To isolate CSF EVs, CSF samples (250 μl) were centrifuged at 3000 x g to remove cellular debris and supernatants were incubated overnight at 4°C with ExoQuick reagent (System Biosciences, Inc., Mountain View, CA) according to manufacturer’s instructions. The suspensions were centrifuged at 1500 x g for 30 minutes and CSF EV pellets were suspended in 20 μl PBS for TEM or RIPA buffer (Triton X-100 1%, NaCl 150 mM, sodium deoxycholate 0.5%, Tris-HCL 50 mM, SDS 0.1%, pH 7.4) for ELISA and western blotting. The supernatants (EV-depleted CSF) were stored at −80°C. EV concentrations and sizes were measured by nanoparticle tracking analysis (NTA) (Particle Metrix, Germany) either directly from CSF samples (diluted 1:500) or from isolated EV fractions (diluted 1:5000). Transmission electron microscopy (TEM) was performed using a Tecnai G2 Spirit BioTWIN instrument equipped with an AMT 2k CCD camera (Harvard University TEM core).
Nanoparticle tracking analysis
Manufactured by Particle Metrix
Sourced in Germany
Nanoparticle tracking analysis (NTA) is a laboratory technique used to analyze the size and concentration of nanoparticles in a liquid sample. The system tracks the Brownian motion of individual nanoparticles and uses this information to determine their size distribution and concentration.
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Lab products found in correlation
Exoquick reagent, by System Biosciences (2 mentions)
Amt 2k ccd camera, by Thermo Fisher Scientific (1 mentions)
Tecnai g2 spirit biotwin instrument, by Thermo Fisher Scientific (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Tecnai g2 spirit biotwin, by Thermo Fisher Scientific (1 mentions)
Zetaview analysis software, by Particle Metrix (1 mentions)
Zetaview, by Particle Metrix (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
10 protocols using nanoparticle tracking analysis
1
Isolation and Characterization of CSF Extracellular Vesicles
2
Isolation and Characterization of CSF Extracellular Vesicles
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To isolate CSF EVs, CSF samples (250 μl) were centrifuged at 3000g to remove cellular debris and supernatants were incubated overnight at 4°C with ExoQuick reagent (System Biosciences, Inc., Mountain View, California, USA) according to manufacturer's instructions. The suspensions were centrifuged at 1500g for 30 min and CSF EV pellets were suspended in 20 μl PBS for transmission electron microscopy (TEM) or RIPA buffer (Triton X-100 1%, NaCl 150 mmol/l, sodium deoxycholate 0.5%, Tris-HCL 50 mmol/l, SDS 0.1%, pH 7.4) for ELISA and western blotting. The supernatants (EV-depleted CSF) were stored at −80°C. EV concentrations and sizes were measured by nanoparticle tracking analysis (NTA) (Particle Metrix, Germany) either directly from CSF samples (diluted 1 : 500) or from isolated EV fractions (diluted 1 : 5000). TEM was performed using a Tecnai G2 Spirit BioTWIN instrument (FEI company, Hillsboro, Oregon, USA) equipped with an AMT 2k CCD camera (Harvard University TEM core).
3
Isolation and Characterization of Extracellular Vesicles from Glioblastoma Cells
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EVs were isolated from supernatants of mouse GBM cell lines SB28 and GL261, and human derived GBM cell lines LN18, Ge738, Ge982, Ge975, Ge904 and Ge835. The cells (2.5 × 105) were cultured in 15 ml of medium in T75 flasks (TPP) in triplicates for 24 h and the media was collected for EV isolation The isolation procedure was based on a previously described protocol [51 ] with modifications. Briefly, culture medium was centrifuged at 300 g for 10 min to pellet the cells and large cell debris. The supernatant was then centrifuged for 10 min at 2000 g to remove dead cells and small cell debris. Finally, EVs were pelleted by ultracentrifugation at 100,000 g for 70 min and washed with PBS once, then pelleted again by ultracentrifugation at 100,000 g for another 70 min. The EV containing pellet was resuspended in PBS for subsequent tests. Size and concentration of EVs were quantified using Nanoparticle Tracking Analysis (NTA) (Particle Metrix, Germany). For further functional experiments, EV:recipient cell ratios were normalized to 3000 nanoparticles (measured by NTA) per recipient cell.
4
Nanoparticle Tracking Analysis of BEVs
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BEVs isolated by UC or ϵ-PL were diluted 3000-fold with distilled water. The particle size distribution of BEVs was determined using nanoparticle tracking analysis (Particle Metrix, Germany). All particle-size analyses used the same set of parameters to ensure comparable results.
5
Exosome Isolation and Characterization
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The conditioned media (CM) of SW620 cells was collected. After that, exosomes were isolated using the GETTM Exosome Isolation Kit (GeneExosome technologies). Nanoparticle-tracking analysis (Particle Metrix, Meerbusch, Germany) was applied to determine the size of exosomes. Next, a transmission electron microscopy (TEM) was used to visualize the morphology of exosomes as described previously [22 (link)].
