Volume: 7 Issue: 16 —BIO-PROTOCOL

Cancer Biology

Antisense Oligonucleotide-mediated Knockdown in Mammary Tumor Organoids Authors: Sarah D. Diermeier and David L. Spector, date: 08/20/2017, view: 6201, Q&A: 0

Primary mammary tumor organoids grown in 3D are an excellent system to study tumor biology. They resemble the organization and physiology of native epithelia more closely than cancer cell lines grown in 2D, and additionally model interactions with the ECM (Boj et al., 2015; Clevers, 2016; Shamir and Ewald, 2014). Mammary tumor organoids ...

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Mouse Model of Dextran Sodium Sulfate (DSS)-induced Colitis Authors: Srustidhar Das, Surinder K. Batra and Satyanarayana Rachagani, date: 08/20/2017, view: 11844, Q&A: 0

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the intestinal tract and is primarily comprised of Crohn’s disease (CD) and ulcerative colitis (UC). Several murine models that include both chemical induced and genetic models have been developed that mimic some aspects of either CD or UC. These models have been instrumental in ...

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Rapid Profiling Cell Cycle by Flow Cytometry Using Concurrent Staining of DNA and Mitotic Markers Authors: Yuqing Shen, Paolo Vignali and Ruoning Wang, date: 08/20/2017, view: 7252, Q&A: 1

The flow cytometric quantitation of DNA content by DNA-binding fluorochrome, propidium iodide (PI) is the most widely used method for cell cycle analysis. However, the commonly used methods are time-consuming and labor-intensive and are incompatible with staining of mitotic markers by fluorescent-labeled antibodies. Here, we report an optimized ...

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Teratoma Formation Assay for Assessing Pluripotency and Tumorigenicity of Pluripotent Stem Cells Authors: Shingo Miyawaki, Yohei Okada, Hideyuki Okano and Kyoko Miura, date: 08/20/2017, view: 9701, Q&A: 0

Pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) form teratomas when transplanted into immunodeficient mice. As teratomas contain all three germ layers (endoderm, mesoderm, ectoderm), teratoma formation assay is widely used as an index of pluripotency (Evans and Kaufman, 1981; Hentze et al. ...

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Cell Biology

Isolation and Analysis of Stromal Cell Populations from Mouse Lymph Nodes Authors: Yannick O. Alexandre and Scott N Mueller, date: 08/20/2017, view: 6396, Q&A: 0

Our protocol describes a simple procedure for isolating stromal cells from lymph nodes (LN). LN are disrupted then enzymatically digested with collagenase and dispase to produce a single cell suspension that can be stained with fluorescently labelled antibodies and analysed by flow cytometry. This protocol will enable identification of ...

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Improved Oviduct Transfer Surgery for Genetically Modified Rat Production Authors: Laura J. Lambert, Larry W. Johnson, Daniel Kennedy, Joan Cadillac and Robert A. Kesterson, date: 08/20/2017, view: 6356, Q&A: 0

Rat embryo transfer surgeries are becoming more common with targeted nucleases increasing the demand for rat models. This protocol details pre-surgical preparation, improved surgical techniques for placing embryos into the oviduct, and post-surgical care of rats to parturition. Direct application of mouse oviduct transfer protocols results in ...

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Immunology

Isolation and Analysis of Stromal Vascular Cells from Visceral Adipose Tissue Authors: Jessica Vu and Wei Ying, date: 08/20/2017, view: 8398, Q&A: 0

The obesity epidemic is the underlying driver of the type 2 diabetes mellitus epidemic. A remarkable accumulation of various pro-inflammatory immune cells in adipose tissues is a hallmark of obesity and leads to pathogenesis of tissue inflammation and insulin resistance. Here, we describe a detailed protocol to isolate adipose tissue stromal ...

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Macrophage Survival Assay Using High Content Microscopy Authors: Remzi Onur Eren and Nicolas Fasel, date: 08/20/2017, view: 4667, Q&A: 0

Macrophages maintain tissue homoeostasis by regulating inflammation and tissue repair mechanisms. Thus, the fate of macrophages has an impact on the state of the tissue. The aim of this protocol is to quantify macrophage survival using high content microscopy and image processing software. Here, we describe a high-content image based protocol to ...

