Visceral and subcutaneous adipose samples collected from obese and lean patients intra-operatively were promptly cultured using a published protocol (12 (link)). To isolate exosomes, visceral and subcutaneous adipose tissue were washed with PBS and cut into 4 mm3 pieces, transferred to twelve-well plates containing 3 mL/well of Dulbecco's modified Eagles medium (Invitrogen, Carlsbad, CA) supplemented with 50 µg/mL gentamicin. The culture supernatant was centrifuged at 3,000g for 15 minutes to remove cells and cellular debris. Exosomes were then isolated from cultured supernatants using ExoQuick-TC Precipitation Solution (System Biosciences, Mountain View, CA) and filtered through a 200 nm filter (Sarstedt, Nümbrecht, Germany).
Exoquick tc precipitation solution
Manufactured by System Biosciences
Sourced in United States, Germany
ExoQuick-TC Precipitation Solution is a laboratory product designed for the isolation and concentration of extracellular vesicles (EVs), including exosomes, from cell culture media or other biological fluids. The solution facilitates the precipitation of EVs, allowing for their subsequent collection and further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Exoquick tc, by System Biosciences (3 mentions)
200 nm filter, by Sarstedt (2 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Exoquick precipitation solution, by System Biosciences (2 mentions)
Zetaview, by Particle Metrix (1 mentions)
Bca kit, by Beyotime (1 mentions)
Cd9 exoelisa kit, by System Biosciences (1 mentions)
Exosome depleted fbs, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 15 centrifugal filter, by Merck Group (1 mentions)
Dextran coated magnetic particles, by STEMCELL (1 mentions)
11 protocols using exoquick tc precipitation solution
1
Isolation of Adipose Tissue Exosomes
2
Exosome Isolation and Characterization
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ExoQuick-TC precipitation solution (System Biosciences, Mountain View, CA, UAS) was used to isolate exosomes from the culture medium according to the manufacturer's instructions. A BCA kit (Beyotime, Shanghai, China) was used to measure the concentration of exosomes. To ensure the isolation of exosomes, the protein expression of TSG101 and CD63 was assessed by western blotting. Transmission electron microscopy (TEM; Tokyo, Japan) was used to identify the size and shape of the exosomes. The particle size of the exosomes was determined using nanoparticle tracking analysis (NTA; Zetaview, Particle Metrix Inc., Bavaria, Germany).
3
Exosome Isolation from Microdialysate
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Exosomes were isolated from microdialysate using the ExoQuick-TC precipitation solution (System Biosciences, CA, USA). The microdialysates were centrifuged at 3000 xg for 15 minutes and the ExoQuick-TC precipitation solution was added to the supernatant at 1:5 and incubated overnight at 4°C. Thereafter, the mixture was centrifuged at 1500 xg for 30 minutes at 4°C and then at 1500 xg for 5 minutes to remove all traces of fluid. The number of exosome particles was quantified by using the CD9 ExoELISA™ kit (System Biosciences, CA, USA) according to the manufacturer’s instruction.
4
Exosome Isolation from Lymphoma Cells
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Exosomes were isolated from culture medium using ExoQuick-TC precipitation solution (System Biosciences, Mountain View, CA) according to the manufacturer's instructions as we isolated exosomes from lymphoma cell lines in our previous study 28 . RPMI 8226 cells were cultured for 2 to 4 weeks under hypoxic (2% O2 and 1% O2) or normoxic (21% O2) conditions and then culture medium was replaced with 10% exosome-depleted FBS (Gibco) for 72 h. Culture medium was collected and differentially centrifuged at 300 g for 10 min, 2000 g for 10 min and 10,000 g at 4 °C for 30 min and then passed through a 0.22-μm filter to remove cell debris. Clarified cell culture medium was mixed with ExoQuick-TC solution and incubated at 4 °C overnight before centrifugation twice at 1,500 g for 30 min and 5 min, respectively, in order to remove the supernatant. The pellet was resuspended in 100 μL of phosphate-buffered saline (PBS).
5
Isolation and Characterization of Exosomes from CAFs and NFs
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Exosomes were isolated from the culture supernatants of CAFs or NFs using ExoQuick-TC Precipitation Solution (System Bioscience, Palo Alto, CA, USA). Briefly, the supernatants were pre-cleaned by successive centrifugations at 2000 g for 10 min and 10,000 g for 30 min and filtration through 0.22 μm filters. Subsequently, ultracentrifugation at 110, 000 g for 2 h was carried out for exosome collection. Then, the ExoQuick Solution was added to the media followed by centrifugation at 1500 g for 30 min. Afterwards, the exosomes were suspended in phosphate buffer solution. To characterize the isolated exosomes, the expression of CD63, CD81, Hsp70, TSG101, TFIIB, and LaminA/C was detected by Western blot analysis. Moreover, the expression of CD63 in exosomes was further verified by flow cytometry. The exosomes were incubated with CD63-Alexa647 antibody (BioLegend, San Diego, CA, USA) for 2 h followed by detection on a flow cytometer. To observe the presence and morphology of the prepared exosomes, transmission electron microscopy (TEM) was applied. In brief, the fixed exosomes were dropped onto copper grid, immersed in 2% phosphotungstic acid, dried for 10 min, and observed under the TEM (Hitachi, Tokyo, Japan) at 100 kV.
