We used an ultracentrifugation method to isolate exosomes from plasma (2 to 10 mL). The detailed isolation procedure is shown in S1A Fig . The expected exosomes size range was confirmed by electron microscopy using antibodies directed against CD63 and CD81 as previously described and by particle size analysis with a NanoSight NS300 Instrument (Malvern instruments)[7 (link)].
Ns300 instrument
Manufactured by Malvern Panalytical
Sourced in United Kingdom
The NS300 Instrument is a laboratory device designed for particle size analysis. It utilizes the principle of dynamic light scattering to measure the size distribution of particles suspended in a liquid or gas. The instrument provides accurate and reproducible measurements of particle size, size distribution, and concentration, making it a valuable tool for various industries and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Scrc 4000, by American Type Culture Collection (1 mentions)
P8340, by Merck Group (1 mentions)
532 nm laser, by Malvern Panalytical (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Osmium tetroxide solution, by Merck Group (1 mentions)
Jem 1400 flash transmission electron microscope, by JEOL (1 mentions)
Scmos camera, by Malvern Panalytical (1 mentions)
11 protocols using ns300 instrument
1
Exosome Isolation from Plasma
2
Nanoparticle Characterization Using NanoSight NS300
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The NanoSight NS300 Instrument (Malvern) provides an easy-to-use, reproducible platform for nanocrystals characterization. The NanoSight NS300 uses the technology of Nanoparticle Tracking Analysis (NTA). This unique technology utilizes the properties of both light scattering and Brownian motion in order to obtain the size distribution and concentration measurement of particles in liquid suspension. All measurements were performed at room temperature. The software used for capturing and analyzing the data was the NTA 3.1 Build 3.1.46. The samples were measured for 60 s with manual shutter and gain adjustments. The error bars displayed on the NTA graphs were obtained by the standard deviation of five different measurements of each sample. The mean size and SD values obtained by the NTA software correspond to the arithmetic values calculated with the sizes of all the particles analyzed by the software.
3
Characterizing Extracellular Vesicles for Virus Mimicry
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Extracellular vesicles (EVs) isolated from cell cultures were used to evaluate material properties on membrane particle absorption and mimic virus particles. EVs were isolated and characterized from ASC52-telo (SCRC-4000, ATCC, Manassas, VA, USA) cell culture similarly to previously described protocols [38 (link),39 (link)]. Single-channel devices were washed with 70% ethanol overnight, dried, and washed with sterile 1× PBS overnight. Then, 20 µL of 1.02 × 108 EVs/mL in 0.02 µm filtered 1× PBS were injected into channels and incubated for 1 h at +37 °C. After incubation, EV solutions from channels were collected and measured by nanoparticle tracking analysis (NTA) with an NS 300 instrument (Malvern, Philadelphia, PA, USA) equipped with green (532 nm) laser and scientific Complementary metal–oxide–semiconductor (sCMOS) camera and compared with input sample. Here, 0.02 µm filtered 1× PBS was used as a negative control. Measurements were performed on five 30 s videos that were recorded using camera level 12. The data were analyzed using NTA software v3.0 with the detection threshold 8 and screen gain at 5. Experiments were performed in biological duplicates and measured in technical duplicates. p-value was calculated by the Mann–Whitney test.
4
Exosome Characterization by TEM and NTA
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Exosomes suspended in 100 μl of PBS were then fixed with 5% glutaraldehyde (G6257, Merck, Darmstadt, Germany) and maintained at 4°C until transmission electron microscopy (TEM) analysis. According to the TEM sample preparation procedure, a drop of exosome sample was placed on a carbon-coated copper grid and immersed it in 2% phosphotungstic acid solution (pH 7.0; P4006, Merck) for 30s. The preparations were observed with a transmission electron microscope (G1403, Merck). Exosome quantification was determined via Nanosight Nanoparticle Tracking Analysis (NTA) as previously described (19 (link)). Briefly, PBS was utilized for resuspending the extracted exosomes. Subsequently, a syrine filter (SLHN033, Merck) was applied for filtering the exosomes. Ultimately, the size distribution of the diluted exosome samples was analyzed with a NanoSight NS300 instrument (Malvern PANalytical, Xuhui District, Shanghai, China). For determination of the exosome marker proteins (CD63 and TSG101) and calnexin (as negative control), the proteins were first extracted using extraction buffer with a protease inhibitor cocktail (P8340, Merck). The extracted proteins were then subjected to western blot analysis.
