Volume: 6 Issue: 23 —BIO-PROTOCOL

Cancer Biology

DNA Damage Induction by Laser Microirradiation Authors: Marianna Tampere and Oliver Mortusewicz, date: 12/05/2016, view: 11829, Q&A: 1

Genome instability can lead to cell death, senescence and cancerous transformation. Specific repair pathways have evolved to prevent accumulation of DNA lesions. Studying these highly dynamic and specific repair pathways requires precise spatial and temporal resolution, which can be achieved through a combination of laser microirradiaiton and live ...

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Lentiviral shRNA Screen to Identify Epithelial Integrity Regulating Genes in MCF10A 3D Culture Authors: Elsa Marques and Juha Klefström, date: 12/05/2016, view: 6076, Q&A: 0

MCF10A 3D culture system provides a reductionist model of glandular mammary epithelium which is widely used to study development of glandular architecture, the role of cell polarity and epithelial integrity in control of epithelial cell functions, and mechanisms of breast cancer. Here we describe how to use shRNA screening approach to identify ...

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Cell Biology

Lymphocyte Isolation, Th17 Cell Differentiation, Activation, and Staining Authors: Pawan Kumar and Jay K Kolls, date: 12/05/2016, view: 7405, Q&A: 1

In vitro Th17 (α, β T helper cell which produce IL-17A, IL-17F and IL-22) differentiation has been routinely used for functional T cells studies. Here we describe a method for Th17 cell differentiation.

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Analysis of Myosin II Minifilament Orientation at Epithelial Zonula Adherens Authors: Magdalene Michael, Xuan Liang and Guillermo A. Gomez, date: 12/05/2016, view: 5062, Q&A: 0

Non-muscle myosin II (NMII) form bipolar filaments, which bind F-actin to exert cellular contractility during physiological processes (Vicente-Manzanares et al., 2009). Using a combinatorial approach to fluorescently label both N- and C-termini of the NMII heavy chain, recent works have demonstrated the ability to visualize NMII bipolar ...

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Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays Authors: Ashwin D’Souza, Paulomi Sanghavi, Ashim Rai, Divya Pathak and Roop Mallik, date: 12/05/2016, view: 5715, Q&A: 0

We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined ...

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Developmental Biology

Vascular Smooth Muscle Cell Isolation and Culture from Mouse Aorta Authors: Callie S. Kwartler, Ping Zhou, Shao-Qing Kuang, Xue-Yan Duan, Limin Gong and Dianna M. Milewicz, date: 12/05/2016, view: 12319, Q&A: 0

Vascular smooth muscle cells (SMC) in the ascending thoracic aorta arise from neural crest cells, whereas SMCs in the descending aorta are derived from the presomitic mesoderm. SMCs play important roles in cardiovascular development and aortic aneurysm formation. This protocol describes the detailed process for explanting ascending and descending ...

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Intracellular Assessment of ATP Levels in Caenorhabditis elegans Authors: Konstantinos Palikaras and Nektarios Tavernarakis, date: 12/05/2016, view: 6296, Q&A: 0

Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP ...

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Measuring Oxygen Consumption Rate in Caenorhabditis elegans Authors: Konstantinos Palikaras and Nektarios Tavernarakis, date: 12/05/2016, view: 6341, Q&A: 0

The rate of oxygen consumption is a vital marker indicating cellular function during lifetime under normal or metabolically challenged conditions. It is used broadly to study mitochondrial function (Artal-Sanz and Tavernarakis, 2009; Palikaras et al., 2015; Ryu et al., 2016) or investigate factors mediating the switch from ...

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Microbiology

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection Authors: Jean Kaoru Millet and Gary R. Whittaker, date: 12/05/2016, view: 5785, Q&A: 0

Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway ...

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In vitro Autophosphorylation and Phosphotransfer Assay of Cyanobacterial Histidine Kinase 2 Author: Iskander M. Ibrahim, date: 12/05/2016, view: 6423, Q&A: 0

This is a detailed protocol of an autophosphorylation and phosphotransfer activities of Synechocystis sp. PCC 6803 full-length Histidine Kinase 2 (Hik2) protein described by Ibrahim et al., 2016. In this protocol, radioactively labelled ATP was used to study an autophosphorylation and phosphotransfer activity of the full-length ...

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Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues Authors: Beth Mann, Lip Nam Loh, Geli Gao and Elaine Tuomanen, date: 12/05/2016, view: 5900, Q&A: 0

Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the body, including brain, heart, placenta and ...