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Establishment of a Human Cell Line Persistently Infected with Sendai Virus Authors: Christopher Coakley, Cara Peter, Stephanie Fabry and Saurabh Chattopadhyay, date: 08/20/2017, view: 5284, Q&A: 0

Interferon regulatory transcription factor 3 (IRF3) is a transcription factor that upon activation by virus infection promotes the synthesis of antiviral genes, such as the interferons (Hiscott, 2007). In addition to inducing genes, IRF3 triggers antiviral apoptosis by RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA), which is ...

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Microbiology

Detection of Pathogens and Ampicillin-resistance Genes Using Multiplex Padlock Probes Authors: Rick Conzemius and Ivan Barišić, date: 08/20/2017, view: 5249, Q&A: 0

Diagnostic assays for pathogen identification and characterization are limited either by the number of simultaneously detectable targets, which rely on multiplexing methods, or by time constraints due to cultivation-based techniques. We recently presented a 100-plex method for human pathogen characterization to identify 75 bacterial and fungal ...

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Superoxide Dismutase (SOD) and Catalase (CAT) Activity Assay Protocols for Caenorhabditis elegans Authors: Jing Zhang, Rui Chen, Zhenyang Yu and Lili Xue, date: 08/20/2017, view: 24717, Q&A: 0

Assays for superoxide dismutase (SOD) and catalase (CAT) activities are widely employed to indicate antioxidant responses underlying the toxic effects of test chemicals. Yet, earlier studies mainly described the procedures as performed according to manufacturer’s instructions without modifications that are specific to any organisms. The present ...

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Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography Authors: Juan Andres Imelio, Nicole Larrieux, Ariel Edgardo Mechaly, Felipe Trajtenberg and Alejandro Buschiazzo, date: 08/20/2017, view: 4551, Q&A: 0

We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR ...

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An Optimized Method for the Production Using PEI, Titration and Neutralization of SARS-CoV Spike Luciferase Pseudotypes Authors: George Carnell, Keith Grehan, Francesca Ferrara, Eleonora Molesti and Nigel Temperton, date: 08/20/2017, view: 8849, Q&A: 0

The protocol outlined represents a cost-effective, rapid and reliable method for the generation of high-titre viral pseudotype particles with the wild-type SARS-CoV spike protein on a lentiviral vector core using the widely available transfection reagent PEI. This protocol is optimized for transfection in 6-well plates; however it can be readily ...

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Protocol for HeLa Cells Infection with Escherichia coli Strains Producing Colibactin and Quantification of the Induced DNA-damage Authors: Nadège Bossuet-Greif, Marcy Belloy, Michèle Boury, Eric Oswald and Jean-Philippe Nougayrede, date: 08/20/2017, view: 7707, Q&A: 0

Strains of Escherichia coli bearing the pks genomic island synthesize the genotoxin colibactin. Exposure of eukaryotic cells to E. coli producing colibactin induces DNA damages, ultimately leading to cell cycle arrest, senescence and death. Here we describe a simple method to demonstrate the genotoxicity of bacteria ...

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Molecular Biology

Assessment of Modulation of Protein Stability Using Pulse-chase Method Author: Mohamed Elgendy, date: 08/20/2017, view: 7295, Q&A: 0

Pulse-chase technique is a method widely used to assess protein or mRNA stability. The principle of pulse-chase relies on labeling proteins or mRNA produced during a short period of time called ‘pulse’ and then following the rate of disappearance of those labeled proteins over a period of time called ‘chase’. This technique thus allows ...

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Neuroscience

A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics Authors: Julia F. Alterman, Andrew H. Coles, Lauren M. Hall, Neil Aronin, Anastasia Khvorova and Marie-Cécile Didiot, date: 08/20/2017, view: 6414, Q&A: 0

Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in ...

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Assessment of Thermal Pain Sensation in Rats and Mice Using the Hargreaves Test Authors: Menghon Cheah, James W Fawcett and Melissa R Andrews, date: 08/20/2017, view: 7640, Q&A: 0

The Hargreaves test is specifically designed to assess thermal pain sensation in rodents such as rats and mice. This test has been used in experiments involving pain sensitization or recovery of thermal pain response following neural injury and regeneration. We present here a step-by-step protocol highlighted with important notes to guide ...

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An ex vivo Perifusion Method for Quantitative Determination of Neuropeptide Release from Mouse Hypothalamic Explants Authors: Ophélia Le Thuc, Jacques Noël and Carole Rovère, date: 08/20/2017, view: 4816, Q&A: 0

The hypothalamus is a primary brain area which, in mammals, regulates several physiological functions that are all related to maintaining general homeostasis, by linking the central nervous system (CNS) and the periphery. The hypothalamus itself can be considered an endocrine brain region of some sort as it hosts in its different nuclei several ...

