In recent years there has been an increasing interest in the research of submicrometer particles released to the environment by cells.
Microvesicles (MV) can be defined as a heterogeneous group of materials with distinct names: microparticles, ectosomes, exosomes, exosome like vesicles, shed vesicles, oncosomes all of them give a rough idea of the variety of biological sources. (Exosomes: 50-100 nm; Microvesicles: 100-1000 nm)
Particularly, exosomes have caught the attention of scientist from different areas due to the potential source of information contained inside these particles. This information could be crucial for instance as a new tool to diagnosis purposes or biomarker studies, the investigation of their role as a signal mediators or in different biological processes, or even as a novel treatment strategies. Nevertheless, the weak scattering properties of this material along with their small size turn their analysis into a challenging task using conventional techniques. Moreover, knowing the particle concentration in the sample is as important as the size or particle size distribution of the sample.
The isolation of exosomes is the key stage, although the sample analysis has also to be considered as an important stage. For instance, you could waste much time to isolate microvesicles but if the selected technique to analyze the sample presents limitations, you could not take advantage of all the potential information.
One of the most employed techniques is conventional flow cytometry, although the size of particles aimed to analysis could be problematic, that is, the technique could underestimate the concentration of vesicles. Another important point to be taken into account is the heterogeneous refractive index of the particles and their unknown exact value. Therefore, a reliable estimation of the particle refractive index is required to get a good conversion from the optical scatter signal to size. Furthermore, a reference material is needed to calibrate the instrument having a refractive index as close as possible to the real sample (usually polystyrene beads are employed). Normally the minimum size detectable using flow cytometry is above 200-300 nm depending on the type of instrument
At Nanovex Biotechnologies facilities there is available a Nanosight LM10 instrument equipped with a laser of 405 nm which could perform Nanoparticle Tracking Analysis (NTA) of particles below 1 µm based on the analysis of Brownian motion.
NTA allows monitoring the hydrodynamic diameter of the exosomes straightaway in solution, besides obtaining at the same time an estimation of particle concentration. NTA can provide both sample concentration and size, besides it has a high resolving power. Moreover, NTA analysis is not intensity-weighted to larger particles, but it is a technique in which the size of the particles is obtained from the measurement of the dynamic Brownian motion, the information is not extracted from the amount of light scattered by the particles in contrast to the basis of flow cytometry. Another important advantage of NTA is the fluorescence capability of the system, which enables to label specifically the particles.
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