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Biochemistry

Active Cdk5 Immunoprecipitation and Kinase Assay Authors:  Andrew N. Bankston, Li Ku and Yue Feng, date: 07/05/2017, view: 2423, Q&A: 0
Cdk5 activity is regulated by the amounts of two activator proteins, p35 and p39 (Tsai et al., 1994; Zheng et al., 1998; Humbert et al., 2000). The p35-Cdk5 and p39-Cdk5 complexes have differing sensitivity to salt and detergent concentrations (Hisanaga and Saito, 2003; Sato et al., 2007; Yamada et al., ...
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Formaldehyde Fixation of Extracellular Matrix Protein Layers for Enhanced Primary Cell Growth Authors:  Natalia V. Andreeva and Alexander V. Belyavsky, date: 07/05/2017, view: 2055, Q&A: 0
Coating tissue culture vessels with the components of the extracellular matrix such as fibronectin and collagens provides a more natural environment for primary cells in vitro and stimulates their proliferation. However, the effects of such protein layers are usually rather modest, which might be explained by the loss immobilized proteins ...
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Cancer Biology

Tumorigenicity Assay in Nude Mice Authors:  Feng Du, Xiaodi Zhao and Daiming Fan, date: 07/05/2017, view: 4156, Q&A: 0
Tumorigenicity refers to the ability of cultured cells to develop viable tumors in immune-deficient animals. The goal of this protocol is to illustrate tumorigenicity assay by subcutaneous tumor-cell-transplantation in nude mice. Target cells are transplanted to 6-week-old nude mice subcutaneously and the tumor growth is monitored over a period of ...
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Generation of a Cellular Reporter for Functional BRD4 Inhibition Authors:  Sara Sdelci and Stefan Kubicek, date: 07/05/2017, view: 1715, Q&A: 0
The ubiquitously expressed bromodomain-containing protein 4 (BRD4) is an epigenetic reader, which recruits transcriptional regulatory complexes to acetylated chromatin. Because of its role in enhancing proliferation, BRD4 has become a therapeutic target in oncology, as the inhibition of this protein leads to the reduction of the growth of many ...
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A Novel Mouse Skin Graft Model of Vascular Tumors Driven by Akt1 Authors:  Thuy L. Phung and Sriram Ayyaswamy, date: 07/05/2017, view: 2962, Q&A: 0
To investigate whether endothelial Akt1 activation is sufficient to induce vascular tumor formation in the skin, we have developed a skin graft model in which a skin fragment from transgenic donor mice with inducible and endothelial cell-specific overexpression of activated Akt1 (myrAkt1) is grafted into the skin of wild type recipient mice. The ...
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Cell Biology

Photothrombotic Induction of Capillary Ischemia in the Mouse Cortex during in vivo Two-Photon Imaging Authors:  Robert G. Underly and Andy Y. Shih, date: 07/05/2017, view: 2216, Q&A: 0
Photothrombosis of blood vessels refers to the activation of a circulating photosensitive dye with a green light to induce clotting in vivo (Watson et al., 1985). Previous studies have described how a focused green laser could be used to noninvasively occlude pial arterioles and venules at the brain surface (Schaffer et al. ...
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Endpoint or Kinetic Measurement of Hydrogen Sulfide Production Capacity in Tissue Extracts Authors:  Christopher Hine and James R. Mitchell, date: 07/05/2017, view: 2585, Q&A: 0
Hydrogen sulfide (H2S) gas is produced in cells and tissues via various enzymatic processes. H2S is an important signaling molecule in numerous biological processes, and deficiencies in endogenous H2S production are linked to cardiovascular and other health complications. Quantitation of steady-state H2S ...
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Developmental Biology

Oxidative Stress Assays (arsenite and tBHP) in Caenorhabditis elegans Authors:  Collin Yvès Ewald, John M. Hourihan and T. Keith Blackwell, date: 07/05/2017, view: 3173, Q&A: 0
Cells and organisms face constant exposure to reactive oxygen species (ROS), either from the environment or as a by-product from internal metabolic processes. To prevent cellular damage from ROS, cells have evolved detoxification mechanisms. The activation of these detoxification mechanisms and their downstream responses represent an overlapping ...
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Ex vivo Ooplasmic Extract from Developing Drosophila Oocytes for Quantitative TIRF Microscopy Analysis Authors:  Imre Gáspár and Anne Ephrussi, date: 07/05/2017, view: 2205, Q&A: 0
Understanding the dynamic behavior and the continuously changing composition of macromolecular complexes, subcellular structures and organelles is one of areas of active research in both cell and developmental biology, as these changes directly relate to function and subsequently to the development and homeostasis of the organism. Here, we ...
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Immunology

