ИСТИНА

Introduction: In this report, we summarize our experience of nanoparticle tracking analysis (NTA) measurements of various samples including inorganic nanoparticles, liposomes and extracellular vesicles to get the quantitative and reproducible results for particle sizes and concentrations. Methods: Measurements were made with Nanosight LM10 instrument (405 nm laser and EMCCD camera) and Nanosight NS500 instrument (532 nm laser and EMCCD camera). Results: The general idea of proper work with NTA is that all NTA samples are extremely diluted solutions. Typical concentration for NTA of 10^8 particles/ml corresponds to 1.7 pM (picomoles of particles per litre). Two main concerns should be kept in mind: avoiding losing the substance and avoiding contamination. Particle free water is a key factor for good NTA measurement. Water of a good and sustained quality could be prepared with well-maintained MilliQ (or analogous) systems fed with preliminary purified water. Large amounts of particle free buffers could be prepared by slow filtration of 10× buffer concentrate via sterile 0.22 mkm syringe filter with Nylon membrane (which not only retain particles >220 nm, but also adsorbs smaller ones) followed by dilution of this concentrate by particle free water. Intense shaking/vortexing of diluted samples should be avoided as it causes the release of particles from test tube/vial walls with sizes 30–600 nm and concentrations up to 10^9 part./ml. Both polypropylene (PP) and glass test tube are affected. The effect is batch dependent: 2 different batches of the same PP test tubes release different concentrations of particles during intense shaking/vortexing. Thus samples for NTA should be mixed gently. If shaking/vortexing is unavoidable, it should be done with concentrated solutions (>10^12 part./ml) with a blank buffer control to estimate contamination level. The last frequent mistake is tips/tubes handling. Touching tips with gloves or opening the test tubes in such a way that the gloved thumb touches the inner part of the tube cap causes the contamination as well. Conclusions: A proper design of the experiment is vital for correct and reproducible NTA measurements. Wrong sample preparation and handling could lead to highly distorted size distribution and concentration of particles being studied. Journal of Extracellular Vesicles 2015, 4: 28165 - http://dx.doi.org/10.3402/jev.v4.28165