The task of differentiating between Extracellular Vesicles (EVs) and other nanoparticles (e.g. Nanobubbles, protein aggregates, inorganic salt precipitates) typically found within a biological sample can be very challenging.
Here we report a unique technology for rapid determination of membranous particles along with an analysis of multiple EV biomarkers. In spite of the experimental sophistication, the operational costs of our instrument are kept to a minimum.
NTA is a well-established method to analyze the size & concentration of EVs with sizes ranging between 50 – 200 nm (Giebel and Helmbrecht, 2017; Soo et al. 2012). Unfortunately, the standard scattered-light methodology is not able to discriminate EVs from other ultrafine particles in the same size range (see figure 1).
To overcome the limitations of standard NTA, we developed a series of NTA instruments which are optimized not only for scatter light but also for fluorescence NTA (Melzer et al., 2019). Each NTA system is equipped with up to four excitation lasers along with their corresponding emission filters. Our multi-laser systems are driven by software that provides rapid and automated exchange between:
(i) the detection modes (scatter vs. fluorescence),
(ii) the different laser sources, and
(iii) the corresponding emission filters (See instrument page).
Our instruments make it possible to measure the size, concentration, zeta potential, and fluorescence intensity (up to four detection channels), all within a single sample volume (figure 2). Furthermore, our new patent-pending anti-bleaching technology allows the use of fast bleaching fluorophores like e.g. FITC and Calcin.
Giebel B., Helmbrecht C. Methods to Analyze EVs; Methods Mol Biol. 2017;1545:1-20.
Soo C.Y., Song Y., Zheng Y., Campbell E.C., Riches A.C., Gunn-Moore F. and Powis S.J. Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells. Immunology. 2012, 136:192–197. doi:10.1111/j.1365-2567.2012.03569.
Melzer C, Rehn V, Yang Y, Bähre H, von der Ohe J, Hass R. Taxol-Loaded MSC-Derived Exosomes Provide a Therapeutic Vehicle to Target Metastatic Breast Cancer and Other Carcinoma Cells. Cancers (Basel). 2019 Jun 9;11(6). pii: E798. doi: 10.3390/cancers11060798.
After successfully showing C-NTA of double-stained MSC-EVs in our previous study (https://bit.ly/43GG0Yk) and confirming
the results with image flow cytometry, this note describes C-NTA of multi-stained MSC-EVs using a PMX-430 QUATT NTA
system with four lasers.
In this new application note we describe a quick determination of the physical titer of coronaviruses by fluorescence-based Nanoparticle Tracking Analysis and specific spike antibody recognition.
In this report, we describe the quantification of colocalization ratios on double-stained MSC-derived EVs using fluorochrome conjugated antibodies against the
cell surface antigens CD9 and CD41, which also serve as EV marker proteins.
In addition to using Nano Particle Tracking Analysis (NTA) to measurement the size distribution and concentration of EV samples, both Microfluidic Resistive Pulse Sensing (MRPS) and the Single Particle Interference Reflectance Image Sensor (SP-IRIS) methods have been widely used as an alternative means of characterizing EVs. In this note, we relay two specific cases for using the ZetaView® NTA system to achieve relative “high-throughput” analysis of many EV samples, along estimations of throughput for MRPS & SPIRIS methods for an equivalent number of samples; further, we establish realistic estimates for the high cost of ownership for operating MRPS & SP-IRIS systems as a result of the cost of consumables as well as the substantially greater amount of time spent to run the same number of samples.
Along with generally accepted methods are some “tricks of the trade” such as the addition of Tween® or BSA; however, some of those additions are problematic.
Determination of the titer of viruses and bacteriophages is an indispensable key technology in virological research and for diagnostic purposes. Depending on the method used, the measurements are either qualitative or quantitative, very time-consuming, and do not provide information about integrity or aggregation behaviour of the virus particles. Nanoparticle Tracking Analysis with the Particle Metrix ZetaView® instrument allows the user to perform a rapid concentration determination of virus particles. Using the bacteriophage Phi6 as an example, we show how titer, purity and integrity of phage particles can be measured quickly and reliably using the fluorescence detection capability of the Particle Metrix ZetaView® instrument.
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