The pellet from EVs isolated by ultracentrifugation was suspended in PBS and visualised in a NanoSight LM14 instrument (Malvern Panalytical). An amount of 3–5 videos of 60 s were acquired per sample and only included if exceeding 500 particle tracks. EVs/ml were calculated as average of these technical replicates and normalised to the number of cells counted at endpoint to calculate EVs/cell.
Nanosight lm14
Manufactured by Malvern Panalytical
Sourced in United Kingdom, Germany
The NanoSight LM14 is a nanoparticle characterization instrument that uses Nanoparticle Tracking Analysis (NTA) technology to measure the size, concentration, and movement of nanoparticles in liquid suspensions. The instrument utilizes a laser light source and a high-sensitivity camera to track the Brownian motion of individual nanoparticles, which is then used to determine their hydrodynamic size and concentration.
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Lab products found in correlation
Exoquick tc, by System Biosciences (3 mentions)
Micro bca assay, by Thermo Fisher Scientific (1 mentions)
Nta v2, by Malvern Panalytical (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Sw60ti, by Beckman Coulter (1 mentions)
Ab134045, by Abcam (1 mentions)
Ab79559, by Abcam (1 mentions)
Ab108415, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
H 9500, by Hitachi (1 mentions)
Silica microspheres, by Polysciences (1 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphoethanolamine, by Avanti Polar Lipids (1 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phospho l serine pops, by Avanti Polar Lipids (1 mentions)
32 protocols using nanosight lm14
1
Quantifying EV Concentration and Yield
2
Adenovirus Propagation and Characterization
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SKOV3 (human ovarian carcinoma: ATCC® HTB-77 TM) cells were cultured in RPMI-1640 medium (Invitrogen, USA) supplemented with 2 mM L-glutamine (Invitrogen, Paisley, UK), 10% FBS (PAA Laboratories, Leonding, Austria) and 1% penicillin-streptomycin (Invitrogen, Paisley, UK) at 37 °C and 5% CO2. HEK-293 cells (human embryonic kidney: ATCC® CRL-1573 TM) were grown in MEM (Invitrogen, USA) with 2 mM L-glutamine, 10% FBS, 1 mM sodium pyruvate and 1% penicillin-streptomycin (Invitrogen, USA) at 37 °C and 5% CO2. Vectors (HAdV-5 luc and LacZ [β-galactosidase]; HAdV-5T* luc and LacZ containing the following mutations I421G, T423N, E424S, L426Y, E451Q in HVR7 and T270P and E271G in HVR5 showing reduced FX-binding capacity [8 (link)]) were propagated in HEK-293 cells and purified by CsCl gradient centrifugation [15 (link)]. Viral particles were determined by micro BCA assay (Life Technologies, Camarillo, CA, USA) using the formula 1 mg protein = 4 × 109 virus particles (vp). Laser-based nanoparticle tracking analysis (NTA) was used to characterize the size of adenoviral particles from pure preparations with Nanosight NTA v2.3 software in a NanoSight LM14 (Malvern Panalytical, Malvern, UK). Plaque forming units (pfu)/mL were calculated by end-point dilution assay [15 (link)]. The luc vectors were used for all the in vitro experiments, whereas LacZ vectors were used for the in vivo analyses.
3
Isolation and Characterization of Extracellular Vesicles
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EVs were isolated from cell culture supernatants, X-VIVO 15 medium, and SF by differential centrifugation at 4 °C. Samples were centrifuged at 450 × g for 5 min, followed by centrifugation at 2000 × g for 10 min. After centrifugation at 10,000 × g for 30 min, the supernatant was subjected to ultracentrifugation (110,000 × g, 70 min, in an SW 60 Ti swinging-bucket rotor, Beckman Coulter). The EV-enriched pellet (hereinafter named EVs) was washed and resuspended in PBS. Particle concentration and size were determined using the NTA instrument NanoSight LM14 (Malvern Panalytical) equipped with a 638 nm laser and a Marlin F-033B IRF camera (Allied Vision Technologies), operated with NTA 3.0 software.
4
Characterization of Extracellular Vesicles
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M-EV and exosomes were characterized by nanoparticle tracking analysis (NTA) using a NanoSight LM14 instrument (Malvern Instruments, UK). The particle suspensions were diluted 50 times with PBS for optimal analysis. The morphology was visualized using a transmission electron microscope (TEM, H-9500, Hitachi). First, EV were dropped on copper grids coated with carbon film (Zhongjingkeyi Technology, Beijing, China) and allowed to diffuse onto the grid for 2 min at room temperature. Then the samples were stained for contrast with 2% uranyl acetate for 30 s. For western blot, M-EV and exosomes isolated from DC2.4 cells and human serum, respectively were lysed in RIPA buffer supplemented with complete protease inhibitor cocktail tablets (Roche). Samples were run on SDS-PAGE gels and transferred onto PVDF membranes (Bio-Rad). The blots were incubated with primary antibodies to CD47 rabbit monoclonal antibody (ab108415, Abcam, Cambridge, MA, USA), CD63 rabbit monoclonal antibody (ab134045, Abcam, Cambridge, MA, USA), or CD 81 mouse monoclonal antibody (ab79559, Abcam, Cambridge, MA, USA) overnight at 4°C. Secondary antibodies (1:5000; System Bioscience, Mountain View, CA) were incubated with the membranes for 30 min at 37°C. Signals were detected using chemiluminescent reagents from Pierce, according to the manufacturer’s instructions.
