A total of 1 mL of the conditioned media (MEM supplemented with N2 supplement) of rat primary cortical neural cultures (seeded in 150 mm culture dishes at a density of 4.8 × 106 cells/dish and 25 mL of media) was subsequently centrifuged at 200× g and 2000× g for 10 min each in order to remove dead cells and debris. The supernatant was transferred to a new Eppendorf tube and centrifuged at 10,000× g and 4 °C for 30 min. The supernatant was diluted 1:10 in filtered PBS 1× to obtain a concentration in the range of 108 and 109 vesicles/mL, and the concentration of vesicles present in the sample was analyzed by Nanoparticle Tracking Analysis (NTA) with a NanoSigh NS500 instrument (Malvern Panalytical, Malvern, UK). The instrument was equipped with a 488 nm laser, a high sensitivity CMOS camera. Both the concentration of the vesicles present in the media of neural cultures at different DIV, as well as the mean size, were analyzed using the nta2.3 software (Malvern Panalytical) after filming 3 × 60 s long videos.
Nanosigh ns500 instrument
Manufactured by Malvern Panalytical
Sourced in United Kingdom
The NanoSight NS500 instrument is a nanoparticle tracking analysis (NTA) system designed to measure the size, concentration, and movement of nanoparticles in liquid suspension. The instrument uses a laser beam to illumine the particles, and a high-sensitivity camera captures the light scattered by the individual particles, allowing their Brownian motion to be tracked and analyzed. The NanoSight NS500 provides detailed information on the size distribution and concentration of nanoparticles in the range of 10 to 2,000 nanometers.
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Lab products found in correlation
2 protocols using nanosigh ns500 instrument
1
Isolation and Characterization of Extracellular Vesicles from Rat Cortical Cultures
2
Isolation and Characterization of Neuronal Extracellular Vesicles
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1 ml of the conditioned medium (MEM supplemented with N2 supplement) of rat primary cortical neurons seeded in a six multi-well plate (300,000 cells/well; 1.5 ml per well), was subsequently centrifuged at 200g and 2,000g for 10 min each to remove dead cells and debris. The supernatant was transferred to a fresh Eppendorf tube and centrifuged at 10,000g and 4°C for 30 min. The supernatant was diluted 1:10 in filtered PBS 1× to obtain a concentration in the range of 1–10 × 108 vesicles/ml, and the concentration of vesicles present in the sample was analyzed by Nanoparticle Tracking Analysis by using a NanoSigh NS500 instrument (Malvern Instruments). The instrument was equipped with a 488-nm laser, a high-sensitivity complementary metal-oxide-semiconductor camera, and a syringe pump. Both the concentration of the vesicles present in the media of cortical neurons at different DIV, as well as the mean size, were analyzed using the nta2.3 software (Malvern Instruments) after filming three 60-s videos.
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