Thermal Stability of Heterotrimeric pMHC Proteins as Determined by Circular Dichroism Spectroscopy Authors:  Anna Fuller, Aaron Wall, Michael D Crowther, Angharad Lloyd, Alexei Zhurov, Andrew K. Sewell, David K. Cole and Konrad Beck, date: 07/05/2017, view: 2039, Q&A: 0
T cell receptor (TCR) recognition of foreign peptide fragments, presented by peptide major histocompatibility complex (pMHC), governs T-cell mediated protection against pathogens and cancer. Many factors govern T-cell sensitivity, including the affinity of the TCR-pMHC interaction and the stability of pMHC on the surface of antigen presenting ...
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Qualitative and Quantitative Assay for Detection of Circulating Autoantibodies against Human Aortic Antigen Authors:  Brent Veerman and Ritu Chakravarti, date: 07/05/2017, view: 2268, Q&A: 0
Increased amount of autoantibodies in human sera are the hallmark of autoimmune diseases (Wang et al., 2015). In case of known antigen, detection of autoantibodies is done using laboratory based methods. However, in most autoimmune diseases, knowledge of self-antigen is still vague. We have developed an ELISA-based quantitative assay to ...
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EAE Induction by Passive Transfer of MOG-specific CD4+ T Cells Authors:  Yuki Tanaka, Yasunobu Arima, Kotaro Higuchi, Takuto Ohki, Mohamed Elfeky, Mitsutoshi Ota, Daisuke Kamimura and Masaaki Murakami, date: 07/05/2017, view: 2325, Q&A: 0
Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which is a chronic inflammatory disease of the central nervous system (CNS). It is characterized by focal demyelination and inflammatory responses mediated by myelin-specific autoreactive CD4+ T cells. Using a passive transfer model of EAE in ...
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Vaginal HSV-2 Infection and Tissue Analysis Authors:  Marie Beck Iversen, Søren Riis Paludan and Christian Kanstrup Holm, date: 07/05/2017, view: 2156, Q&A: 0
The vaginal murine HSV-2 infection model is very useful in studying mucosal immunity against HSV-2 (Overall et al., 1975; Renis et al., 1976; Parr and Parr, 2003). Histologically, the vagina of Depo-Provera-treated mice is lined by a single layer of mucus secreting columnar epithelial cells overlying two to three layers of ...
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In vitro Demonstration and Quantification of Neutrophil Extracellular Trap Formation Authors:  Dongsheng Jiang, Mona Saffarzadeh and Karin Scharffetter-Kochanek, date: 07/05/2017, view: 3120, Q&A: 0
In the recent decade, neutrophil extracellular traps (NETs) have been identified and confirmed as a new anti-microbial weapon of neutrophils. In this protocol, we describe easy methods to demonstrate NET formation by immunofluorescence staining of extracellular chromatin fiber with anti-DNA/Histone H1 antibody and quantification of NETs by using a ...
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Microbiology

Assaying Glycogen and Trehalose in Yeast Authors:  Yuping Chen and Bruce Futcher, date: 07/05/2017, view: 2090, Q&A: 0
Organisms store carbohydrates in several forms. In yeast, carbohydrates are stored in glycogen (a multi-branched polysaccharide) and in trehalose (a disaccharide). As in other organisms, the amount of stored carbohydrate varies dramatically with physiological state, and accordingly, an assay of stored carbohydrate can help reveal physiological ...
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Bacterial Survival in Dictyostelium Authors:  Regin Rønn, Xiuli Hao, Freja Lüthje, Nadezhda A. German, Xuanji Li, Fuyi Huang, Javan Kisaka, David Huffman, Hend A. Alwathnani, Yong-Guan Zhu and Christopher Rensing, date: 07/05/2017, view: 2051, Q&A: 0
We performed an assay to test the ability of different E. coli strains to survive inside amoebal cells after ingestion. In the assay we incubated bacteria together with cells of Dictyostelium discoideum for six hours. After co-incubation most of the uningested bacteria were removed by centrifugation and the remaining uningested ...
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Determination of NO and CSF Levels Produced by Bacillus subtilis Authors:  Sebastián Cogliati, Facundo Rodriguez Ayala, Carlos Bauman, Marco Bartolini, Cecilia Leñini, Juan Manuel Villalba, Federico Argañaraz and Roberto Grau, date: 07/05/2017, view: 2111, Q&A: 0
The cell-to-cell communication and division of labour that occurs inside a beneficial biofilm produce significant differences in gene expression compared with the gene expression pattern of cells grew under planktonic conditions. In this sense, the levels of NO (nitric oxide) and CSF (Competence Sporulation Stimulating Factor) produced in Bacillus ...
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Molecular Biology

Multiplex Gene Editing via CRISPR/Cas9 System in Sheep Authors:  Yiyuan Niu, Yi Ding, Xiaolong Wang and Yulin Chen, date: 07/05/2017, view: 2543, Q&A: 0
Sheep is a major large animal model for studying development and disease in biomedical research. We utilized CRISPR/Cas9 system successfully to modify multiple genes in sheep. Here we provide a detailed protocol for one-cell-stage embryo manipulation by co-injecting Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and ...
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Neuroscience

