We used 500 µL of frozen human plasma to obtain EVs for protein analysis. EVs were precipitated by using ExoQuick™ Plasma Prep and Exosome Precipitation Kit (EXOQ5TM-1, System Biosciences Inc., Mountainview, CA, United States), which allow EV isolation at low gravitational centrifugal forces (Peterson et al., 2015 (link)). Plasma samples were first treated with thrombin and incubated at room temperature for 5–10 min. Then, samples were centrifuged at 10,000 rpm for 5 minutes and the supernatant was transferred into a clean tube for EV isolation. After this, we added 126 µL of ExoQuick solution to each thrombin-treated plasma sample, mixing well by inverting the tube, and incubated the resulting mixture for 30 min at 4°C. Tubes were kept upright during incubation. Then, vials were centrifuged at 1,500 × g for 30 min, according to the manufacturer’s instructions. After the centrifugation, EVs appeared as pellets at the bottom of the tube. The supernatant was aspirated from each tube, and each pellet was resuspended in 500 µL of 1 x phosphate-buffered saline (PBS). Samples were then stored at −80°C. We used TSG101 (EV and exosome marker) ELISA to confirm the presence of EVs in the samples.
Exoquick plasma prep and exosome precipitation kit
Manufactured by System Biosciences
Sourced in United States
The ExoQuick Plasma prep and Exosome precipitation kit is a laboratory tool designed to isolate and concentrate exosomes from plasma samples. The kit utilizes a proprietary polymer-based precipitation method to selectively precipitate exosomes, allowing for their separation from the sample. The isolated exosomes can then be used for further analysis or downstream applications.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Total exosome isolation kit from plasma, by Thermo Fisher Scientific (3 mentions)
Centrifugal filter unit, by Merck Group (3 mentions)
Dnase 1, by Solarbio (3 mentions)
Epcam, by Cusabio (3 mentions)
Pd l1, by Cusabio (3 mentions)
Vascular endothelial growth factor (vegf), by Cusabio (3 mentions)
Exosome isolation kit, by Qiagen (2 mentions)
Centrifuge 5430 r, by Eppendorf (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Sta procoag ppl, by Stago (1 mentions)
Lsrfortessa, by BD (1 mentions)
Fei tecnai g2 spirit, by Thermo Fisher Scientific (1 mentions)
Ns300, by Malvern Panalytical (1 mentions)
Nta 3, by Malvern Panalytical (1 mentions)
Pvdf filter, by Merck Group (1 mentions)
Nanosight ns300 instrument, by Malvern Panalytical (1 mentions)
Nta 3.0 analytical software, by Malvern Panalytical (1 mentions)
0.22 μm pvdf filter, by Merck Group (1 mentions)
Bioanalyzer system, by Agilent Technologies (1 mentions)
18 protocols using exoquick plasma prep and exosome precipitation kit
1
Isolation and Characterization of Extracellular Vesicles from Human Plasma
2
Plasma Exosome Isolation and Characterization
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Plasma was isolated within 2 h of blood draw by centrifugation of whole blood at 1,200 g for 10 min, followed by another centrifugation of the supernatant at 2,000 g for 10 min. The supernatant was taken as plasma and stored at −80°C for future use. Two hundred microliter of plasma from each case was used. Prior to exosome precipitation, plasma samples were treated with RNase A to remove free circulating RNAs and then RNase A was inactivated with RNase inhibitor. Exosomes were isolated using the Total Exosome Isolation Kit (from plasma) (Invitrogen™) following manufacturer's instructions. For the validation cohort at Mayo Clinic, US, exosomes were isolated using ExoQuick Plasma prep and Exosome precipitation kit (System Biosciences) without RNase pre-treatment.
3
Isolation of Exosomes from Astronaut Blood
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Exosomes were isolated from blood plasma samples of 3 astronauts (Sample ID: D12, D13, and D14) at L-10 and R+3 using the ExoQuick Plasma prep and exosome precipitation kit (Cat # EXOQ5TM, System Biosciences, CA, USA), as previously described (7 (link)). Briefly, thrombin was added to the plasma and incubated for 5 min at room temperature. Next, samples were centrifuged at 10,000 rpm for 5 min, and the supernatant was incubated with the exosome precipitation solution for 30 min at 4°C. Samples were centrifuged for 30 min at 1,500 × g at 4°C, and the pellet was dissolved in 100 μl of sterile 1 × PBS.
4
Exosome Isolation and Characterization
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Exosome isolation was conducted as previously described [19 (link)]. After cultured for three days, cell supernatants were collected and centrifuged for removal of cell debris. ExoQuick Plasma prep and Exosome precipitation kit (System Biosciences, Palo Alto, CA, USA) was applied for exosome extraction. For exosomal RNA and protein extraction, exosomes were pretreated with RNase or Proteinase K, respectively. The protein concentration in exosomes was examined by Pierce® BCA Protein Assay kit (Thermo Scientific).
