The immunocapture-based ELISA for extracellular vesicles (EVs)/exosomes, originally described in 2009 by Logozzi and colleagues, allows to capture, detect, characterize and quantify extracellular vesicles in both human body fluids and cell culture supernatants. It is based on the use of two antibodies directed one against a typical exosomal housekeeping protein and the second against either another exosomal housekeeping protein or a potential disease marker the first antibody is used for the capture of exosomes, the second for the quantification and characterization of the captured vesicles. In fact, with this method it is possible both to characterize and count exosomes and to detect the presence of disease, including tumor, biomarkers. this website This needs of course to preliminary obtain an EVs purification from the clinical sample; the most agreed method to get to an EVs purification is the repeated rounds of ultracentrifugation, that, while far to be perfect, is the methodological approach allowing to not exclude Ee.Fluorescent labeling of extracellular vesicles (EVs) enables studying their uptake and influence on individual cells, biodistribution as well as facilitates their characterization using high-resolution flow cytometry at a single EV level. Here we describe the importance of fluorescent labeling, the available fluorescent dyes and labeling approaches, the characteristics of an ideal dye, and the available techniques for post-labeling purification. We discuss the importance of preserving the size of EVs for uptake, biodistribution, and characterization studies and focus on the effect of common lipophilic PKH and luminal CFSE dyes on the size of EVs. Lastly, we present an example protocol for luminal labeling of EVs and characterization of the effect of labeling on the size of EVs using nanoparticles tracking analysis (NTA).Extracellular vesicles (EVs) have been recognized as relevant players in cell-cell communication. To fully explore their potential as carriers of biological information in clinical settings, protocols capable of dealing with minute amounts of proteins, lipids, and nucleic acids present in their cargo are a requirement. Here we delve into a protocol to decipher the total transcriptome of EVs, from undetectable amounts of EVs-derived RNA from clinical samples.The liquid biopsy preserves a noninvasive technique to analyze promising biomarkers in cell-free bodyfluids, mainly in cell-free plasma. The most cells secrete extracellular vesicles into the extracellular place which can be isolated, analyzed easily due to the wide range of different protocols and commercial kits. The mitochondrial DNA isolated from biofluids can serve as new view in early diagnosis of various diseases (e.g. cancers, cardiovascular diseases). In this chapter, possible protocols of mitochondrial DNA copy number quantification are discussed presenting some ways to determine the mtDNA level of extracellular vesicles in different diseases.Exosomes are extracellular vesicles secreted by cells and involved in intercellular communications among close and distant cells. Exosomes encapsulate and carry biomolecules as cargo to the recipient cells. They contain nucleic acids (DNA, RNA, microRNA) proteins and lipids. Each exosomal components may be isolated and be studied by specific techniques. In this chapter, different methods will be described to isolate DNA from exosomes, since it is important in shaping the response of the recipient cells following the exosome uptake in multiple scenarios, including physiological and pathological conditions. Moreover, the exosomal DNA may be a novel biomarker for diagnosis, disease progression and patient’s treatment response.Molecular imaging methods are powerful tools for gaining insight into the cellular organization of living cells. To understand the biogenesis and uptake of extracellular vesicles (EVs) as well as to engineer cell-derived vesicles for targeted drug delivery and therapy, genetic labeling with fluorescent proteins has increasingly been used to determine the structures, locations, and dynamics of EVs in vitro and in vivo. Here, we report a genetic method for the stable labeling of EVs to study their biogenesis and uptake in living human cells. Fusing a green fluorescent protein (GFP) with either the endogenous CD63 (CD63-GFP) or a vesicular stomatitis virus envelope glycoprotein, VSVG (VSVG-GFP), we successfully obtained distinct fluorescence signals in the cytoplasm, revealing the biogenesis of EVs in post-transfected cells. We describe experimental procedures in detail for EV isolation, imaging, and cellular uptake using both confocal microscopy and flow cytometry. We also provide a perspective on how genetic labeling methods can be used to study EV biology, characterization of engineered EVs, and development of EV-based nano-medicine.There are perinatal characteristics, such as gestational age, reproducibly associated with the risk for pediatric asthma. Identification of biologic processes influenced by these characteristics could facilitate risk stratification or new therapeutic targets. We hypothesized that transcriptional changes associated with multiple epidemiologic risk factors would be mediators of pediatric asthma risk. Using publicly available transcriptomic data from cord blood mononuclear cells, transcription of genes involved in myeloid differentiation was observed to be inversely associated with a pediatric asthma risk stratification based on multiple perinatal risk factors. This gene signature was validated in an independent prospective cohort and was specifically associated with genes localizing to neutrophil-specific granules. Further validation demonstrated that umbilical cord blood serum concentration of PGLYRP-1, a specific granule protein, was inversely associated with mid-childhood current asthma and early-teen FEV1/FVCx100. Thus, neutrophil-specific granule abundance at birth predicts risk for pediatric asthma and pulmonary function in adolescence.Since the discovery of muscle in the 19th century, myosins as molecular motors have been extensively studied. However, in the last decade, a new functional super-relaxed (SRX) state of myosin has been discovered, which has a 10-fold slower ATP turnover rate than the already-known non-actin-bound, disordered relaxed (DRX) state. These two states are in dynamic equilibrium under resting muscle conditions and are thought to be significant contributors to adaptive thermogenesis in skeletal muscle and can act as a reserve pool that may be recruited when there is a sustained demand for increased cardiac muscle power. This report provides an evolutionary perspective of how striated muscle contraction is regulated by modulating this myosin DRX↔SRX state equilibrium. We further discuss this equilibrium with respect to different physiological and pathophysiological perturbations, including insults causing hypertrophic cardiomyopathy, and small-molecule effectors that modulate muscle contractility in diseased pathology.