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Current Protocols in Cell Biology

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https://www.readbyqxmd.com/read/30178917/genome-editing-in-mice-using-crispr-cas9-technology
#1
Bradford Hall, Andrew Cho, Advait Limaye, Kyoungin Cho, Jaspal Khillan, Ashok B Kulkarni
CRISPR/Cas9 technology has revolutionized genome editing in mice, allowing for simple and rapid development of knockouts and knockins. CRISPR relies on small guide RNAs that direct the RNA-guided nuclease Cas9 to a designated genomic site using ∼20 bp of corresponding sequence. Cas9 then creates a double-strand break in the targeted loci that is either patched in an error-prone fashion to produce a frame-shift mutation, a knockout, or is repaired by recombination with donor DNA containing homology arms, a knockin...
September 4, 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/30102462/triggered-cell-cell-fusion-assay-for-cytoplasmic-and-organelle-intermixing-studies
#2
Daniel Feliciano, Jonathon Nixon-Abell, Jennifer Lippincott-Schwartz
Different multicellular organisms undergo cell-cell fusion to form functional syncytia that support specialized functions necessary for proper development and survival. For years, monitoring the structural consequences of this process using live-cell imaging has been challenging due to the unpredictable timing of cell fusion events in tissue systems. Here we present a triggered vesicular stomatitis virus G-protein (VSV-G)-mediated cell-cell fusion assay that can be used to synchronize fusion between cells. This allows the study of cellular changes that occur during cell fusion...
August 13, 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/30085418/quantifying-the-proteolytic-cleavage-of-plasma-membrane-proteins-in-living-cells
#3
Martino Calamai, Francesco Saverio Pavone
The standard approach to study the activity of proteases consists of lysing cells and measuring the changes in the fluorescence properties of a synthetic substrate after cleavage in vitro. Here, a general protocol that uses a bi-fluorescent chimeric construct of a known substrate protein that follows the proteolytic processing in living cells is described. This approach is useful, in particular, to search for pharmacological conditions altering the cleavage rate of a certain protease, or to investigate the biological factors influencing a certain proteolytic mechanism...
August 7, 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/30085404/imaging-membrane-repair-in-single-cells-using-correlative-light-and-electron-microscopy
#4
Coralie Croissant, Flora Bouvet, Sisareuth Tan, Anthony Bouter
Many cells possess the ability to repair plasma membrane disruption in physiological conditions. Growing evidence indicates a correlation between membrane repair and many human diseases. For example, a negative correlation is observed in muscle where failure to reseal sarcolemma may contribute to the development of muscular dystrophies. Instead, a positive correlation is observed in cancer cells where membrane repair may be exacerbated during metastasis. Here we describe a protocol that combines laser technology for membrane damage, immunostaining with gold nanoparticles and imaging by fluorescence microscopy and transmission electron microscopy (TEM), which allows the characterization of the molecular machinery involved in membrane repair...
August 7, 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/30070431/expansion-microscopy-protocols-for-imaging-proteins-and-rna-in-cells-and-tissues
#5
Shoh M Asano, Ruixuan Gao, Asmamaw T Wassie, Paul W Tillberg, Fei Chen, Edward S Boyden
Expansion microscopy (ExM) is a recently developed technique that enables nanoscale-resolution imaging of preserved cells and tissues on conventional diffraction-limited microscopes via isotropic physical expansion of the specimens before imaging. In ExM, biomolecules and/or fluorescent labels in the specimen are linked to a dense, expandable polymer matrix synthesized evenly throughout the specimen, which undergoes 3-dimensional expansion by ∼4.5 fold linearly when immersed in water. Since our first report, versions of ExM optimized for visualization of proteins, RNA, and other biomolecules have emerged...
August 2, 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/30028910/single-cell-time-lapse-reactive-oxygen-species-detection-in-e-coli
#6
Zhilin Yang, Heejun Choi
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...
July 20, 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/30133963/preparation-and-culture-of-human-liver-resident-immune-cells
#7
Hao Feng, Baochi Ou, Wei Dong, Wolfgang E Thasler
Co-cultivation of tumor cells and liver resident immune cells or other non-parenchymal cells (NPCs) from the same donor is important for the study of cancer metastasis. So far, little is known about the mechanism of tumor cell or pathogen clearance, leukocyte infiltration, and immune cell recruitment in the human liver. To investigate these processes in vitro, the use of primary human hepatocytes and non-parenchymal cell, especially immune cell, co-culture systems play essential roles in the establishment of cell-cell and cell-extracellular matrix communications similar to native liver tissues...
