HeLa cells (2 × 106 cells) were seeded into 100-mm dishes in α-MEM (10 mL) containing 10% FBS and incubated for 1 day at 37 °C in 5% CO2. The cells were washed with serum-free α-MEM (five times, 5 mL) and incubated in α-MEM (10 mL) containing 10% exosome-free FBS for 2 days. The cell culture medium was collected, and the secreted EVs were isolated by the ultracentrifugation method. The collected cell culture medium was centrifuged (300 × g) for 10 min at 4 °C to remove the cell debris. The supernatant was centrifuged (2,000 × g) for 10 min at 4 °C and centrifuged again (10,000 × g) for 30 min at 4 °C to remove the microvesicles. The supernatant was then centrifuged twice (150,000 × g) for 70 min at 4 °C using an ultracentrifuge (Himac CP65β, Hitachi Koki, Tokyo, Japan), and the pellet was collected in PBS. The concentrations of the isolated EVs are described in terms of their protein concentrations, which were determined by BCA protein assays. The particle size distribution of the EVs was measured by a NanoSight LM10 with NTA2.3 Analytical Software (Malvern Panalytical, Malvern, UK). The encapsulation of FITC-labeled dextran into EVs was conducted as previously described (Nakase et al., 2015 (link)).
Nta 2.3 analytical software
Manufactured by Malvern Panalytical
Sourced in United Kingdom
The NTA 2.3 analytical software from Malvern Panalytical is a tool for particle size and concentration analysis. It provides precise measurements of particle size distribution and particle concentration in liquid samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanosight lm10, by Malvern Panalytical (4 mentions)
Nanosight ns300, by Malvern Panalytical (2 mentions)
Nanosight ns500, by Malvern Panalytical (2 mentions)
Himac cp65β, by Hitachi (1 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
Nanosight ns300 instrument, by Malvern Panalytical (1 mentions)
Glacios, by Thermo Fisher Scientific (1 mentions)
488 nm laser, by Malvern Panalytical (1 mentions)
Prism 9, by GraphPad (1 mentions)
Zetasizer nano za, by Malvern Panalytical (1 mentions)
Nanosight lm10 instrument, by Malvern Panalytical (1 mentions)
10 protocols using nta 2.3 analytical software
1
Isolation and Characterization of Extracellular Vesicles from HeLa Cells
2
Cryo-EM and NTA Characterization of Exosomes
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Cryo-EM was performed to directly visualize the exosomes. Briefly, 3 μL of the exosome suspension was applied to Quantifoil holey carbon EM grids (R1.2/1.3, 200 mesh; EMS). The EM grids were then glow-discharged for 60 s at 15 mA before sample application. Furthermore, the grids were blotted with Vitrobot Mark IV (FEI, Hillsboro, OT, USA) for 3 s with 100% relative humidity at 4 °C. The samples were then imaged using Glacios (FEI) at an acceleration voltage of 200 kV with a Falcon IV direct electron detector (FEI). Images were captured at 120,000× magnification and −3.0-μm defocus.
The size distribution of exosomes was determined via NTA using a Nanosight NS300 instrument with NTA2.3 analytical software (Malvern Panalytical Ltd., Malvern, UK).
The size distribution of exosomes was determined via NTA using a Nanosight NS300 instrument with NTA2.3 analytical software (Malvern Panalytical Ltd., Malvern, UK).
3
Characterizing Extracellular Vesicle Properties
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The zeta potential and particle size of the EVs diluted in PBS (20 μg·mL−1) were determined using the zeta potential and particle size analyzer ELSZ‐DN2 (Otsuka Electronics, Osaka, Japan), according to the manufacturer's instructions. The particle number of EVs was measured by NanoSight LM10 with NTA2.3 Analytical software (Malvern Panalytical, Malvern, UK).
4
Exosome Characterization Using NanoSight
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The isolated exosomes were resuspended in PBS and homogenized, followed by filtration through a 0.22 μm filter. The exosomes were tested using NanoSight NS300 (Malvern Instruments, Malvern, UK), and the results were analyzed using NTA 2.3 Analytical Software (Malvern Instruments). The tests were repeated three times for each sample.
