Rview–Body fluids contain cell-derived extracellular vesicles (EVs), which can suppress and improve the immune method and contribute to the improvement of systemic autoimmune illness. To investigate the role of EVs in immunology, flow cytometry (FCM) could be the technologies of choice for determining the concentration of EVs expressing specific antigens. However, due to the fact EVs are substantially smaller sized and dimmer than cells, EV detection and data interpretation are challenging, top to misconceptions. One example is, on the one particular hand, it really is generally overlooked that FCM does not Neurotrophin-3 Proteins Recombinant Proteins detect the complete size array of EVs. Alternatively, it’s normally incorrectly thought that FCM is incapable of detecting EVs smaller than the wavelength of light. The aim of this section would be to briefly address some frequent misconceptions of EV FCM and to provide recommendations to stop potential artifacts arising from APRIL Proteins Biological Activity sample preparation, staining, assay protocol, and data analysis.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptEur J Immunol. Author manuscript; obtainable in PMC 2020 July ten.Cossarizza et al.Page4.two Introduction–Blood as well as other body fluids contain cell-derived extracellular vesicles (EVs), which is the umbrella term for all varieties of cell-derived vesicles which includes microvesicles and exosomes. Figure 34A shows a transmission electron microscopy (TEM) image of EVs, which could be seen as subcellular cargo containers transporting biomolecules, such as transmembrane receptors and genetic information, to target cells. From an immunological point of view, EVs are interesting mainly because EVs transport ligands which can suppress the immune system, improve the immune response by antigen presentation, and contribute for the improvement of systemic autoimmune disease [250]. See also Chapter V Section two Organisms, cells, organelles, chromosomes, and extracellular vesicles. 4.three EV analyses by flow cytometry–EV FCM is especially valuable to establish the number concentration of certain EV forms in (body) fluids. However, the compact size of EVs complicates FCM analyses. Figure 34B shows a size distribution of EVs from human urine based on TEM and resistive pulse sensing. Common properties of an EV size distribution are a smallest diameter of 50 nm, a peak below 400 nm, and a decreasing concentration with rising diameter for EVs bigger than the peak diameter [251, 25557]. Therefore, most EVs are smaller sized than the illumination wavelength () commonly utilized in FCM. A basic misconception is that EVs smaller sized than the illumination wavelength cannot be detected by FCM. Based on the Rayleigh criterion, EVs smaller than roughly half the illumination wavelength cannot be distinguished by classical light microscopy [258]. Having said that, even the smallest EVs do scatter light of longer wavelengths and can be detected by FCM, provided that single EVs are illuminated along with the flow cytometer has nanoparticle sensitivity. In practice, most flow cytometers don’t have nanoparticle sensitivity: a current standardization study showed that only six of 46 tested flow cytometers within the field had been capable to detect EVs as small as 300 nm [259]. To explain how the size of EVs have an effect on their light scattering intensity, Fig. 34C shows the FSC measured by FCM (A60-Micro, Apogee Flow Systems, UK) versus the diameter of plateletderived EVs and platelets exposing integrin three (CD61) from human plasma and, for comparison, of polystyrene particles. The diameters of EVs, platelets, and polystyrene portion.