5
Exosome Isolation from Astronaut Plasma
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Exosomes were isolated from blood plasma samples of five astronauts (S12, S30, S31, S34, and S39) at L-10 and R + 3 using the ExoQuick Plasma prep and Exosome precipitation kit (Cat # EXOQ5TM, System Biosciences, CA, United States), as previously described (26 ). Briefly, thrombin was added to the plasma and incubated for 5 min at room temperature (RT). Next, samples were centrifuged at 10,000 rpm for 5 min, and the supernatant was incubated with the exosome precipitation solution for 30 min at 4°C. Next, samples were centrifuged for 30 min at 1,500 × g at 4°C. Finally, the supernatant was aspirated, and the beige-colored pellet was dissolved in 100 μl of sterile 1× PBS.
6
Exosome Isolation from Plasma and Media
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For the extraction of exosomes from human plasma and cell culture media, first, cells were removed (300g for 10 min) and the supernatant was centrifuged at 2000g for 10 min to discard dead cells. Then, the supernatant or plasma was collected and centrifuged at 12,000g for 30 min to remove cell debris. Afterward, the supernatant or plasma was filtered through a centrifugal filter unit (Millipore, Billerica, MA, USA) to remove the apoptotic bodies and microvesicles. Next the supernatant or plasma was collected and ultracentrifuged at 110,000g for 2 hours to precipitate the exosomes. Subsequently, the exosomes were washed with phosphate-buffered saline (PBS) to remove contaminated proteins and the ultracentrifugation at 110,000g for 70 min was used to collect the exosomes. For the extraction of exosomes from plasma of mice, plasma was filtered through a centrifugal filter unit (Millipore), and ExoQuick plasma prep and exosome precipitation kit (System Biosciences) was used according to the instruction of the manufacturer.
7
Exosome Isolation and Characterization Protocol
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The ExoQuick Plasma prep and Exosome precipitation kit (from System Biosciences, PM-EXOQ5TM-1). DNase I was obtained from solarbio (Beijing, China; www.solarbio.com). The rGO-MNPS was purchased from Nanjing Xianfeng, as a reduced graphene oxide modified magnetic nanoparticles. The PD-L1, CA125, CD63, EPCAM and VEGF were purchased from Cusabio Biotech Co.Ltd. (www.cusabio.cn/). The PD-L1 aptamer (5'-ACG GGC CAC ATC AAC TCA TTG ATA GAC AAT GCG TCC ACT GCC CGT-3'-FAM) was synthesized by Sangon Biological Engineering Technology Co., Ltd. (Shanghai, China; www.sangon. com), and purified using high performance liquid chromatography. All reagents were diluted to the required concentration with working buffer (10 mM Tris, 100 mM NaNO 3 pH 7.4) before usage. The ultrapure water obtained from a millipore water purification system (18.2 MX•cm resistivity, Milli-Q Direct 8).
8
Isolation and Characterization of Small Extracellular Vesicles
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Serum, plasma, and peripheral blood mononuclear cells (PBMCs) were obtained from each participant. All samples were processed within one hour of blood drawing. Monocytes were isolated from PBMCs from all participants, and platelets were obtained from HD whole blood. sEVs were isolated from plasma using the ExoQuick Plasma prep and Exosome precipitation kit (System Biosciences SBI, Palo Alto, CA, USA) and characterised by NanoSight, Cryo-TEM, western blot and flow cytometry analysis (LSR Fortessa, BD Biosciences, Erembodegem, Belgium, Table S5 ). sEV quantification was determined via the FluoroCet Exosome Quantification Kit (System Biosciences SBI, Palo Alto, CA, USA) and sEV procoagulant activity by STA-Procoag-PPL (Stago, Asnieres, France). Details in Supporting Information (SI) .
9
Exosome Isolation and Tumor Formation Assay
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Exosomes were isolated from cell culture medium by differential centrifugation. Cells and other debris were removed by centrifugation at 300 g and 3,000 g. Shedding vesicles were removed from supernatant by centrifuged at 10,000 g. Finally, supernatant was centrifuged at 110,000g and exosomes were obtained. Isolation of exosomes from serum was performed using ExoQuick Plasma prep and Exosome precipitation kit (SBI, USA).
Tumor formation assay in nude mice NCG (NOD-Prkdc em26Cd52 IL2 rgem26Cd22 /Gpt) mice were purchased from NBRI of Nanjing University (Nanjing, China), which were maintained in a pathogen-free facility. Cells overexpressing SHNG7 were trypsin digested, washed with PBS, and then resuspended in PBS. Then 200µl of the suspended cells (1×10 7 ) were injected into the armpit or peritoneal cavity of each mouse.3-4 weeks later, the mice were sacri ced and tumor weight or metastasis number were examined. All the animal experiments were performed with the approval of the Shandong First Medical University Animal Care and Use Committee.
Tumor formation assay in nude mice NCG (NOD-Prkdc em26Cd52 IL2 rgem26Cd22 /Gpt) mice were purchased from NBRI of Nanjing University (Nanjing, China), which were maintained in a pathogen-free facility. Cells overexpressing SHNG7 were trypsin digested, washed with PBS, and then resuspended in PBS. Then 200µl of the suspended cells (1×10 7 ) were injected into the armpit or peritoneal cavity of each mouse.3-4 weeks later, the mice were sacri ced and tumor weight or metastasis number were examined. All the animal experiments were performed with the approval of the Shandong First Medical University Animal Care and Use Committee.
10
Exosome Isolation from Cancer Patient Plasma
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