6
Exosome Isolation from Mesenchymal Stem Cells
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The MSCs at passage three or higher were used for preparing conditioned medium (CM). The exosomes were isolated from CM according to the International Society of Extracellular Vesicles recommendations. In brief, 150 mL of CM was collected from 150 mm plates (n=10), each containing 13 million MSCs after 48 hours of culture in CM containing exo-depleted FBS. Cell debris was removed by centrifuging at 300× g for 5 minutes, then 2,000× g for 20 minutes, and finally 16,000× g for 1 hour. The cleared CM was passed through a 0.22 µm filter and concentrated using 100 kDa molecular weight Amicon Ultra-15 Centrifugal Filter (Merck Millipore, Billerica, MA, USA). The filtered supernatants were incubated with the appropriate volume of Exoquick-TC precipitation solution according to the manufacturer’s instructions (System Biosciences) for 16 hours at 4°C, and then centrifuged for 30 minutes at 1,500× g to pellet exosomes. Some studies have indicated that there are no significant differences in exosome population isolated by the Exoquick protocol compared with ultracentrifugation methods.13 (link),14 (link)
7
Isolation of Adipose Tissue Exosomes
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Visceral and subcutaneous adipose samples collected from obese and lean patients intra-operatively were promptly cultured using a published protocol (12 (link)). To isolate exosomes, visceral and subcutaneous adipose tissue were washed with PBS and cut into 4 mm3 pieces, transferred to twelve-well plates containing 3 mL/well of Dulbecco's modified Eagles medium (Invitrogen, Carlsbad, CA) supplemented with 50 µg/mL gentamicin. The culture supernatant was centrifuged at 3,000g for 15 minutes to remove cells and cellular debris. Exosomes were then isolated from cultured supernatants using ExoQuick-TC Precipitation Solution (System Biosciences, Mountain View, CA) and filtered through a 200 nm filter (Sarstedt, Nümbrecht, Germany).
8
Exosome Isolation from Cell Culture
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EV isolation was performed using ExoQuick-TC precipitation solution (System Biosciences) from conditioned culture media according to the manufacturer’s specifications. Briefly, 10 mL culture media from confluent HBMEC cultures was centrifuged at 3000 g for 15 min to remove cells and debris, and then mixed thoroughly with 2 mL of Exo-Quick precipitation solution and incubated overnight at 4 °C. The next day, samples were centrifuged at 1500 g for 30 min, and the supernatants were removed and centrifuged again at 1500 g for 5 min. The EV pellets were stored at –80 °C and used for proteomics analysis. Separate EV samples were prepared for EV surface and total proteomics.
9
Isolation and Enrichment of Adipose-Derived Small Extracellular Vesicles
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600 μL of serum and 500 μL of CSF per subject were thawed over ice and then centrifuged at 3000× g for 15 min to remove cellular debris. The supernatant was filtered through a 0.2 µm filter. Small extracellular vesicles were obtained from the serum using Exoquick Precipitation Solution (System Biosciences, Mountain View, CA, USA) and from the CSF using Exoquick TC Precipitation Solution (System Biosciences, Mountain View, CA, USA), per the manufacturer’s protocol. Ad-sEVs were selected for using fatty acid binding protein 4 (FABP4) antibody, a sensitive and specific marker for ad-sEVs, and dextran-coated magnetic particles (StemCell Technologies, Vancouver, BC, Canada).
10
Exosome Isolation from Mouse Serum
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At 16 h after surgery, 0.5 mL of whole blood were collected from each sham-operated and CLP-treated mouse into tubes containing anticoagulant. After incubation at room temperature for 15 min, the samples were centrifuged at 3,000 × g for 10 min. The white blood cells were slowly removed from the corresponding layers, and serum samples were extracted. Exosomes were isolated with the exosome isolation reagent, ExoQuick-TC (System Biosciences, Palo Alto, CA, USA). Briefly, the supernatants were transferred to sterile tubes containing 63 μL of ExoQuick-TC Precipitation Solution (System Biosciences), mixed, and incubated for at least 12 h at 4°C. After incubation, the samples were centrifuged at 1,500 × g for 30 min at 4°C. The white pellet containing exosomes was resuspended in 500 μL of buffer.
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