volumes according to the manufacturer's instructions. All exosomes were stored at − 80 °C immediately after isolation until further analysis.
5
Exosome Isolation from BMMSCs
6
Plasma Exosome Isolation and Characterization
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Plasma samples were used for exosome isolation with Umibio® exosome isolation kits (Umibio, Shanghai, China). In brief, an initial spin was performed at 3000× g, 4 °C, for 10 min and 10,000× g, 4 °C, for 20 min for each sample, to remove cells and debris. The corresponding amounts of reagents were added proportionally to the starting sample volume, according to the manufacturer’s instructions. Mixtures were vortexed and incubated at 4 °C for up to 2 h and then centrifuged at 10,000× g, 4 °C, for 60 min to precipitate exosome pellets. Pellets were resuspended with 1 × PBS and purified with Exosome Purification Filter at 3000× g, 4 °C, for 10 min. All exosomes were stored at −80 °C immediately after isolation until further analysis. Exosome particle size and concentration were determined using nanoparticle tracking analysis (NTA) at VivaCell Biosceinces with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) (Table S1 ). Western blot was performed to detect exosome biomarkers including CD63 and TSG101 (Abcam, Cambridge, MA, USA). Transmission electron microscopy was performed to visualize exosomes, and the negative staining method was used [12 (link)] (Figure S2 ).
7
Exosome Isolation and Characterization
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According to the manufacturer's instructions, we used Umibio® exosome isolation kits (Umibio, Cat. No: UR52121, China). In short, each sample was centrifuged at 3000 g 4 ℃ for 10 minutes and then at 10000 g 4 ℃ for 20 minutes to remove cells and debris. According to the manufacturer's instructions, we added the corresponding amount of reagent proportional to the volume of the starting sample. The mixture was well mixed, incubated at 4 ℃ for 2 hours, then centrifuged at 10000 g at 4 ℃ for 60 minutes to precipitate exosomes. The precipitate was resuspended with 1 x PBS and puri ed by exosome puri cation lter at 3000 g 4 ℃ for 10 minutes. The initial volume of exosome particles was 5 ml and the resuspension volume was 200 μ L. All exosomes were stored at -80 ℃ immediately after extraction until further analysis. Zetaview PMX 110 (particle metrix, meerbusch, Germany) and transmission electron microscopy (TEM, jeol, jem-1230, TEM, Peabody, MA) were used to measure the exon size by nanoparticle tracking analysis (NTA).
8
Exosome Isolation from M2 Macrophages
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For exosome identification, the exosomes were separated by Exosome Isolation Kit (UR52121; Umibio; Shanghai, China) following with the manufacturer's instructions. Briefly, the DMEMs medium was replaced with exosome-depleted FBS (Gibco) when M2 macrophages reached 80 % confluence, and then 48 h later, the supernatant was collected. Subsequently, the supernatant was centrifuged at 3000×g, 4 °C for 10 min and then at 10,000×g, 4 °C for 20 min to eliminate cells and debris in the precipitate. The supernatant was incubated with Kit reagents in proportion at 4 °C for up to 2 h and centrifuged at 10,000×g, 4 °C for 60 min. The exosome in the precipitate was washed once with 1 × PBS. After centrifugation at 3000×g for 10 min at 4 °C, the exosome pellet was dissolved in 100 μl PBS and stored at −80 °C before use. For M2-exo used in functional assays, differential ultracentrifugation was used for M2-Exo isolation. Briefly, the M2 macrophages culture supernatant was centrifuged at 3000×g, 4 °C for 10 min and then at 10,000×g, 4 °C for 20 min to eliminate cells and debris in the precipitate. After filtering through a 0.22-μm filter, the supernatant was ultracentrifuged at 110,000×g for 2 h at 4 °C (Optima XPN-100 Ultracentrifuge, Beckman Coulter, USA) to acquire M2-Exo.
9
Exosome Isolation and Purification
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The collected conditioned media was centrifuged at 2000 g for 20 min at 4°C to remove dead cells and cell debris then transferred to Amicon® Ultra-15 centrifugal filter device (100 kD, Millipore) and concentrated by centrifugation at 5000 g for 30 min at 4°C. After filtration through a 0.22 μm filter, exosomes were isolated using Exosome Isolation Kit (Umibio) and resuspended in PBS. The extracted coarse exosomes were transferred to Exosome Purification Filter Column (Umibio) and centrifuged at 3000 g for 10 min at 4°C to obtain the purified Exosome particles for validation or subsequent experiments.
10
Isolation and Characterization of Escherichia coli Extracellular Vesicles
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Briefly, the E. coli (LF82) was cultured in lysogeny broth media at 37°C for 24 h. The EVs were extracted and purified by an exosome isolation kit (Umibio, Cat. No: UR52121, China) according to the manufacturer’s instructions. The culture supernatant was collected after 3,000 g and 4°C for 10 minutes, and then mixed with exosome concentration solution (Umibio). The mixtures were vortexed and incubated at 4°C for up to 2 hours and then centrifuged at 10,000 g and 4°C for 60 minutes to precipitate EVs pellets. Pellets of EVs were resuspended in 1×PBS and purified an exosome purification filter at 3,000 g and 4°C for 10 minutes. We measured the particle size and concentration of EVs via nanoparticle tracking analysis (NTA) at Viva Cell Biosciences with Zeta View PMX 110 (Particle Metrix, Meerbusch, Germany) and the corresponding software Zeta View 8.04.02. The ultrastructure and size of EVs were analyzed by transmission electron microscopy (Hitachi, HT7800, Japan). The EV pellet was dissolved in lysis buffer for WB analysis with a marker of E. coli-derived EVs. The EVs were stored at −80°C immediately after isolation for further processing.
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