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Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria Authors: Monica Benincasa, Quentin Barrière, Giulia Runti, Olivier Pierre, Mick Bourge, Marco Scocchi and Peter Mergaert, date: 12/05/2016, view: 7231, Q&A: 0

Antimicrobial peptides (AMPs) can target the bacterial envelope or alternatively have intracellular targets. The latter requires uptake of the peptide by the bacterial cells. The bacterial internalization of an AMP can be evaluated by a fluorescence-based method that couples the use of the fluorescently labelled AMP to the fluorescence quencher ...

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Mouse Model of Dengue Virus Infection with Serotypes 1 and 2 Clinical Isolates Authors: Satoru Watanabe , Kitti Wing Ki Chan and Subhash G. Vasudevan, date: 12/05/2016, view: 6137, Q&A: 0

Dengue is a global public health threat caused by infection with any of the 4 related dengue virus serotypes (DENV1-4). Clinical manifestations range from self-limiting febrile illness, known as dengue fever (DF), to life-threatening severe diseases, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Most cases of DHF/DSS are ...

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Measurement of Intracellular Calcium Concentration in Pseudomonas aeruginosa Authors: Manita Guragain, Anthony K. Campbell and Marianna A. Patrauchan, date: 12/05/2016, view: 5271, Q&A: 0

Characterization of the molecular mechanisms of calcium (Ca²⁺) regulation of bacterial physiology and virulence requires tools enabling measuring and monitoring the intracellular levels of free calcium (Ca²⁺_in). Here, we describe a protocol optimized to use a recombinantly expressed Ca²⁺-binding protein, ...

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Pyocyanin Extraction and Quantitative Analysis in Swarming Pseudomonas aeruginosa Authors: Michelle M. King, Manita Guragain, Svetlana A. Sarkisova and Marianna A. Patrauchan, date: 12/05/2016, view: 8806, Q&A: 0

This protocol describes the quantification of pyocyanin extracted from swarming colonies of Pseudomonas aeruginosa. Pyocyanin is a secondary metabolite and a major virulence factor, whose production is inducible and varies highly under different growth conditions. The protocol is based on the earlier developed chloroform/HCl extraction of ...

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Neuroscience

Various Modes of Spinal Cord Injury to Study Regeneration in Adult Zebrafish Authors: Subhra Prakash Hui and Sukla Ghosh, date: 12/05/2016, view: 7649, Q&A: 0

Spinal cord injury (SCI) in mammals leads to failure of both sensory and motor functions, due to lack of axonal regrowth below the level of injury as well as inability to replace lost neural cells and to stimulate neurogenesis. In contrast, fish and amphibians are capable of regenerating a variety of their organs like limb/fin, jaw, heart and ...

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MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation Authors: Roberta Antonelli and Paola Zacchi, date: 12/05/2016, view: 5006, Q&A: 0

Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific protein, to identify interacting partners in ...

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Delayed Spatial Win-shift Test on Radial Arm Maze Authors: Simone N. De Luca, Luba Sominsky and Sarah J. Spencer, date: 12/05/2016, view: 6968, Q&A: 0

The radial arm maze (RAM) is used to assess reference and working memory in rodents. This task relies on the rodent’s ability to orientate itself in the maze using extra-maze visual cues. This test can be used to investigate whether a rodent’s cognition is improved or impaired under a variety of experimental conditions. Here, we describe one way ...

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Plant Science

Isolating and Measuring the Growth and Morphology of Pro-embryogenic Masses in Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae) Authors: Jackellinne Caetano Douétts-Peres, Vanildo Silveira, Marco Antonio Lopes Cruz and Claudete Santa-Catarina, date: 12/05/2016, view: 5192, Q&A: 0

Embryogenic suspension cultures of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae) can be used as a model to test the effects of compounds added to the culture medium on the cellular growth and morphology of Pro-Embryogenic Masses (PEMs). PEMs are formed by embryogenic and suspensor-type cells. To measure changes in the cellular ...

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Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes Authors: Mariana Edith Tellechea, Javier Garcia-Pardo, Juliana Cotabarren, Daniela Lufrano, Laura Bakas, Francesc Xavier Avilés, Walter David Obregon, Julia Lorenzo and Sebastián Tanco, date: 12/05/2016, view: 5569, Q&A: 0

Metallocarboxypeptidases (MCP) are zinc-dependent exopeptidases that catalyze the hydrolysis of C-terminal amide bonds in proteins and peptides. They are involved in a wide range of physiological processes and have recently emerged as relevant drug targets in biomedicine (Arolas et al., 2007). In this context, the study and discovery of ...