5
Exosome Size and Concentration Analysis
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The concentration and size distribution of isolated exosomes were measured with a NanoSight LM14 instrument (Malvern Instruments, Malvern, UK). Data were analyzed with Nanoparticle Tracking Analysis (NTA) software (v.3.2). The analysis settings were optimized and kept constant between samples. Readings were taken in triplicate by capturing video during 45 s at 30 frames per second (fps), at a camera level set to 13. Silica microspheres were used as a size standard (100 nm, Polysciences). Purified exosome samples were diluted 25–50 times with sterile and filtered PBS yielding particle concentrations in the region of 0.5–1.5 × 109 particles per milliliter in accordance with the manufacturer’s recommendations.
6
Synthetic Liposome Preparation and Characterization
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Synthetic liposomes were prepared using a modified version of a previously described method52 . Briefly, chloroform suspended 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) (Avanti Polar Lipids, Alabaster, AL) were combined to form lipid mixtures of the described molar ratios. Lipid solutions were then dried to a thin film under a slow N2 flow and vacuum desiccated for 1 hour. Lipid films were resuspended in HBS buffer (10 mM HEPES, 0.15 M NaCl, pH 7.4) containing 2 mM CaCl2. Homogenously sized liposomes were formed by extruding the lipid solutions through polycarbonate track etched membranes (GE Healthcare, Pittsburgh, PA) with pore sizes of 30, 100, and 400 nm using a LiposoFast FL-50 extruder (Avestin, Ottawa, Canada). Extruded liposome sizes were characterized by nanoparticle tracking analyses using a NanoSight LM14 (Malvern Instruments, Malvern, United Kingdom).
7
Characterization of Extracellular Vesicle Size
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The size and particle concentrations of the purified EVs were assessed by Brownian microscopy using a NanoSight LM14 analyzer (Malvern Panalytical, Malvern, UK). Briefly, the samples were prediluted with PBS to a total volume of 500 µL and infused into a cuvette (d = 500 µm) at a temperature of 22 °C. Illumination with a laser beam (λ = 532 nm) caused light scattering of particles present in the solution. Scattered light was collected with a 20× microscope lens and captured by a CMOS camera. A total of three videos (length of 30 s each, 25 frames per second) were recorded and processed by Nanotracking Analysis Software, with the analysis based on tracking individual particles in the acquired videos. The obtained mean square displacement as well as the solvent viscosity (phosphate-buffered saline (PBS): 0.93 mPa s) enabled particle diameter calculation.
8
Exosome Size Distribution Analysis
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Precise assessment of exosome size distribution and concentrations was evaluated through nanoparticle tracking analysis. Briefly, the eluate samples were diluted further in buffer XE and gently agitated to ensure a complete homogeneity. The samples were then loaded into a clean 1 mL glass cuvette before being placed inside the NanoSight LM14 (Malvern Panalytical, Wocestershire, UK) for analysis. A refractive index of 1.349 was selected based on the buffer XE used with the absorption value of 0.1. Raw data containing the values of X intensities were first analyzed in Microsoft Excel prior further statistical analysis in GraphPad Prism v9 software.
9
Nanoparticle Tracking Analysis of RVLPs
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Concentration, size distribution, and mode size of RVLPs were determined by NTA using the Nanosight LM14 instrument (Malvern Panalytical, Kassel, Germany) equipped with a 638-nm laser and a Marlin F-033B IRF camera. Collected RVLP samples were diluted 50- to 1000-fold in PBS prior to NTA analysis. Five 30-s movies were recorded on camera level 16, and then analyzed with detection threshold 6 in NTA 3.2 Build 16. All NTA EV size data are presented as mode values.
10
EV Quantification via FACS and Nanosight
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Quantification of EV was carried out with the FACSAriaIIIu (BD Biosciences, Heidelberg, Germany). Submicron Bead Calibration Kit (0.2/0.5/0.76 μm; Bangs Laboratories, Fishers, IN, USA) and Flow Cytometry Size Calibration Kit (1/2 μm; Thermo Fisher Scientific, Waltham, MA, USA) were used to define the counting gate up to 1 μm. Sure Count Particle Standard 3 μm (Bangs Laboratories) were placed within the plot area for subsequent quantification of EV. Additionally, 0.1 μm filtered PBS and Count Particles diluted in 0.1 μm filtered PBS were evaluated for contamination with submicron particles.
To evaluate the impact of “swarm detection” of small particles in FACS [27 (link)] we additionally employed NanoSight measurements for EV concentration and size (NanoSight LM 14, Malvern Instruments GmbH, Herrenberg, Germany). Data were acquired in 10 repeats for 10 sec each per EV sample (n = 6).
To evaluate the impact of “swarm detection” of small particles in FACS [27 (link)] we additionally employed NanoSight measurements for EV concentration and size (NanoSight LM 14, Malvern Instruments GmbH, Herrenberg, Germany). Data were acquired in 10 repeats for 10 sec each per EV sample (n = 6).
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