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Theranostics 2017Exosomes are one type of membrane vesicles secreted into extracellular space by most types of cells. In addition to performing many biological functions particularly in... (Review)
Review
Exosomes are one type of membrane vesicles secreted into extracellular space by most types of cells. In addition to performing many biological functions particularly in cell-cell communication, cumulative evidence has suggested that several biological entities in exosomes like proteins and microRNAs are closely associated with the pathogenesis of most human malignancies and they may serve as invaluable biomarkers for disease diagnosis, prognosis, and therapy. This provides a commanding impetus and growing demands for simple, efficient, and affordable techniques to isolate exosomes. Capitalizing on the physicochemical and biochemical properties of exosomes, a number of techniques have been developed for the isolation of exosomes. This article summarizes the advances in exosome isolation techniques with an emphasis on their isolation mechanism, performance, challenges, and prospects. We hope that this article will provide an overview of exosome isolation techniques, opening up new perspectives towards the development more innovative strategies and devices for more time saving, cost effective, and efficient isolations of exosomes from a wide range of biological matrices.
Topics: Cytological Techniques; Exosomes; Humans
PubMed: 28255367
DOI: 10.7150/thno.18133 -
Journal of Laboratory Automation Apr 2015Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell... (Review)
Review
Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial and epithelial monolayers. TEER values are strong indicators of the integrity of the cellular barriers before they are evaluated for transport of drugs or chemicals. TEER measurements can be performed in real time without cell damage and generally are based on measuring ohmic resistance or measuring impedance across a wide spectrum of frequencies. The measurements for various cell types have been reported with commercially available measurement systems and also with custom-built microfluidic implementations. Some of the barrier models that have been widely characterized using TEER include the blood-brain barrier (BBB), gastrointestinal (GI) tract, and pulmonary models. Variations in these values can arise due to factors such as temperature, medium formulation, and passage number of cells. The aim of this article is to review the different TEER measurement techniques and analyze their strengths and weaknesses, determine the significance of TEER in drug toxicity studies, examine the various in vitro models and microfluidic organs-on-chips implementations using TEER measurements in some widely studied barrier models (BBB, GI tract, and pulmonary), and discuss the various factors that can affect TEER measurements.
Topics: Animals; Cytological Techniques; Electric Impedance; Electrophysiological Phenomena; Endothelial Cells; Epithelial Cells; Humans
PubMed: 25586998
DOI: 10.1177/2211068214561025 -
Journal of Visualized Experiments : JoVE Jun 2014Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods...
Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.
Topics: Animals; Cell Movement; Chemotaxis; Cytological Techniques; Melanoma, Experimental; Mice; NIH 3T3 Cells; Neoplasm Invasiveness
PubMed: 24962652
DOI: 10.3791/51046 -
Journal of Visualized Experiments : JoVE Jul 2012Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons,...
Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.
Topics: Animals; Cells, Cultured; Cytological Techniques; Dissection; Embryo, Mammalian; Female; Hippocampus; Mice; Mice, Inbred C57BL; Neurons; Pregnancy; Rats
PubMed: 22871921
DOI: 10.3791/3634 -
Nature Protocols Feb 2015Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop...
Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, thus allowing the study of fibrogenesis by genetic approaches in transgenic mice. However, mouse HSC isolation is commonly hampered by low yield and purity. Here we present an easy-to-perform protocol for high-purity and high-yield isolation of quiescent and activated HSCs in mice, based on retrograde pronase-collagenase perfusion of the liver and subsequent density-gradient centrifugation. We describe an optional add-on protocol for ultrapure HSC isolation from normal and fibrotic livers via subsequent flow cytometric sorting, thus providing a validated method to determine gene expression changes during HSC activation devoid of cell culture artifacts or contamination with other cells. The described isolation procedure takes ∼4 h to complete.
