A protocol for exosome isolation and characterization_ evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods (pdf download available)

Exosomes are 40-150 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of tumorigenic proteins, mRNA and miRNA. Database 1 to 1 relationship Exosomes are important regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest their importance for diagnostic and therapeutic applications, and as drug delivery vehicles. Database testing Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques.

Database graph In this chapter, we reveal the protocol and key insights into the isolation, purification and characterization of exosomes, distinct from shed microvesicles and apoptotic blebs. Database naming conventions Using the colorectal cancer cell line LIM1863 as a cell model, a comprehensive evaluation of exosome isolation methods including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM-coated magnetic beads (IAC-Exos) were examined. Database entity All exosome isolation methodologies contained 40-150 nm vesicles based on electron microscopy, and positive for exosome markers (Alix, TSG101, HSP70) based on immunoblotting. Database developer This protocol employed a proteomic profiling approach to characterize the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method in exosome isolation. Data recovery plan Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, IAC-Exos was shown to be the most effective method to isolate exosomes. Data recovery kansas city However, the use of density-based separation (DG-Exos) provides significant advantages for exosome isolation when the use of immunoaffinity capture is limited (due to antibody availability and suitability of exosome markers).

[Show abstract] [Hide abstract] ABSTRACT: Extracellular vesicles (EVs), including exosomes (30-150 nm) and microvesicles (100-1,500 nm) play important roles in mediating cell-cell communication. N k database Such particles package distinct cargo elements including lipids, proteins, mRNAs, miRNAs and DNA, that vary depending on the cell of origin and its phenotype. Data recovery 2016 This cargo can be horizontally transferred to target cells where its components can reprogram the recipient cell to modify its function. 510 k database fda EVs have been identified within the uterine cavity of women, sheep and mice, where they contribute to the microenvironment of sperm transport, and of blastocyst and endometrial preparation for implantation. Database programmer It is likely that exosomes and microvesicles carry different cargo and coordinate different roles in this intrauterine environment. Data recovery osx Understanding and defining these subtypes of EVs is important for future functional studies and clinical translation. Database integrity Here we critically review the various purification and validation procedures for EV analysis, discuss what is known of endometrial-derived exosome cargo, and that they are hormonally regulated. Database backup The current knowledge of the functions of uterine exosomes, with respect to sperm transport and function, and of their actions on trophectodermal cells to promote implantation are summarized and evaluated in their physiological context. Hollywood u database Given the potential importance of this form of cell-cell interactions within the reproductive tract, the critical issues discussed will guide new insights in this rapidly expanding field.