The concentration and size distribution of the sEV samples were measured right away (Fresh sEV, Ctrl sEV, w/o FBS sEV and w/ wash sEV) or after being stored at −80 °C (Frozen sEV) using a NanoSight NS300 instrument with 532 nm laser (Malvern Panalytical Ltd., Malvern, UK) coupled with NTA software 3.4 (Malvern Panalytical Ltd.). The samples were diluted in particle-free PBS before measurement. The following settings were used in all of the measurements: syringe pump flow rate: 30 units; temperature: 24 °C; camera level: 16; capture duration: 60 s/video; number of captures: 3; screen gain: 1; and detection threshold: 4.
Nta software 3
Manufactured by Malvern Panalytical
Sourced in United Kingdom
NTA software 3.0 is a software package developed by Malvern Panalytical for the analysis of nanoparticle tracking and characterization. The software provides tools for the measurement and analysis of particle size, concentration, and motion in liquid samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanosight ns300, by Malvern Panalytical (5 mentions)
Ns300, by Malvern Panalytical (3 mentions)
532 nm laser, by Malvern Panalytical (2 mentions)
Nanosight ns300 system, by Malvern Panalytical (2 mentions)
Scmos camera, by Hamamatsu Photonics (2 mentions)
Nanosight model lm14, by Malvern Panalytical (2 mentions)
Millex vv, by Merck Group (2 mentions)
Bx51wi, by Olympus (1 mentions)
Prism 9, by GraphPad (1 mentions)
15 protocols using nta software 3
1
Nanoparticle Characterization of sEV
2
Multimodal Microscopy of Nanoparticles
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The images were acquired using an upright microscope (BX51WI, Olympus, Japan) and a charge-coupled device camera (CoolSNAP HQ2, Photometrics, USA). The disc’s rotational speed was measured using a smartphone camera with 30 to 240 frames per second. The data and figures were analyzed using ImageJ (National Institutes of Health, USA). The nanoparticle size distribution was analyzed using an NS500 running NTA software 3.4 by Malvern Panalytical.
3
Characterizing DPSC and BM-MSC Extracellular Vesicles
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Particle size and concentration of DPSC and BM-MSC EVs were measured by a NanoSight NS300 device equipped with a 532 nm laser (Malvern Panalytical, Worcester, UK) based on the light scattering of particles in suspension undergoing Brownian movement. EV suspensions were diluted with PBS over a range of concentrations to obtain between 10 and 100 particles per frame. Each sample was measured five times for 60 s at 25 °C with manual shutter at camera level 16. Data were analysed by NTA software 3.2 (Malvern) with manual gain adjustments and detection threshold 6–21.
4
EV Size and Concentration Analysis
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EV size and concentration profiles were determined using the NanoSight NS300 system (Malvern Ltd, Sysmex Belgium N.V.) equipped with a 532 nm laser. EV suspensions were diluted with PBS over a range of concentrations to obtain between 10 and 100 particles per frame. Samples were injected into the sample chamber and measured five times for 60 s with a syringe pump speed of 80 and camera level and thresholds were adjusted for the individual samples. Acquisitions were captured and analysed using NTA software 3.2 (NanoSight. Malvern Ltd). EV concentration was reported as particles/mL and EV size in mean values.
5
Nanoparticle Characterization of Extracellular Vesicles
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The mean size, concentration and size distribution of EV were quantified using the NanoSight NS300 system (Malvern Ltd, Sysmex Belgium N.V.) equipped with a 532 nm laser. EV suspensions were diluted with PBS over a range of concentrations to obtain between 10 and 100 particles per frame. The camera level was set to 14. Samples were injected into the sample chamber and measured five times for 60 s with a syringe pump speed of 75 µl/s. Acquisitions were captured and analyzed using NTA software 3.2 (NanoSight. Malvern Ltd) and the threshold was set to 9. Five measurements were taken for each sample (at least nine biological replicates), mean and standard deviation were calculated and plotted using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). EV concentration was reported as particles/mL and EV size (nm) in mean values.
6
Cryo-EM and Nanoparticle Tracking Analysis of Extracellular Vesicles
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Cryo‐electron microscopy (Tecnai G2 F30) on EVs (2 μg) was performed as described (Rai, Fang, et al., 2021 (link); Xu et al., 2015 ). Vesicle particle size was determined using a NanoSight NS300, Nanoparticle tracking analysis (NTA) (Malvern) system fitted with a NS300 flow‐cell top plate with a 405 nm laser as described (Rai, Greening, et al., 2021 (link)). Samples (1 μg μl–1) were diluted in 500 μl PBS (1:10,000) and injected using 1 ml syringes (BD Biosciences) (detection threshold = 10, flowrate = 50, temperature = 25°C). Each analysis consisted of 60 s video captures. Data was analysed using NTA software 3.0 (Malvern).
7
Nanoparticle Tracking Analysis of Purified MVs
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Purified MV samples were analyzed by NTA using the Malvern Panalytical NanoSight NS300 with a 405-nm laser instrument (Malvern Instruments, UK). Five microliters from the samples were diluted in 695-µL filtered PBS, and three 30-second videos were recorded using a camera level of 12–15 with a frame rate of 30 frames per second. The data were analyzed using NTA software 3.0 (Malvern Instruments) which was optimized to first identify and then track each particle on a frame-by-frame basis.21 (link) The detection threshold was optimized for each sample and screen gain at 10 to track as many particles as possible with minimal background.22 (link)
8
Exosome Characterization by NanoSight
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The size and the concentration of exosomes were determined using a NanoSight NS300 instrument (Malvern Instruments Ltd., Malvern, United Kingdom) based on nanoparticle tracking analysis (NTA). NTA utilizes the properties of both light scattering and Brownian motion to obtain the particle size distribution of samples in liquid suspension. Briefly, exosome samples from Nef-transfected and mock (empty vector)-transfected HEK293T cells were diluted 1:100 in PBS, and exosomes were tracked on the NanoSight NS300 instrument. The samples were loaded by means of the use of a constant pressure syringe pump controller. Videos were recorded for 60 s two times, at camera setting 13, and were analyzed with NTA software 3.0 (Malvern instruments Ltd., Malvern, United Kingdom).
9
Cryo-EM and NTA Analysis of EVs
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Cryo-electron microscopy (Tecnai G2 F30) on EVs (2 µg) was performed as described18 (link). Vesicle particle size was determined using a NanoSight NS300, Nanoparticle tracking analysis (NTA) (Malvern) system fitted with a NS300 flow-cell top plate with a 405 nm laser. Samples (1 µg µl−1) were diluted in 500 µl PBS (1:10,000) and injected using 1 ml syringes (BD Biosciences) (detection threshold = 10, flowrate = 50, temperature = 25 °C). Each analysis consisted of 60 s video captures. Data was analysed using NTA software 3.0 (Malvern).
10
EV Concentration and Size Analysis by NTA
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NTA was used to assess the concentration and size distribution of the isolated EV (Gardiner et al., 2013 (link)). EV samples were analysed using Nanosight model LM14 (Malvern Instruments Ltd, Malvern, UK) equipped with blue (404 nm, 70 mW) laser and SCMOS camera (Hamamatsu photonics K.K., Hamamatsu, Japan). To obtain 40–100 particles/view samples were diluted in 0.1 μm filtered (Millex VV, Millipore) DPBS. Five 30‐s videos were recorded using camera level 14 and analysed with NTA software 3.0 (Malvern Instruments Ltd) with the detection threshold 5 and screen gain at 10.
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