5
Exosome Size and Concentration Analysis
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After being collected by the Total Exosome Isolation Reagent as above and diluted 2000 times in particle-free PBS, the exosomes were injected into the NanoSight sample pool. Next, the exosome size distribution and concentration were analyzed using a NanoSight NS300 Instrument (Malvern Instruments, Malvern, UK) equipped with NTA 2.3 software. Sixty-second videos were recorded, and exosomes were sized and counted using the NTA 2.3 software.
6
EV Characterization by Nanoparticle Tracking
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EV concentration and size were analyzed using a Nanosight NS300 instrument with a 532-nm laser (Malvern). Images were captured using an sCMOS camera, with a gain of 1.0, and camera level of 13. EVs purified by size-exclusion chromatography (SEC) were diluted 200-fold in phosphate-buffered saline (PBS) and injected using a Nanosight autopump (Malvern) in script mode commanding a set temperature of 22 °C, an infusion rate of 25 μl/min, and video capture of five consecutive 30-s videos with a 5-s delay. Data were captured and analyzed using NTA Analytical Software suite version 3.1 (Malvern) with a detection threshold of 5.0. The instrument was calibrated using 100 nm silicone beads. Samples that were below 20 particles per frame or above 100 particles per frame were re-diluted to a concentration within this range.
7
Exosome Characterization by NanoSight
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Exosome size distribution and concentration were investigated on the NanoSight NS300 Instrument according to the manufacturer’s protocol (Malvern Instrument, Malvern, Worcestershire, UK). Briefly, 3 to 5 videos of 30 s were taken for each sample diluted in PBS. A pool of 6 populations of fibroblasts-derived exosomes was analysed for each culture method. Capture parameters were set at a rate of 30 frames per seconds and particle movement was analysed with the NTA software (v 2.3; NanoSight Ltd, Amesbury, Wiltshire, UK). Nanoparticle size distribution curve, refractive index and the relative nanoparticle concentration of a particular size were recorded, with the cumulative percentage of nanoparticles.
8
EV Size and Concentration Analysis
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EV size distribution and concentration were measured with a NanoSight NS300 Instrument (Malvern Panalytical, Malvern, United Kingdom). The samples were diluted 1:100 and 1:1000 in sterile-filtered PBS and analyzed. The measurements were based on 5 duration videos of one minute long, screen gain 9.3, camera level 11, detection threshold 3, and screen gain 10. Data analysis was performed using the Nanoparticle Tracking Analysis (NTA) software.
9
OMV Characterization by NanoSight NS300
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A NanoSight NS300 instrument (Malvern Ltd, Worchestershire, UK) was used for measurement of OMV particle concentration and size as described68 (link). Briefly, samples were diluted 1:50 in 20 mM Tris-HCl pH 8.0 buffer and loaded into the sample chamber. Videos were recorded for 60 s and size of individual OMVs and total number of OMV particles were analyzed by Nanoparticle Tracking Analysis software (NanoSight Ltd., UK). All measurements were performed at room temperature. Bicinchoninic Acid (BCA) Assay kit (Thermo Scientific Pierce, Rockford, IL, USA) was used to measure OMV concentration.
10
Nanoparticle Analysis of Extracellular Vesicles
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For size and concentration measurements, 5–10 μg of EVs were resuspended in 1 mL of PBS and measured with a NanoSight NS300 instrument (Malvern, UK).
For TEM, 10 µL (100 µg/mL) of purified EVs were placed on non-glow-discharged carbon-coated Formvar/Carbon grids (PolySciences) and negatively stained for 1 min with 10 µL of 2% osmium tetroxide solution (Sigma-Aldrich). Negative stain solution was removed by wicking onto filter paper, and TEM images were obtained using a JEM-1400 Flash transmission electron microscope (Jeol, Japan).
For TEM, 10 µL (100 µg/mL) of purified EVs were placed on non-glow-discharged carbon-coated Formvar/Carbon grids (PolySciences) and negatively stained for 1 min with 10 µL of 2% osmium tetroxide solution (Sigma-Aldrich). Negative stain solution was removed by wicking onto filter paper, and TEM images were obtained using a JEM-1400 Flash transmission electron microscope (Jeol, Japan).
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