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Current Protocols in Cell Biology

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https://www.readbyqxmd.com/read/29927085/proteoglycan-isolation-and-analysis
#1
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.
June 21, 2018: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29924489/reconstitution-of-mitotic-chromatids-in-vitro
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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/29227555/combining-fluorescence-and-bioluminescence-microscopy-to-study-the-series-of-events-from-cellular-signal-transduction-to-gene-expression
#9
Kazuhito Goda, Takeo Takahashi, Hirobumi Suzuki
The molecular interactions and translocation of signal transduction factors in individual cells can be imaged by fluorescence microscopy. Alternatively, downstream promoter activity in single cells can be imaged by bioluminescence microscopy. However, the same stimuli can lead to different gene expression responses in individual cells. For this reason, it is desirable to simultaneously image signal transduction and gene expression events in the same cells. Here, we describe a method that combines fluorescence and bioluminescence microscopy to image protein kinase C (PKC) translocation from the cytosol to the plasma membrane and the expression of nuclear factor kappa-light polypeptide B (NF-κB)-regulated genes...
December 11, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29227554/simple-and-rapid-tissue-clearing-method-for-three-dimensional-histology-of-the-pancreas
#10
Hang Sheung Wong, Patrick Ka Kit Yeung, Hei Ming Lai, Karen Siu Ling Lam, Wu Wutian, Sookja Kim Chung
Previously, high-resolution three-dimensional imaging of a whole and intact pancreas was not possible, since light is scattered when it passes through cell compartments with different refractive indices. CLARITY is one of the tissue clearing techniques that has yielded success with the central nervous system. To preserve tissue integrity after delipidation, conventional protocols embed tissue in an acrylamide-based hydrogel, which involves the use of specialized equipment. Recently, we determined that the hydrogel-embedding step could be simplified and replaced by passive tissue fixation in 4% paraformaldehyde (PFA)...
December 11, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29227553/fluorescence-lifetime-imaging-of-a-caspase-3-apoptosis-reporter
#11
Johanna M Buschhaus, Anne E Gibbons, Kathryn E Luker, Gary D Luker
Caspase-3 is a proteolytic enzyme that functions as a key effector in apoptotic cell death. Determining activity of caspase-3 provides critical information about cancer cell viability and response to treatment. To measure apoptosis in intact cells and living mice, a fluorescence imaging reporter that detects caspase-3 activity by Förster resonance energy transfer (FRET) was used. Changes in FRET by fluorescence lifetime imaging microscopy (FLIM) were measured. Unlike FRET measurements based on fluorescence intensity, lifetime measurements are independent of reporter concentration and scattering of light in tissue, making FLIM a robust method for imaging in 3D environments...
December 11, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29227552/microfluidics-assisted-tirf-imaging-to-study-single-actin-filament-dynamics
#12
Shashank Shekhar
Dynamic assembly of actin filaments is essential for many cellular processes. The rates of assembly and disassembly of actin filaments are intricately controlled by regulatory proteins that interact with the ends and the sides of filaments and with actin monomers. TIRF-based single-filament imaging techniques have proven instrumental in uncovering mechanisms of actin regulation. In this unit, novel single-filament approaches using microfluidics-assisted TIRF imaging are described. These methods can be used to grow anchored actin filaments aligned in a flow, thus making the analysis much easier as compared to open flow cell approaches...
December 11, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29227551/high-purity-isolation-and-sensitive-quantification-of-extracellular-vesicles-using-affinity-to-tim4
#13
Takeshi Yoshida, Takamasa Ishidome, Rikinari Hanayama
Almost all types of cells secrete extracellular vesicles (EVs), including exosomes and microvesicles. EVs carry various proteins, lipids, mRNAs, and microRNAs, and may participate in many aspects of physiological and pathophysiological processes. Various studies are currently being conducted to develop therapeutic and diagnostic methods targeting or utilizing EVs. Therefore, developing ideal methods for isolating and quantifying EVs is an active area of research. EVs express phosphatidylserine on their outer lipid bilayer...