September 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/30126079/cell-adhesion-activity-of-peptides-conjugated-to-polysaccharides
#8
Kentaro Hozumi, Motoyoshi Nomizu
Many cell-adhesive peptides have been identified from extracellular matrix (ECM) proteins, such as collagen, fibronectin, laminin, and vitronectin. ECM proteins have various cell-adhesive sequences. Most peptides demonstrate cell-adhesive activity when simply coated on a tissue culture plate, but solubility, conformation, and coating efficiency of the peptides can significantly alter their biological function. Evaluation of peptide cell-adhesive activity using peptide-conjugated polysaccharide constructs is a useful strategy for overcoming peptide solubility and conformation problems...
September 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/30058773/hepatitis-c-virus-infection-of-cultured-cells-and-primary-human-hepatocytes
#9
Brianna Lowey, Qisheng Li
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...
September 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/30044540/isolation-of-human-placental-mast-cells
#10
Soraya Mezouar, Amira Ben Amara, Joana Vitte, Jean-Louis Mege
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...
September 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29927085/proteoglycan-isolation-and-analysis
#11
Anne Woods, John R Couchman
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.
September 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29924489/reconstitution-of-mitotic-chromatids-in-vitro
#12
Keishi Shintomi, Tatsuya Hirano
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...
June 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29924488/transcription-factor-mediated-differentiation-of-human-ipscs-into-neurons
#13
Michael S Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E Ward
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...
June 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29924487/using-acutely-dissociated-and-purified-oligodendrocyte-precursor-cells-for-high-throughput-drug-screening-to-identify-compounds-that-promote-oligodendrocyte-differentiation
#14
Karen Lariosa-Willingham, Dmitri Leonoudakis
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...
June 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29924486/monitoring-the-effects-of-pharmacological-reagents-on-mitochondrial-morphology
#15
Dong Fu, Jennifer Lippincott-Schwartz
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...
June 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29924485/isolation-and-enrichment-of-bacteriophages-by-membrane-filtration-immobilization-technique
#16
Gaurav S Ghugare, Vijay D Nimkande, Krishna Khairnar
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...
June 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29924484/multispectral-live-cell-imaging
#17
Sarah Cohen, Alex M Valm, Jennifer Lippincott-Schwartz
Fluorescent proteins and vital dyes are invaluable tools for studying dynamic processes within living cells. However, the ability to distinguish more than a few different fluorescent reporters in a single sample is limited by the spectral overlap of available fluorophores. Here, we present a protocol for imaging live cells labeled with six fluorophores simultaneously. A confocal microscope with a spectral detector is used to acquire images, and linear unmixing algorithms are applied to identify the fluorophores present in each pixel of the image...
June 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29924483/self-pressurized-rapid-freezing-as-cryo-fixation-method-for-electron-microscopy-and-cryopreservation-of-living-cells
#18
Jan Huebinger, Markus Grabenbauer
Reduction or complete prevention of ice crystal formation during freezing of biological specimens is mandatory for two important biological applications: (1) cryopreservation of living cells or tissues for long-term storage, and (2) cryo-fixation for ultrastructural investigations by electron microscopy. Here, a protocol that is fast, easy-to-use, and suitable for both cryo-fixation and cryopreservation is described. Samples are rapidly cooled in tightly sealed metal tubes of high thermal diffusivity and then plunged into a liquid cryogen...
June 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/30040188/elimination-of-mitochondrial-dna-from-mammalian-cells
#19
Natalya Khozhukhar, Domenico Spadafora, Yelitza Rodriguez, Mikhail Alexeyev
To cope with DNA damage, mitochondria developed a pathway by which severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules are abandoned and degraded, and new molecules are resynthesized using intact templates, if available. In this unit, we describe a method that harnesses this pathway to completely eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1). We also provide an alternate protocol for mtDNA depletion using combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC)...
March 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/30040186/a-streamlined-sample-preparation-method-for-mass-spectrometric-analysis
#20
Yun Xiong, Ying Zhang, Jun Yao, Guoquan Yan, Haojie Lu
Mass spectrometry-based proteomic technology experienced remarkable advancement in the past decades. However, their application was still hampered by the complexity of sample preparation. Conventional strategies for sample preparation incorporate multiple time-consuming steps, including cell lysis, protein extraction, protease cleavage, and desalting. Thus, we explored a simplified method (the cell-absorb method) during which living cells were absorbed into vacuum-dried polyacrylamide gel and directly digested in gel into peptides for subsequent LC-MS/MS analysis...
March 2018: Current Protocols in Cell Biology
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