6
Isolation and Characterization of ADSC-Derived Exosomes
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Supernatant was collected after culturing ADSCs in Dulbecco’s modified Eagle’s medium and nutrient mixture F-12 medium for 48 h. The supernatant was subjected to differential centrifugation at 300 × g for 5 min, 2000 × g for 15 min and 10 000 × g for 35 min. Then, the supernatant was filtered through a 0.22-μm filter (Millipore, MA, USA). Finally, the supernatant was transferred to a 100,000 molecular weight cut off (MWCO) ultrafiltration tube (Sartorius, Hamburg, Germany) and centrifuged at 3000 × g for 1.5 h. The supernatant was removed and the pellet was resuspended as ADSC-Exos with an appropriate amount of phosphate-buffered saline (PBS).
Exosome morphology was examined via transmission electron microscopy (TEM). Particle size distribution was determined via nanoparticle tracking analysis ( Particle Metrix, Germany). The presence of exosomal markers, including alix (rabbit IgG, Proteintech, Wuhan, China), CD9 (rabbit IgG, Proteintech), CD63 (rabbit IgG, Proteintech) and calnexin (rabbit IgG, Proteintech), was confirmed via western blot analysis as described below.
Exosome morphology was examined via transmission electron microscopy (TEM). Particle size distribution was determined via nanoparticle tracking analysis ( Particle Metrix, Germany). The presence of exosomal markers, including alix (rabbit IgG, Proteintech, Wuhan, China), CD9 (rabbit IgG, Proteintech), CD63 (rabbit IgG, Proteintech) and calnexin (rabbit IgG, Proteintech), was confirmed via western blot analysis as described below.
7
Exosome Isolation and Characterization
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Briefly, the cultured medium was removed and washed twice with PBS when hucMSC reached 60%–70% confluence. MSCs were then cultured with de-exosome medium for two days. The supernatants were collected and centrifuged at 300
g for 10 min at 4 ℃ and centrifuged at 2000
g for 10 min to remove dead cells. Then, supernatants were centrifuged at 10 000
g for 30 min to remove cell debris and supernatant was then centrifuged at 150 000
g for 90 min to acquire exosome pellets. The concentration and particle size were measured by nanoparticle tracking analysis (Particle Metrix, Germany). All procedures were performed at 4 ℃. Exosomes were incubated in 2% paraformaldehyde solution overnight for transmission electron microscopy analysis. Two microliters of the exosome solution were transferred onto a carbon-coated copper grid. Then, the grid was observed using a transmission electron microscope (Tecnai G2 Spirit BioTwin, FEI, Hillsboro, OR, USA).
g for 10 min at 4 ℃ and centrifuged at 2000
g for 10 min to remove dead cells. Then, supernatants were centrifuged at 10 000
g for 30 min to remove cell debris and supernatant was then centrifuged at 150 000
g for 90 min to acquire exosome pellets. The concentration and particle size were measured by nanoparticle tracking analysis (Particle Metrix, Germany). All procedures were performed at 4 ℃. Exosomes were incubated in 2% paraformaldehyde solution overnight for transmission electron microscopy analysis. Two microliters of the exosome solution were transferred onto a carbon-coated copper grid. Then, the grid was observed using a transmission electron microscope (Tecnai G2 Spirit BioTwin, FEI, Hillsboro, OR, USA).
8
Characterizing Islet Exosome Properties
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The concentration and size distribution of the islet-derived exosomes were measured using nanoparticle tracking analysis (ParticleMetrix, Inning am Ammersee, Germany) and analyzed using Zetaview Analysis software. Briefly, human islet-derived exosomes were isolated using the methods described above and maintained at −80 °C until batched analysis. On the day of analysis, samples were thawed over ice and diluted in distilled water (1:3000). One milliliter of diluted sample was injected for the measurement of exosome size and concentration using Zetaview software.
9
Extracellular Vesicle Abundance Analysis
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EV abundance in plasma samples was analyzed via Nanoparticle Tracking Analysis with the ZetaView (Particle Metrix) as described previously.27 (link) Data were expressed as percentage of control values.
10
Isolation of Extracellular Vesicles from Ovarian Cancer Cells
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EVs were collected from the supernatant of ovarian cancer cells cultivated in EV-depleted medium (overnight centrifugation at 100,000 x g) using sequential ultracentrifugation 32 (link). The centrifugation protocol included consecutive pre-centrifugation steps at 300 x g (10 min), 2,000 x g (10 min) and 3,500 x g (20 min) for clearance of cells and cellular debris before ultracentrifugation at 10,000 x g (60 min) and/or 100,000 x g (90 min) using an SW41Ti or Type 45Ti rotor for at least twice with resuspension in PBS or HBSS. The amount of EV protein was quantified by Nanodrop 1000 (Thermo Scientific, Schwerte, Germany) and/or using a BCA assay (Pierce). The number of particles was determined by Nanoparticle Tracking Analysis (Particle Metrix).
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