Topics: Animals; Centrifugation, Density Gradient; Collagenases; Cytological Techniques; Flow Cytometry; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Mice, Transgenic; Perfusion
PubMed: 25612230
DOI: 10.1038/nprot.2015.017 -
The Journal of Cell Biology Sep 2014Systems cell biology melds high-throughput experimentation with quantitative analysis and modeling to understand many critical processes that contribute to cellular... (Review)
Review
Systems cell biology melds high-throughput experimentation with quantitative analysis and modeling to understand many critical processes that contribute to cellular organization and dynamics. Recently, there have been several advances in technology and in the application of modeling approaches that enable the exploration of the dynamic properties of cells. Merging technology and computation offers an opportunity to objectively address unsolved cellular mechanisms, and has revealed emergent properties and helped to gain a more comprehensive and fundamental understanding of cell biology.
Topics: Biomedical Research; Cell Biology; Cytological Techniques; High-Throughput Nucleotide Sequencing; Models, Biological; Proteomics; Research Design; Systems Biology
PubMed: 25225336
DOI: 10.1083/jcb.201405027 -
Cytometry. Part a : the Journal of the... Sep 2016
Topics: Animals; Cell Separation; Cytological Techniques; Flow Cytometry; Graft vs Host Disease; Humans
PubMed: 27657547
DOI: 10.1002/cyto.a.22976 -
Cytopathology : Official Journal of the... Sep 2021The application of next generation sequencing (NGS) technology to cytological samples has significantly modified molecular cytopathology practice. Cytological samples... (Review)
Review
The application of next generation sequencing (NGS) technology to cytological samples has significantly modified molecular cytopathology practice. Cytological samples represent a valid source of high-quality DNA for NGS analysis, especially for predicting patients' response to targeted treatments and for refining the risk of malignancy in indeterminate cytological diagnoses. However, several pre-analytical factors may influence the reliability of NGS clinical analysis. Here, we briefly review the challenges of NGS in cytology practice, focusing on those pre-analytical factors that may negatively affect NGS success rates and routine diagnostic applications. Finally, we address the future directions of the field.
Topics: Cytodiagnosis; Cytological Techniques; High-Throughput Nucleotide Sequencing; Humans; Neoplasms
PubMed: 33792981
DOI: 10.1111/cyt.12974 -
BMC Biology Sep 2018Array tomography encompasses light and electron microscopy modalities that offer unparalleled opportunities to explore three-dimensional cellular architectures in...
Array tomography encompasses light and electron microscopy modalities that offer unparalleled opportunities to explore three-dimensional cellular architectures in extremely fine structural and molecular detail. Fluorescence array tomography achieves much higher resolution and molecular multiplexing than most other fluorescence microscopy methods, while electron array tomography can capture three-dimensional ultrastructure much more easily and rapidly than traditional serial-section electron microscopy methods. A correlative fluorescence/electron microscopy mode of array tomography furthermore offers a unique capacity to merge the molecular discrimination strengths of multichannel fluorescence microscopy with the ultrastructural imaging strengths of electron microscopy. This essay samples the first decade of array tomography, highlighting applications in neuroscience.
Topics: Cytological Techniques; Electron Microscope Tomography; Imaging, Three-Dimensional; Microscopy, Electron, Scanning; Microscopy, Fluorescence
PubMed: 30189863
DOI: 10.1186/s12915-018-0560-1 -
The Veterinary Clinics of North... Jan 2017Synovial fluid analysis is a key component of the minimum database needed to diagnose and manage primary and secondary articular joint disorders. Unfortunately,... (Review)
Review
Synovial fluid analysis is a key component of the minimum database needed to diagnose and manage primary and secondary articular joint disorders. Unfortunately, preanalytical variables can drastically alter samples submitted for evaluation to veterinary laboratories and it is considered the stage at which most laboratory error occurs. This article addresses common sources of preanalytical variability and error that are seen in veterinary medicine. With consistent quality control and reporting of specimens, downstream clinical decision making and management of patients can be accelerated and improved.
Topics: Animals; Cytological Techniques; Specimen Handling; Synovial Fluid
PubMed: 27720280
DOI: 10.1016/j.cvsm.2016.07.007