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Current Protocols in Cell Biology

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https://www.readbyqxmd.com/read/29227555/combining-fluorescence-and-bioluminescence-microscopy-to-study-the-series-of-events-from-cellular-signal-transduction-to-gene-expression
#1
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
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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
#9
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
#10
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
#11
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
#12
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
https://www.readbyqxmd.com/read/28862338/automated-tracking-of-cell-migration-with-rapid-data-analysis
#13
Brian J DuChez
Cell migration is essential for many biological processes including development, wound healing, and metastasis. However, studying cell migration often requires the time-consuming and labor-intensive task of manually tracking cells. To accelerate the task of obtaining coordinate positions of migrating cells, we have developed a graphical user interface (GUI) capable of automating the tracking of fluorescently labeled nuclei. This GUI provides an intuitive user interface that makes automated tracking accessible to researchers with no image-processing experience or familiarity with particle-tracking approaches...
September 1, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28627757/super-resolution-microscopy-and-single-protein-tracking-in-live-bacteria-using-a-genetically-encoded-photostable-fluoromodule
#14
Saumya Saurabh, Adam M Perez, Colin J Comerci, Lucy Shapiro, W E Moerner
Visualization of dynamic protein structures in live cells is crucial for understanding the mechanisms governing biological processes. Fluorescence microscopy is a sensitive tool for this purpose. In order to image proteins in live bacteria using fluorescence microscopy, one typically genetically fuses the protein of interest to a photostable fluorescent tag. Several labeling schemes are available to accomplish this. Particularly, hybrid tags that combine a fluorescent or fluorogenic dye with a genetically encoded protein (such as enzymatic labels) have been used successfully in multiple cell types...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28627756/microcontact-peeling-a-cell-micropatterning-technique-for-circumventing-direct-adsorption-of-proteins-to-hydrophobic-pdms
#15
Sho Yokoyama, Tsubasa S Matsui, Shinji Deguchi
Microcontact printing (μCPr) is one of the most popular techniques used for cell micropatterning. In conventional μCPr, a polydimethylsiloxane (PDMS) stamp with microfeatures is used to adsorb extracellular matrix (ECM) proteins onto the featured surface and transfer them onto particular areas of a cell culture substrate. However, some types of functional proteins other than ECM have been reported to denature upon direct adsorption to hydrophobic PDMS. Here we describe a detailed protocol of an alternative technique--microcontact peeling (μCPe)--that allows for cell micropatterning while circumventing the step of adsorbing proteins to bare PDMS...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28627755/labeling-dna-replication-foci-to-visualize-chromosome-territories-in-vivo
#16
Apolinar Maya-Mendoza, Dean A Jackson
While a detailed understanding of chromatin dynamics is needed to explain how higher-order chromatin organization influences nuclear function, the molecular principles that regulate chromatin mobility in mammalian nuclei remain largely unknown. Here we describe experimental tools to follow chromatin dynamics by labeling DNA during S phase. Using these methods, we have found that foci labeled during early and mid/late S phase have significantly different dynamic behavior. Spatially constrained heterochromatic foci restrict long-range transformations of the chromosome territory (CT) structure while providing a structural framework on which highly mobile euchromatic foci undergo positional oscillations that drive local changes in the chromosome shape...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28627754/labeling-and-magnetic-resonance-imaging-of-exosomes-isolated-from-adipose-stem-cells
#17
Alice Busato, Roberta Bonafede, Pietro Bontempi, Ilaria Scambi, Lorenzo Schiaffino, Donatella Benati, Manuela Malatesta, Andrea Sbarbati, Pasquina Marzola, Raffaella Mariotti
Adipose stem cells (ASC) represent a promising therapeutic approach for neurodegenerative diseases. Most biological effects of ASC are probably mediated by extracellular vesicles, such as exosomes, which influence the surrounding cells. Current development of exosome therapies requires efficient and noninvasive methods to localize, monitor, and track the exosomes. Among imaging methods used for this purpose, magnetic resonance imaging (MRI) has advantages: high spatial resolution, rapid in vivo acquisition, and radiation-free operation...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28627753/traction-force-microscopy-in-3-dimensional-extracellular-matrix-networks
#18
M Cóndor, J Steinwachs, C Mark, J M García-Aznar, B Fabry
Cell migration through a three-dimensional (3-D) matrix depends strongly on the ability of cells to generate traction forces. To overcome the steric hindrance of the matrix, cells need to generate sufficiently high traction forces but also need to distribute these forces spatially in a migration-promoting way. This unit describes a protocol to measure spatial maps of cell traction forces in 3-D biopolymer networks such as collagen, fibrin, or Matrigel. Traction forces are computed from the relationship between measured force-induced matrix deformations surrounding the cell and the known mechanical properties of the matrix...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28627752/patterning-on-topography-for-generation-of-cell-culture-substrates-with-independent-nanoscale-control-of-chemical-and-topographical-extracellular-matrix-cues
#19
Emily N Sevcik, John M Szymanski, Quentin Jallerat, Adam W Feinberg
The cell microenvironment plays an important role in many biological processes, including development and disease progression. Key to this is the extracellular matrix (ECM), a complex biopolymer network serving as the primary insoluble signaling network for physical, chemical, and mechanical cues. In vitro, the ability to engineer the ECM at the micro- and nanoscales is a critical tool to systematically interrogate the influence of ECM properties on cellular responses. Specifically, both topographical and chemical surface patterning has been shown to direct cell alignment and tissue architecture on biomaterial surfaces, however, it has proven challenging to independently control these surface properties...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28256723/immunoblotting-and-immunodetection
#20
Duojiao Ni, Peng Xu, Sean Gallagher
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents...
March 3, 2017: Current Protocols in Cell Biology
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