EV extraction and L1CAM enrichment has been described previously [9 (link)]. Briefly, 500 µL plasma samples were thawed at 4°C and each treated with 15 µL thrombin to remove fibrinogen. EVs were precipitated using polyethylene glycol (SBI ExoQuick, cat. no. EXOQ5TM–1, System Biosciences, Palo Alto, CA, USA). This fraction represents the total EV extraction. Enrichment of NEEs was accomplished by the addition of mouse anti-human CD171 (L1 cell adhesion molecule, L1CAM, neural adhesion protein) mono-clonal antibody (cat. no. eBIO5G3, 5G3, 13-1719-82, Biotin, eBioscienceTM Antibodies, Thermo Fisher Scientific, Waltham, MA, USA) followed by the addition of streptavidin-agarose resin (cat. no. 53116, Pierce Streptavidin Plus UltraLink Resin, Thermo Fisher Scientific) [10 (link)]. Centrifugation of this mixture created the supernatant fraction which represents the total heterogeneous EV population minus the EVs with L1CAM neural surface proteins, a fraction which we designate as T-N. The pellet containing the NEEs was resuspended in 0.1 M glycine-HCl (pH 2.5) and the solution strongly vortexed and centrifuged to release the EVs from the streptavidin beads. The supernatant was recovered and neutralized with 1 M TRIS–HCl pH 8.0 to create the NEE fraction.
Exoq5 1
Manufactured by System Biosciences
Sourced in United States
The EXOQ5TM-1 is a high-performance exosome isolation system designed for efficient and reliable purification of extracellular vesicles from biological samples. It utilizes a proprietary technology to extract exosomes with high purity and yield.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Exosome precipitation kit, by System Biosciences (2 mentions)
Pierce streptavidin plus ultralink resin, by Thermo Fisher Scientific (1 mentions)
Exoquick plasma prep, by System Biosciences (1 mentions)
Zetaview, by Particle Metrix (1 mentions)
H 7500, by Hitachi (1 mentions)
Exotc50a 1, by System Biosciences (1 mentions)
6 protocols using exoq5 1
1
Neural Extracellular Vesicle Isolation
2
Exosome Isolation from THP-1 Cells
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3
Isolation and Characterization of Platelet-Rich Plasma-Derived Exosomes
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PRP-Exos were isolated from PRP through the combination of polymerization precipitation and ultracentrifugation. Crude PRP-Exos were initially precipitated via polymerization precipitation using ExoQuick (EXOQ5TM-1, ExoQuick Plasma prep and Exosome precipitation kit, System Biosciences, CA, USA). To enhance purity by removing impurities such as lipoproteins, we resuspended the PRP-Exos pellet in PBS and subjected it to ultracentrifugation (4 °C, 100,000×g, 70 min). The purified PRP-Exos pellet was subsequently stored at −80 °C.
The final size of exosome particles was determined through NTA using ZetaView (Particle Metrix, Meerbusch, Germany). Exosome morphology was characterized using transmission electron microscopy (Hitachi, H7500, Tokyo, Japan). The concentration of exosomes was quantified using a PierceTM BCA Protein Assay Kit (#23227, Thermo Scientific, Rockford, USA). The surface marker molecules of PRP-Exos were identified via western blot analysis, with primary antibodies including anti-CD9 (1:1000, 60232-1-Ig, Proteintech, USA), anti-CD63 (1:5000, 67605-1-Ig, Proteintech, USA), anti-CD81 (1:5000, 66866-1-Ig, Proteintech, USA), and anti-CD41 antibodies (1:4000, 60350-1-Ig, Proteintech, USA). All of the original western blot bands were provided in Supplementary Information (Supplementary Fig.1 ).
The final size of exosome particles was determined through NTA using ZetaView (Particle Metrix, Meerbusch, Germany). Exosome morphology was characterized using transmission electron microscopy (Hitachi, H7500, Tokyo, Japan). The concentration of exosomes was quantified using a PierceTM BCA Protein Assay Kit (#23227, Thermo Scientific, Rockford, USA). The surface marker molecules of PRP-Exos were identified via western blot analysis, with primary antibodies including anti-CD9 (1:1000, 60232-1-Ig, Proteintech, USA), anti-CD63 (1:5000, 67605-1-Ig, Proteintech, USA), anti-CD81 (1:5000, 66866-1-Ig, Proteintech, USA), and anti-CD41 antibodies (1:4000, 60350-1-Ig, Proteintech, USA). All of the original western blot bands were provided in Supplementary Information (Supplementary Fig.
4
Exosome Isolation from Cell Culture and Plasma
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Concerning cells were cultured in DMEM with 10% FBS until they reached 80% confluence. The cell culture medium was removed. The cells were washed once with PBS and then cultured in cell culture medium containing no serum for 48 h. Cell culture medium was centrifuged at 3000 g for 15 min to eliminate cell debris. The exosomes were extracted by EXOTC50A-1 (System Biosciences, Palo Alto, CA, USA) according to manufacturer’s instructions. Exosomes were isolated from plasma specimen by EXOQ5TM-1 (System Biosciences), according to the manufacturer’s instructions.
Briefly, 5 mL of centrifuged supernatant was mixed with 1 mL of ExoQuick-TC/TM solution by inverting the tube several times. The sample was incubated overnight at 4 °C then centrifuged twice at 1500 g for 30 and 5 min, respectively, in order to remove the supernatant. The pellet was re-suspended in sterilized PBS, quantified by bicinchoninic acid (BCA) assays (Thermo Fisher Scientific, Waltham, MA, USA).
Briefly, 5 mL of centrifuged supernatant was mixed with 1 mL of ExoQuick-TC/TM solution by inverting the tube several times. The sample was incubated overnight at 4 °C then centrifuged twice at 1500 g for 30 and 5 min, respectively, in order to remove the supernatant. The pellet was re-suspended in sterilized PBS, quantified by bicinchoninic acid (BCA) assays (Thermo Fisher Scientific, Waltham, MA, USA).
5
Exosome Isolation from A549 Cells
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The cells were treated with PtNPs (10 µM) or C6-cer (10 µM) or CSP (10 µM) or GW4869 (20 µM) for 24 h. Exosomes were prepared from culture supernatants of A549 cells by differential centrifugation according to a previously described method.44 (link),45 (link) Exosomes were also isolated and purified using ExoQuick (EXOQ5TM-1, System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s instructions.
6
Extracellular Vesicle Isolation from Maternal Plasma
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Maternal peripheral venous blood was collected into EDTA-coated tubes and centrifuged at 3200xg for 7 min, to separate the plasma fraction, which was stored at -80°C for future use. Extracellular vesicles (EVs) were isolated from 1ml of plasma by using polymer-based precipitation (EXOQ5TM-1, System Biosciences, Mountain View, CA, USA) by following the manufacturer’s protocol. Briefly, plasma samples were centrifuged at 3000xg for 15 minutes to remove cells and the rest of the cellular debris. The supernatant was collected, incubated with thrombin (final concentration of 5U/ml) for 5 minutes at room temperature to catalyze the conversion of fibrinogen to fibrin which precipitated, and was centrifuged off at 10,000 rpm for 5 minutes. The supernatant, containing EVs, was incubated with ExoQuick exosome precipitation solution for 1h at 4°C (pre-optimized conditions). This incubated solution was centrifuged at 1500xg for 30 minutes, and the obtained supernatant was aspirated and saved for analysis (serving as the negative control), and the pellet containing the precipitated EVs was resuspended in PBS.
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