To characterize ultrastructural morphology of plasmatic EVs obtained from patients, STEM was performed. EVs were isolated as describe above. After collection, EVs were resuspended in 500 μL of XE buffer (QIAGEN, Hilden, Germany), and samples were then adsorbed onto 300-mesh carbon-coated copper grids for 1 min in a humidified chamber at room temperature. Grids with adhered EV were examined with a Zeiss Gemini SEM 500 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with a STEM detector at 20–30 kV.
Buffer xe
Manufactured by Qiagen
Sourced in Germany
Buffer XE is a proprietary buffer solution designed for use in molecular biology applications. It is a key component in various nucleic acid extraction and purification protocols. The core function of Buffer XE is to provide the necessary chemical environment to facilitate the efficient binding, washing, and elution of nucleic acids during these processes.
Automatically generated - may contain errors
Lab products found in correlation
Exoeasy maxi kit, by Qiagen (6 mentions)
Exorneasy midi kit, by Qiagen (2 mentions)
Exorneasy serum plasma midi kit, by Qiagen (2 mentions)
Exorneasy maxi kit, by Qiagen (1 mentions)
Nanosight software nta3, by Malvern Panalytical (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Nanosight ns300, by Malvern Panalytical (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Takara Bio (1 mentions)
Gelred nucleic acid stain, by Biotium (1 mentions)
Optima xpn 100, by Beckman Coulter (1 mentions)
Nanosight ns300 system, by Malvern Panalytical (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Nta 3, by Malvern Panalytical (1 mentions)
Nanosight ns500 instrument, by Malvern Panalytical (1 mentions)
K edta tubes, by Sarstedt (1 mentions)
Myecl imager, by Thermo Fisher Scientific (1 mentions)
Polyacrylamide gel, by Bio-Rad (1 mentions)
Exorneasy serum plasma kit, by Qiagen (1 mentions)
Laemmli sample buffer, by Merck Group (1 mentions)
17 protocols using buffer xe
1
Ultrastructural Analysis of Plasma EVs
2
Nanoparticle Tracking Analysis of Extracellular Vesicles
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The size distribution and concentration of plasmatic EVs was determined using a Malvern NanoSight NS300 Analyzer (Malvern Panalytical Ltd., Malvern, UK) with specific parameters according to the manufacturer’s protocols. EVs were isolated using the ExoRNeasy Maxi Kit (QIAGEN, Hilden, Germany) following manufacturer’s instructions and resuspended in 500 μL of XE buffer (QIAGEN, Hilden, Germany). Captures and analysis were achieved by using the built-in NanoSight Software NTA3.3.301 (Malvern Panalytical Ltd., Malvern, UK). The detection threshold for nanoparticles was fixed at 8 for all tests. Samples were diluted in PBS to a final volume of 1 mL. For each measurement, five consecutive 60 s videos were recorded at 25 °C, using a continuous syringe pump at an infusion rate of 40 units. Particles (EVs) were detected using a 488 nm laser (blue) and a scientific CMOS camera.
3
Isolation of Small Extracellular Vesicles
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Isolation of sEVs using membrane affinity spin columns was performed according to the manufacturer’s instructions (exoEasy Maxi Kit, Qiagen). Briefly, one volume of XBP buffer (Qiagen) was added to clarified plasma and the sample then added to a spin column and centrifuged at 500 × g for 1 min. Next, 10 mL XWP buffer (Qiagen) was added to the spin column and the sample centrifuged at 5000 × g for 5 min. Finally, the sEVs were eluted in 400 μL buffer XE (Qiagen) by sample centrifugation at 500 × g for 5 min, re-application of the eluate to the spin column, and centrifugation at 5000 × g for 5 min. When isolating sEVs from more than 4 mL of clarified plasma (for WB and TEM), we combined multiple eluates and concentrated them to a final volume of 400 μL using Amicon® Ultra-15 Centrifugal Filter Units (100 kDa, Merck). The final eluate was collected and subjected directly to downstream analyses.
