MSC were washed three times with PBS and switched to exosome isolation media; either 20% FBS media that was pre-cleared of exosomes via 18 hour 120,000g centrifugation or OptiMEM (Life Technologies) and were conditioned for 40 hours prior to vesicle isolation [9 (link)]. Microvesicles (MV) were isolated as in previous studies [20 ]. Briefly conditioned media was cleared of cells and cell debris via centrifugation (500g and 1,000g, respectively), then spun at 17,000g pellet to isolate MVs. Exosomes were isolated as in previous studies [20 ]. Briefly, for proteomics studies exosomes were isolated using 0.22 μm filtration to get rid of cells, cell debris and MVs prior to being spun at 120,000g for 2 hours, the pellet was then washed with 39 mL of PBS and spun again at 120,000g for 2 hours. All ultracentrifuge steps were performed with a Ti70 rotor in polyallomer quick seal tubes (Beckman Coulter, Brea, CA http://www.beckmancoulter.com ). Vesicle concentration was determined using detergent compatible protein concentration (DC) assay (BioRad, Hercules, CA, http://www.bio-rad.com ), and size distribution was assessed using NanoSight LM10HS (Malvern, Amesbury, MA, http://www.malvern.com ).
Nanosight lm10 hs
Manufactured by Malvern Panalytical
Sourced in United Kingdom
The NanoSight LM10-HS is a nanoparticle characterization instrument that utilizes nanoparticle tracking analysis (NTA) technology to measure the size and concentration of particles in liquid suspension. The system uses a laser light source to illuminate nanoparticles, and a camera to track the Brownian motion of individual particles, allowing the determination of their hydrodynamic size and concentration.
Automatically generated - may contain errors
Lab products found in correlation
Nta 3, by Malvern Panalytical (2 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Aqueous uranyl acetate, by Ted Pella (1 mentions)
Nta software, by Malvern Panalytical (1 mentions)
G2 f20 s twin, by Thermo Fisher Scientific (1 mentions)
Tecnai, by Thermo Fisher Scientific (1 mentions)
15 protocols using nanosight lm10 hs
1
Isolation and Characterization of Extracellular Vesicles
2
Nanoparticle Characterization by NanoSight
3
Nanoparticle Tracking Analysis of Extracellular Vesicles
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The concentration of isolated EVs, as well as the particle size distribution, were determined by nanoparticle tracking analysis using a NanoSight LM10-HS instrument (Malvern Panalytical Ltd, UK) equipped with a 405 nm, 65 mW laser unit with passive temperature reading and a highly sensitive Andor Luca EMCCD camera. (Andor, Belfast, UK) (Supplementary Figure 6B). All measurements were performed in accordance with ASTM E2834-12 (2018) with camera and video processing settings optimized for EVs as previously described29 (link),30 (link). Each sample was diluted to a concentration of (1.3–2.0) × 108 particles/ml, optimal for measurements. Twelve videos (60 s each) were recorded and processed using NTA 2.3 build 33 software (Malvern Panalytical Ltd, UK). All measurements (5200–6500 tracks total) were pooled to produce a histogram of particle size and total particle concentration corrected for dilution factor.
4
Nanoparticle Quantification via NanoSight
5
Nanoparticle Degradation Kinetics
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Nanoparticle tracking analysis (NTA, Nanosight LM10-HS, Malvern Instruments) was used to determine NP concentration and diameter in various solutions. pH dependent NP degradation was studied in buffers with pH 7.4 (0.01 M phosphate buffer), pH 5.5 and 4.0 (0.01 M acetic acid). Degradation was also measured in cell medium (DMEM, Life Technologies) with FBS, pH 7.5 and human blood serum (a kind gift from professor Asbjørn Nilsen, Medical Faculty, NTNU). The NPs were added to the buffer at 20 µg NPs/ml and incubated for up to 336 h (14 days) at 37 °C. For cell medium and human serum, NPs were added at 5 mg/ml, and further diluted to 20 µg/ml in deionized water before NTA analysis. The buffer or medium were not changed during the incubation.
