Current Protocols in Cell Biology最新文献

Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus in the family Flaviviridae with specific hepatotropism. HCV poses a significant health burden worldwide, frequently causing chronic infections associated with progressive liver damage and various extrahepatic manifestations. In recent years, the development of several permissive cell culture (HCVcc) systems has allowed for in vitro propagation of HCV, study of the viral life cycle and virus–host interactions, and identification of novel antivirals. Here we describe the use of human hepatoma cell lines Huh7 and HepG2/CD81/miR-122, as well as primary human hepatocytes (PHHs), for HCV infection and propagation. We also provide protocols for the quantitative analysis of intracellular and extracellular HCV RNA and detection of HCV core protein expression by immunostaining. In addition, we describe methods to manipulate cellular gene expression, including transfection of small interfering RNAs (siRNAs) targeting HCV host factors or overexpressing cellular microRNAs in hepatocytes, to assess their effects on productive HCV infection and liver pathogenesis. © 2018 by John Wiley & Sons, Inc.

Mast cells have been identified as resident cells of human placental tissue by immunohistological procedures, suggesting that they may play a role in pregnancy. However, the study of placental mast cells requires their isolation. Here, we describe a procedure to isolate placental mast cells from placenta of healthy women. At-term placentas were recovered, and small pieces were excised. After extensive washing, they were digested using enzyme, and cell preparations were centrifuged on a Percoll density gradient. A double positive selection was then performed using magnetic beads covered with CD117 and IgE antibodies. The purity of isolated mast cells was finally analyzed by flow cytometry, and was nearly 90%, demonstrating that our protocol was convenient to obtain fresh placental mast cells in sufficient quantity for research investigations. © 2018 by John Wiley & Sons, Inc.

Detection of reactive oxygen species (ROS) in bacteria has been limited to bulk biochemical assays. Although they are powerful and quantitative tools to understand the overall production of ROS in E. coli, such assays provide limited spatial and temporal information when correlating cellular phenotype with perturbations such as antibiotics or other treatments. We have developed single-cell, time-lapse assays to detect ROS in live E. coli. The assays utilize flow systems on a fluorescence microscope to correlate symptoms aroused from biological or chemical perturbations with the in situ detection of ROS. ROS is detected by fluorogenic dyes that accumulate inside the cell, allowing detection of ROS in single cells in both homogeneous and heterogeneous samples using CellROX Green and Amplex® Red/APEX2. © 2018 by John Wiley & Sons, Inc.

Proteoglycans can be difficult molecules to isolate and analyze due to large mass, charge, and tendency to aggregate or form macromolecular complexes. This unit describes detailed methods for purification of matrix, cell surface, and cytoskeleton-linked proteoglycans. Methods for analysis of glycoaminoglycan size and type and of core protein species are described. © 2018 by John Wiley & Sons, Inc.

Multiple sclerosis (MS) is an autoimmune disease that involves an immune-mediated inflammatory response in the central nervous system and optic nerve resulting in demyelination and neural degeneration, the cause of which is unknown. The adult central nervous system has the capacity to remyelinate axons by generating new oligodendrocytes (OLs). To identify clinical candidate compounds that may promote remyelination, we have developed a high-throughput screening (HTS) assay to identify compounds that promote the differentiation of oligodendrocyte precursor cells (OPCs) into OLs. Using acutely dissociated and purified rat OPCs coupled with immunofluorescent image quantification, we have developed an OL differentiation assay. Building on OPC culturing techniques developed over the past 30 years, we have scaled up the isolation and purification process to generate sufficient quantities for HTS. We then describe the use of these acutely derived OPCs in an assay designed to identify compounds that promote differentiation into OLs. We have validated this assay with a known promoter of differentiation, thyroid hormone, and subsequently used the assay to screen the NIH clinical collection library (Lariosa-Willingham, et al., 2016). © 2018 by John Wiley & Sons, Inc.

The mitotic chromosome, which is composed of a pair of sister chromatids, is a large macromolecular assembly that ensures faithful transmission of genetic information into daughter cells. Despite its fundamental importance, how a nucleosome fiber is folded and assembled into a large-scale chromatid structure remains poorly understood. To address this question, we have established a biochemically tractable system in which mitotic chromatids can be reconstituted in vitro by mixing a simple substrate (sperm nucleus) and a limited number of purified factors. The minimum set of required factors includes core histones, three histone chaperones, topoisomerase II, and condensin I. In this article, we describe a set of protocols for the preparation of key reagents and the setup of reconstitution reactions. We believe that this classical approach of biochemical reconstitution will be of great help to dissect the mechanisms of action of individual factors during mitotic chromatid assembly and to assess the contribution of nucleosome dynamics to this process from a fresh angle. © 2018 by John Wiley & Sons, Inc.

This protocol describes how to apply appropriate pharmacological controls to induce mitochondrial fusion or fission in studies of mitochondria morphology for four different mammalian cell types, HepG2 human liver hepatocellular carcinoma cells, MCF7 human breast adenocarcinoma cells, HEK293 human embryonic kidney cells, and collagen sandwich culture of primary rat hepatocytes. The protocol provides methods of treating cells with these pharmacological controls, staining mitochondria with commercially available MitoTracker Green and TMRE dyes, and imaging the mitochondrial morphology in live cells using a confocal fluorescent microscope. It also describes the cell culture methods needed for this protocol. © 2018 by John Wiley & Sons, Inc.

Accurate modeling of human neuronal cell biology has been a long-standing challenge. However, methods to differentiate human induced pluripotent stem cells (iPSCs) to neurons have recently provided experimentally tractable cell models. Numerous methods that use small molecules to direct iPSCs into neuronal lineages have arisen in recent years. Unfortunately, these methods entail numerous challenges, including poor efficiency, variable cell type heterogeneity, and lengthy, expensive differentiation procedures. We recently developed a new method to generate stable transgenic lines of human iPSCs with doxycycline-inducible transcription factors at safe-harbor loci. Using a simple two-step protocol, these lines can be inducibly differentiated into either cortical (i³Neurons) or lower motor neurons (i³LMN) in a rapid, efficient, and scalable manner (Wang et al., 2017). In this manuscript, we describe a set of protocols to assist investigators in the culture and genetic engineering of iPSC lines to enable transcription factor–mediated differentiation of iPSCs into i³Neurons or i³LMNs, and we present neuronal culture conditions for various experimental applications. © 2018 by John Wiley & Sons, Inc.

The method described here enables rapid bacteriophage isolation and enrichment of host-specific bacteriophages from an environmental sample. This is achieved by using a simple 0.45-µm Millipore membrane where a specific host is immobilized on the membrane and a sample suspected of containing bacteriophages is exposed to the immobilized cells with the help of a membrane filtration unit. This filtration step facilitates host-specific interaction of bacteriophages with the host and maximization of this interaction using a classic membrane filtration method. Under the effect of vacuum from a vacuum pump, a filter assembly provides a chance for every bacteriophage in the sample to interact with the specific host on the membrane filter. Our technique allows retaining specific bacteriophages on the membrane along with its host cells via adsorption; these adsorbed bacteriophages (along with their hosts) on a filter disc are then enriched in regular nutritive broth, tryptone soya broth (TSB), by incubation. With help of a plaque assay method, host-specific phages of various bacterial species can be isolated, segregated, and enriched. © 2018 by John Wiley & Sons, Inc.