5
Nanoparticle Size and Concentration Analysis
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NTA was used to measure particle size and concentration in the samples using a NanoSight NS500 equipped with NTA 2.3 analytical software and a 488 nm laser (Malvern Panalytical, UK). 0.22 µm filtered PBS was used to dilute the samples before they were analyzed. Per sample, five 30-s videos were captured at a camera level of 14–15. For each measurement, the analytic software parameters maintained the same (screen gain 2, detection threshold 6).
6
Nanoparticle Tracking Analysis of EVs
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To determine particle size and concentration in isolated EV samples, NTA was performed with a NanoSight LM10 (Malvern Instruments). EV preparations were first diluted in PBS to meet the optimal concentration of 105 to 108 particles/ml. At least 300 µl of diluted sample was needed for each analysis and was mixed by vortexing before injection into the chamber. Three individual videos were collected, and the resulting counts were averaged for each diluted sample. Triplicates of the same dilution were performed, and the overall average of these dilutions was used as the experimental result for each sample. A minimum of 200 particle tracks were completed for each video, and the data were analyzed using NTA 2.3 analytical software (Malvern Instruments). Data are presented as the average and SD of the triplicate.
7
Nanoparticle Characterization via TEM and NTA
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A transmission electron microscope (TEM Tecnai G2 X-Twin, FEI Company, Hillsboro, OR, USA, voltage 200 kV) was used to characterize both synthesized adsorbents (unmodified and modified with the use of PSA).
Nanoparticle tracking analysis (NTA) was used to analyze magnetite suspensions and assess agglomeration of nanoparticles. Water suspensions of Fe3O4 NPs and Fe3O4/PSA NPs were analyzed using NanoSight NS500 (light source: 405 nm (violet), Malvern Panalytical Ltd., Malvern, United Kingdom) with NTA 2.3 analytical software (version number: MAN0520-01-EN-00 (P0560F), Malvern Panalytical Ltd., Malvern, United Kingdom) to evaluate average sizes of agglomerates in the suspensions. Suspension samples were diluted with deionized water (specific conductance 0.06 S/cm) before NTA analysis. Measurements were made at a temperature of 296.5 K.
Nanoparticle tracking analysis (NTA) was used to analyze magnetite suspensions and assess agglomeration of nanoparticles. Water suspensions of Fe3O4 NPs and Fe3O4/PSA NPs were analyzed using NanoSight NS500 (light source: 405 nm (violet), Malvern Panalytical Ltd., Malvern, United Kingdom) with NTA 2.3 analytical software (version number: MAN0520-01-EN-00 (P0560F), Malvern Panalytical Ltd., Malvern, United Kingdom) to evaluate average sizes of agglomerates in the suspensions. Suspension samples were diluted with deionized water (specific conductance 0.06 S/cm) before NTA analysis. Measurements were made at a temperature of 296.5 K.
8
Characterization of Chol-DsiRNA Polyplexes
9
Nanoparticle Tracking Analysis of Exosomes
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The isolated exosomes were first diluted 100 times with PBS, and homogenized using a vortex mixer. The NTA was performed using Nanosight NS300 (Malvern Instruments). The data was analyzed using NTA 2.3 Analytical Software (Malvern Instruments).
10
Nanoparticle Tracking Analysis of DPCs
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To determine the particle size and concentration of DPCs within 10% KSR medium, we performed nanoparticle tracking analysis (NTA) using a NanoSight LM10 instrument (Malvern Instruments, Malvern, UK). Initially, exosome preparations were diluted in PBS to obtain an ideal concentration of 106 to 108 particles/mL. Each analysis required at least 300 μL of diluted sample, and this was mixed by vortexing before being injected into the chamber. Three videos of diluted samples were recorded and the resulting counts were averaged for each diluted sample (three replicates per sample). The duration of moving particles was 60 s for each video, with a shutter speed of 30 ms and camera gain of 680. The data were analyzed using NTA 2.3 analytical software (Malvern Instruments).
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