December 11, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29227550/live-cell-visualization-of-calcium-flux-in-vibratome-cut-thick-sections-of-viable-tumor-tissue
#14
James Koh, Joyce A Hogue, Julie A Sosa
This unit outlines a live-cell imaging approach developed for visualization of intracellular calcium flux in human parathyroid tumors following stimulation of the calcium-sensing receptor (CASR), a class C G protein-coupled receptor (GPCR). The primary assay readout, intracellular calcium release induced by activation of the inositol triphosphate receptor, is potentially generalizable to multiple other GPCR signaling events that utilize this common downstream signal transduction pathway. Advantages of the approach include: (1) preservation of native tissue context and positional information, allowing direct visualization of intratumoral functional heterogeneity; (2) quantitative documentation of reactivity to a physiological stimulus in an experimentally tractable ex vivo system; and (3) generation of a dynamic, functional classifier of tumor biochemical behavior to augment static marker assessment...
December 11, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29227549/in-vitro-dissection-of-autophagy
#15
Min Zhang, Dawei Liu, Liang Ge
Autophagy is an essential cellular process for bulk degradation of cytoplasmic components through the lysosome. Underlying this process is an intricate interaction between protein factors and the cell endomembrane system, leading to a gradual maturation of the autophagic membrane. This structure sequesters a portion of the cytoplasm by the formation of a double-membrane compartment called the autophagosome. The autophagosome then delivers the cargo to the lysosome to complete degradation. The molecular mechanism accounting for the generation of the autophagic membrane is a longstanding question...
December 11, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28862343/cellular-detection-of-g-quadruplexes-by-optical-imaging-methods
#16
Souheila Amor, Sunny Y Yang, Judy M Y Wong, David Monchaud
G-quadruplexes (G4s) are higher-order nucleic acid structures that fold from guanine (G)-rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of knowledge acquired over the past three decades strongly supports pivotal roles of G4 in the regulation of gene expression at both transcriptional (DNA quadruplexes) and translational levels (RNA quadruplexes). Recent biochemical discoveries uncovered myriad of additional G4 actions: from chromosomal stability to the firing of replication origins, from telomere homeostasis to functional dysregulations underlying genetic diseases (including cancers and neurodegeneration)...
September 1, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28862342/lectin-array-blotting
#17
Raquel Pazos, Juan Echevarria, Alvaro Hernandez, Niels-Christian Reichardt
Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step...
September 1, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28862341/centrifugation-free-magnetic-isolation-of-functional-mitochondria-using-paramagnetic-iron-oxide-nanoparticles
#18
Bhabatosh Banik, Shanta Dhar
Subcellular fractionation techniques are essential for cell biology and drug development studies. The emergence of organelle-targeted nanoparticle (NP) platforms necessitates the isolation of target organelles to study drug delivery and activity. Mitochondria-targeted NPs have attracted the attention of researchers around the globe, since mitochondrial dysfunctions can cause a wide range of diseases. Conventional mitochondria isolation methods involve high-speed centrifugation. The problem with high-speed centrifugation-based isolation of NP-loaded mitochondria is that NPs can pellet even if they are not bound to mitochondria...
September 1, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28862340/in-vitro-reconstitution-of-the-endoplasmic-reticulum
#19
Csilla-Maria Ferencz, Gernot Guigas, Andreas Veres, Brigitte Neumann, Olaf Stemmann, Matthias Weiss
Reconstitution of cellular organelles in vitro offers the possibility to perform quantitative and qualitative experiments in a controlled environment that cannot be done with the same accuracy in living cells. Following a previous report, the subsequent list of protocols describes how to reconstitute and quantify a tubular ER network in vitro based on purified microsomes from culture cells and cytosol from Xenopus laevis egg extracts. Biological material preparation and reconstitution assays require mostly basic laboratory instrumentation and chemicals, and can be executed without any specific training, making them appealing to a wide range of laboratories...
September 1, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28862339/determination-of-membrane-protein-distribution-on-the-nuclear-envelope-by-single-point-single-molecule-frap
#20
Krishna C Mudumbi, Weidong Yang
Nuclear envelope transmembrane proteins (NETs) are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM) in eukaryotic cells. The abnormal distribution of NETs has been associated with many human diseases. However, quantitative determination of the spatial distribution and translocation dynamics of NETs on the ONM and INM is still very limited in currently existing approaches. Here we demonstrate a single-point single-molecule fluorescence recovery after photobleaching (FRAP) microscopy technique that enables quick determination of distribution and translocation rates for NETs in vivo...
September 1, 2017: Current Protocols in Cell Biology
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