4
Extracellular Vesicle Isolation and Characterization from Biological Fluids
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From 1 ml of serum or CSF, we isolated EVs using the exoRNeasy affinity spin column-based system (Qiagen). Serum and CSF from two individuals with SMA, as well as two serum and CSF samples from controls, were eluted in the elution buffer, buffer XE (Qiagen), and sent for nanoparticle tracking analysis. Particle size distribution and concentration measurements were assessed with a Nanosight NS300 (Particle Tracking Analysis) instrument (Malvern Panalytical, Malvern, UK). Vesicles were resuspended in 1x XE and diluted to the working range of the system (10exp6-10exp9 particles/ml). Videos were captured and analyzed with Nanosight NS300 software (version 3.4) using an sCMOS camera.
5
Exosome Treatment and Cellular Uptake
6
Exosome Purification Using exoEasy Kit
7
Plasmid Stability under DNase I Digestion
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One plasmid of 4628 bp (constructed in our laboratory) was dissolved in H2O, Buffer XE (Qiagen) and PBS (Takara), respectively. In each solvent group, 300 ng of plasmid DNA was treated with 1 unit of DNase I (Thermo Fisher Scientific) for 0, 1, 4 h or overnight. The DNase I digestion was stopped according to manufacturer’s instructions. DNA was separated using 1% agarose gel and stained with GelRed nucleic acid stain (Biotium). Gel images were visualized using Tanon-3500 Gel Image System (Tanon).
8
Exosome Isolation and Purification Methods
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Two different methods were performed for exosome isolation. For the affinity membrane-based method, exosomes were isolated from serum or plasma samples using ExoEasy Maxi Kit (Qiagen) according to the manufacturer’s protocol, and then treated with 2 units of DNase I at 37°C for the indicated time in 30 μl of reaction system. The DNase I digestion was stopped according to manufacturer’s instructions. For ultracentrifugation, serum or plasma samples were diluted in 13 ml of PBS and centrifuged at 100,000 × g for 2 h at 4°C in a SW41 Ti rotor using Optima XPN-100 ultracentrifuge (Beckman Coulter). The exosome pellet was resuspended in Buffer XE (Qiagen) and incubated with 10 units DNase I at 37°C for 4 h in 150 μl of reaction system. After a second ultracentrifugation wash step of 80 min, the resulting exosome pellet was then resuspended in Buffer XE. Exosome enumeration and sizing were carried out using NanoSight NS300 system (Malvern). Images were recorded with detection threshold to 5.
9
Exosome Isolation from Plasma Samples
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Plasma samples were pass through a 0.8 μm filter to remove additional cellular fragments and cell debris before exosome isolation (Millipore Millex). Exosomes were collected using the exoEasy Maxi Kit (Qiagen) [7 (link)]. A total of 10 ml buffer XWP were added and centrifuge at 5000g for 5 min to wash exosomes. We used 400 μL of Buffer XE (Qiagen) to dissolve the exosomes, centrifuged at 5000g for 5 min and then collected the eluate. All steps followed the manufacturer’s instructions. Purified exosomes were then stored at − 80 °C until use.
10
Fecal Microvesicle Isolation Protocol
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Frozen feces were allowed to thaw to semi-solid at room temperature and vortexed into slurries with 3mL cold PBS per gram stool (minimum 10mL per isolation). Fecal slurries were clarified by sequential centrifugation at room temperature at 300 xg for 10 min and at 2000 xg for 15 min. Clarified supernatants were filtered at 70 μm and microvesicles isolated using the exoEasy Maxi kit (Qiagen, Germany), with a final elution at 300μL Buffer XE/gram of starting feces. Microvesicle concentration, diameter, and distribution were obtained by NanoSight (Malvern Panalytical, Netherlands). Microvesicles were standardized to 3x1010 microvesicles/mL in Buffer XE (Qiagen) and frozen in individual aliquots for subsequent analyses.
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