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Extraction and Measurement of Abscisic Acid in a Unicellular Red Alga Cyanidioschyzon merolae Authors: Yuki Kobayashi and Kan Tanaka, date: 12/05/2016, view: 6190, Q&A: 0

Abscisic acid (ABA) has been known as a phytohormone of land plants, which is synthesized in response to abiotic stresses and induces various physiological responses, but is also found from eukaryotic algae. Recently, we reported that a unicellular red alga Cyanidioschyzon merolae produced ABA, which prevented cell growth and enhanced ...

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Fusarium graminearum Maize Stalk Infection Assay and Associated Microscopic Observation Protocol Authors: Juan He, Tinglu Yuan and Wei-Hua Tang, date: 12/05/2016, view: 6315, Q&A: 0

The ascomycete fungus Fusarium graminearum (previously also called Gibberella zeae) causes Gibberella stalk rot in maize (Zea mays) and results in lodging and serious yield reduction. To develop methods to assess the fungal growth and symptom development in maize stalks, we present here a protocol of maize stalk ...

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Measurement of ATP Hydrolytic Activity of Plasma Membrane H⁺-ATPase from Arabidopsis thaliana Leaves Authors: Masaki Okumura and Toshinori Kinoshita, date: 12/05/2016, view: 5346, Q&A: 0

Plant plasma membrane H⁺-ATPase, which is a P-type ATPase, couples ATP hydrolysis to H⁺ extrusion and thereby generates an electrochemical gradient across the plasma membrane. The proton gradient is necessary for secondary transport, cell elongation, and membrane potential maintenance. Here we describe a protocol for ...

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Shoot Apical Meristem Size Measurement Authors: Hsuan Chou, Huanzhong Wang and Gerald A. Berkowitz, date: 12/05/2016, view: 5545, Q&A: 0

The shoot apical meristem (SAM) is a collection of cells that continuously renew themselves by cell division and also provide cells to newly developing organs. It has been known that CLAVATA (CLV) 3 peptide regulates a transcription factor WUSCHEL (WUS) to keep numbers of undifferentiated cells constant and maintain the size of the SAM. The ...

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A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9 Authors: Johan Vad-Nielsen, Lin Lin , Kristopher Torp Jensen, Anders Lade Nielsen and Yonglun Luo, date: 12/05/2016, view: 14090, Q&A: 1

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve multiplexed gene editing or regulation. Using ...

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Tandem Purification of His₆-3x FLAG Tagged Proteins for Mass Spectrometry from Arabidopsis Authors: He Huang and Dmitri Anton Nusinow, date: 12/05/2016, view: 7205, Q&A: 0

Tandem affinity purification is a powerful method to identify protein complexes that function in association with a known gene of interest. This protocol describes a methodology to capture proteins tagged with His₆-3x FLAG explicitly for the purpose of on-bead digestion and identification by mass spectrometry. The high sensitivity and ...

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Stem Cell

In vitro Chondrogenic Hypertrophy Induction of Mesenchymal Stem Cells Authors: Sang Young Jeong, Miyoung Lee, Soo Jin Choi, Wonil Oh and Hong Bae Jeon, date: 12/05/2016, view: 6677, Q&A: 0

To investigate underlying mechanism of chondrogenic hypertrophy, we need proper in vitro hypertrophic model of mesenchymal stem cells (MSCs). This protocol describes our defined method for induction of in vitro chondrogenic hypertrophy of human umbilical cord blood-derived MSCs (hUCB-MSCs). By adding thyroid hormone (T3; ...

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Systems Biology

Protocol for Molecular Dynamics Simulations of Proteins Authors: MNV Prasad Gajula, Anuj Kumar and Johny Ijaq, date: 12/05/2016, view: 13737, Q&A: 0

Molecular dynamics (MD) simulations have become one of the most important tools in understanding the behavior of bio-molecules on nanosecond to microsecond time scales. In this protocol, we provide a general approach and standard setup protocol for MD simulations by using the Gromacs MD suite.

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Past Issues in 2016

Volume: 6 Issue: 23

Cancer Biology

Cell Biology

Developmental Biology

Microbiology

Neuroscience

Plant Science

Stem Cell

Systems Biology