Social Observation Task in a Linear Maze for Rats Authors:  Xiang Mou and Daoyun Ji, date: 07/05/2017, view: 1842, Q&A: 0
Animals often learn through observing their conspecifics. However, the mechanisms of them obtaining useful knowledge during observation are beginning to be understood. This protocol describes a novel social observation task to test the ‘local enhancement theory’, which proposes that presence of social subjects in an environment facilitates one’s ...
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Live Imaging of Myogenesis in Indirect Flight Muscles in Drosophila Author:  Dagan Segal, date: 07/05/2017, view: 2117, Q&A: 0
The indirect flight muscles (IFMs) are the largest muscles in the fly, making up the bulk of the adult thorax. IFMs in Drosophila are generated during pupariation by fusion of hundreds of muscle precursor cells (myoblasts) with larval muscle templates (myotubes). Prominent features, including the large number of fusion events, the ...
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Spinal Cord Preparation from Adult Red-eared Turtles for Electrophysiological Recordings during Motor Activity Authors:  Peter C Petersen and Rune W Berg , date: 07/05/2017, view: 2943, Q&A: 0
Although it is known that the generation of movements is performed to a large extent in neuronal circuits located in the spinal cord, the involved mechanisms are still unclear. The turtle as a model system for investigating spinal motor activity has advantages, which far exceeds those of model systems using other animals. The high resistance to ...
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Behavioral and Functional Assays for Investigating Mechanisms of Noxious Cold Detection and Multimodal Sensory Processing in Drosophila Larvae Authors:  Atit A. Patel and Daniel N. Cox, date: 07/05/2017, view: 2455, Q&A: 0
To investigate cellular, molecular and behavioral mechanisms of noxious cold detection, we developed cold plate behavioral assays and quantitative means for evaluating the predominant noxious cold-evoked contraction behavior. To characterize neural activity in response to noxious cold, we implemented a GCaMP6-based calcium imaging assay enabling ...
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Delayed-matching-to-place Task in a Dry Maze to Measure Spatial Working Memory in Mice Authors:  Xi Feng, Karen Krukowski, Timothy Jopson and Susanna Rosi, date: 07/05/2017, view: 2270, Q&A: 0
The delayed-matching-to-place (DMP) dry maze test is a variant of DMP water maze (Steele and Morris, 1999; Faizi et al., 2012) which measures spatial working/episodic-like learning and memory that depends on both hippocampal and cortical functions (Wang and Morris, 2010; Euston et al., 2012). Using this test we can detect normal ...
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Plant Science

A Simple and Rapid Assay for Measuring Phytoalexin Pisatin, an Indicator of Plant Defense Response in Pea (Pisum sativum L.) Authors:  Lee A. Hadwiger and Kiwamu Tanaka, date: 07/05/2017, view: 1661, Q&A: 0
Phytoalexins are antimicrobial substance synthesized in plants upon pathogen infection. Pisatin (Pisum sativum phytoalexin) is the major phytoalexin in pea, while it is also a valuable indicator of plant defense response. Pisatin can be quantitated in various methods from classical organic chemistry to ...
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Determination of Reduced and Total Glutathione Content in Extremophilic Microalga Galdieria phlegrea Authors:  Giovanna Salbitani, Claudia Bottone and Simona Carfagna, date: 07/05/2017, view: 2929, Q&A: 0
Glutathione is an important molecule involved in the primary and secondary metabolism of all organisms. The Glutathione redox status is an indicator of the cellular redox state. Therefore, it is important to have precise methods on hand to determine the glutathione redox status in the cell. In this protocol, we describe an improved ...
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Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9 Authors:  Florian Hahn, Marion Eisenhut, Otho Mantegazza and Andreas P. M. Weber, date: 07/05/2017, view: 5594, Q&A: 0
The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing ...
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Determination of the Effects of Local and Systemic Iron Excess on Lateral Root Initiation in Arabidopsis thaliana Authors:  Guangjie Li, Lin Zhang and Weiming Shi, date: 07/05/2017, view: 1665, Q&A: 0
Root system architecture depends on nutrient availability. A symptom of iron (Fe) toxicity in plants is stunted root growth, yet little is known about the effects of excess Fe on lateral root (LR) development. To better understand how nutrient signals are integrated into root developmental programs, we investigated the morphological response of ...
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Stem Cell

Microvesicle Isolation from Rat Brain Extract Treated Human Mesenchymal Stem Cells Authors:  Ji Yong Lee, Seong-Mi Choi and Han-Soo Kim, date: 07/05/2017, view: 2897, Q&A: 0
Microvesicle (MVs) are submicron-sized membranous vesicles that are either actively released from cells via secretory compartments or shed from cell surface membranes. MVs are generated by many cell types and serve as vehicles that transfer biological information (e.g., protein, mRNA, and miRNA) to distant cells, thereby affecting their ...
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