6
Quantifying Extracellular Vesicle Release
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The number and size distribution of EVs were performed using Nanoparticle Tracking Analysis (NTA). EV samples were diluted in sterile PBS before analysis with NanoSight LM10 HS (Malvern Instruments, Malvern, UK). All samples were measured in triplicate at 25 °C and used the same instrument settings. Three videos of 30 s per sample were analyzed with NanoSight NTA 3.1 software (Malvern, UK) setting the detection threshold at 3. The results presented are the average of the three videos and three independent EV samples. Concentrations and distributions of EVs were then normalized to cell counts and expressed as numbers of EVs released per cell.
7
Nanoparticle Tracking Analysis Protocol
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For particle measurement by NTA we used a NanoSight LM10-HS (Malvern Instruments Ltd, Malvern, UK) with a 405 nm laser and a Luca DL-658 M-OEM EMCCD camera (Andor Technology, Belfast, UK). NanoSight NTA 2.3 was applied for data processing.
The duration of particle tracking was 60 seconds with the camera shutter set to 500, camera gain to 300, and detection threshold to 3. Sample viscosity was linked to the corresponding viscosity for water at the given temperature. Automatic settings were selected for minimum expected particle size, blur, and minimum track length.
Samples were diluted in PBS to a concentration within the linear range for NTA. Samples were loaded manually into the sample chamber using a 1 mL syringe. The microscopic field of view was positioned near to the flare spot in the area where the particles were most clearly visualised. Background signal was subtracted before sequence processing.
Samples were analysed in duplicates and the mean value given. Daily QC procedures were performed as previously described [16 (link)].
The duration of particle tracking was 60 seconds with the camera shutter set to 500, camera gain to 300, and detection threshold to 3. Sample viscosity was linked to the corresponding viscosity for water at the given temperature. Automatic settings were selected for minimum expected particle size, blur, and minimum track length.
Samples were diluted in PBS to a concentration within the linear range for NTA. Samples were loaded manually into the sample chamber using a 1 mL syringe. The microscopic field of view was positioned near to the flare spot in the area where the particles were most clearly visualised. Background signal was subtracted before sequence processing.
Samples were analysed in duplicates and the mean value given. Daily QC procedures were performed as previously described [16 (link)].
8
Characterization of EV Morphology
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EV concentrations in 1× PBS were determined by nanoparticle tracking analysis using NanoSight LM10HS (Malvern Instruments, Malvern, UK). The morphology of EVs were examined by negative staining and electron microscopy. EVs in 1× PBS were fixed in 1% glutaraldehyde and allowed to absorb onto formvar/carbon-coated copper grids for 10 min. Grids were washed with water and stained with 1% aqueous uranyl acetate (Ted Pella Inc., Redding, CA) for 1 min. Excess liquid was gently wicked off and grids were allowed to air dry. Samples were viewed on a JEOL 1200EX transmission electron microscope (JEOL USA, Peabody, MA) equipped with an AMT 8 megapixel camera (Advanced Microscopy Techniques, Woburn, MA).
9
Nanoparticle Tracking Analysis of Exosomes
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Nanoparticle tracking analysis (NTA) was performed on fresh exosome samples in triplicate using a Nanosight LM10-HS (Malvern Instruments, Malvern, UK) configured with a blue (488 nm) laser and sCMOS camera to estimate size distribution and particle concentration [49 (link)]. Samples were diluted to 1–2 µg/mL in PBS, and three videos of 60 s were acquired for each replicate. The camera shutter speed was fixed at 30 ms. NTA3.0 software (Malvern, UK) was used to measure the mode size, i.e., light-scatter (standard) mode, the mean size, and the concentration of particles per 1 mL solution. The mode size is the vesicle size that appears most often and is usually a more accurate representation of the EV size as vesicle aggregates affect the mean size. The camera level was set to 13, and the detection threshold was maintained at 3 to ensure accurate and consistent detection of small particles. The laser chamber was cleaned thoroughly between each sample reading with particle-free water and 70% ethanol.
10
Nanoparticle Concentration Measurement by NTA
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After sEV isolation, particle concentration was measured using a Nanosight LM10HS equipped with a blue laser system (Malvern Instruments Ltd., Malvern, UK). Samples were diluted 25-fold with PBS. Particles were visualized by light scattering using a conventional optical microscope aligned perpendicularly to the beam axis. Data were analyzed using NTA software (Malvern Instruments Ltd.) with a detection threshold set to 7 and temperature approximately 25 °C. The Brownian motion of each particle was tracked between frames to calculate its size using the Stokes–Einstein equation. Each sample was measured three times and averaged values were used for the statistical analysis.
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