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Plant Science

Using Silicon Polymer Impression Technique and Scanning Electron Microscopy to Measure Stomatal Aperture, Morphology, and Density Authors: Hui-Chen Wu, Ya-Chen Huang, Chia-Hung Liu and Tsung-Luo Jinn, date: 08/20/2017, view: 5912, Q&A: 0

The number of stomata on leaves can be affected by intrinsic development programming and various environmental factors, in addition the control of stomatal apertures is extremely important for the plant stress response. In response to elevated temperatures, transpiration occurs through the stomatal apertures, allowing the leaf to cool through ...

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TUNEL Assay to Assess Extent of DNA Fragmentation and Programmed Cell Death in Root Cells under Various Stress Conditions Authors: Amit K. Tripathi, Ashwani Pareek and Sneh Lata Singla-Pareek, date: 08/20/2017, view: 9019, Q&A: 0

DNA damage is one of the common consequences of exposure to various stress conditions. Different methods have been developed to accurately assess DNA damage and fragmentation in cells and tissues exposed to different stress agents. However, owing to the presence of firm cellulosic cell wall and phenolics, plant cells and tissues are not easily ...

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ROS Detection in Botryococcus braunii Colonies with CellROX Green Reagent Authors: Edmundo Lozoya-Gloria, Ivette Cornejo-Corona, Hem R. Thapa, Daniel R. Browne and Timothy P. Devarenne, date: 08/20/2017, view: 5138, Q&A: 0

We analyzed the reactive oxygen species (ROS) accumulation in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO₃, salicylic acid (SA), methyl jasmonate, and acetic acid. A staining assay using the fluorescent dye CellROX Green was used. CellROX Green is a ...

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Quantification of Membrane Damage/Cell Death Using Evan’s Blue Staining Technique Authors: Preethi Vijayaraghavareddy, Vanitha Adhinarayanreddy, Ramu S Vemanna, Sheshshayee Sreeman and Udayakumar Makarla , date: 08/20/2017, view: 11463, Q&A: 0

Membrane damage is a hallmark of both biotic and abiotic stress responses. The membrane determines the ability of a cell to sustain altered environmental conditions and hence can be used as a biomarker to assess stress-induced cell damage or death. We present an easy, quick, cost-effective, staining and spectrophotometric method to assess membrane ...

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Stem Cell

Exit from Pluripotency Assay of Mouse Embryonic Stem Cells Authors: Daniel Cirera-Salinas and Constance Ciaudo, date: 08/20/2017, view: 5623, Q&A: 0

A novel method to assess the dissolution of the core pluripotency transcription-factor circuit of mouse Embryonic Stem Cells (mESCs) has been developed (Ying et al., 2003; Betschinger et al., 2013). In order to efficiently identify genes essential for the break-down of the pluripotency network in mutant mESCs with proliferation ...

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A Co-culture Assay to Determine Efficacy of TNF-α Suppression by Biomechanically Induced Human Bone Marrow Mesenchymal Stem Cells Authors: Miguel F. Diaz, Siobahn M. Evans, Scott D. Olson, Charles S. Cox Jr and Pamela L. Wenzel, date: 08/20/2017, view: 5181, Q&A: 0

The beneficial effects of mesenchymal stem cell (MSC)-based cellular therapies are believed to be mediated primarily by the ability of MSCs to suppress inflammation associated with chronic or acute injury, infection, autoimmunity, and graft-versus-host disease. To specifically address the effects of frictional force caused by blood flow, or wall ...

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Isolation and Expansion of Mesenchymal Stem Cells from Murine Adipose Tissue Authors: Natalia V. Andreeva, Alexandra A. Dalina and Alexander V. Belyavsky, date: 08/20/2017, view: 7949, Q&A: 1

Mesenchymal stem cells (MSCs) are currently intensively studied due to significant promise which they represent for successful implementations of future cell therapy clinical protocols. This in turn emphasizes importance of careful preclinical studies of MSC effects in various murine disease models. The appropriate cell preparations with ...

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Past Issues in 2017

Volume: 7 Issue: 16

Cancer Biology

Cell Biology

Immunology

Microbiology

Molecular Biology

Neuroscience

Plant Science

Stem Cell