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Past Issues in 2016

Volume: 6 Issue: 3

Cancer Biology

Stable Isotope Resolved Metabolomics Studies in ex vivo TIssue Slices Authors: Teresa W-M. Fan, Andrew N. Lane and Richard M. Higashi, date: 02/05/2016, view: 8202, Q&A: 0

An important component of this methodology is to assess the role of the tumor microenvironment on tumor growth and survival. To tackle this problem, we have adapted the original approach of Warburg (Warburg, 1923), by combining thin tissue slices with Stable Isotope Resolved Metabolomics (SIRM) to determine detailed metabolic activity of human ...

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Cell Biology

Mouse Oocyte Isolation, Cultivation and RNA Microinjection Authors: Anna Tetkova and Marketa Hancova, date: 02/05/2016, view: 12915, Q&A: 0

Mammalian oocyte is a highly specialized cell, characterized by synthesis and storage of maternal proteins and RNAs that contributes to the meiotic cell cycle and early embryo development. The fully grown oocyte is transcriptionally quiescent and utilizes only transcripts synthesized and stored during the growing phase. Mouse oocytes are often ...

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Immunology

Mouse BMDC-dependent T Cell Polarization Assays Author: Vijaya Satchidanandam, date: 02/05/2016, view: 11075, Q&A: 0

In response to exposure to antigen, T cells whose T cell receptor (TCR) are capable of recognizing the self MHC-antigen derived peptide complex, respond to the antigen and differentiate into one of several subsets, namely TH1, TH2, TH17, Treg, etc. characterized by the signature cytokine they secrete, namely IFN-γ, IL-4, IL-17 or IL-10, ...

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Cell-based Assays to Monitor AID Activity Authors: Ludivine C. Litzler, Stephen P. Methot, Anne-Marie Patenaude, Astrid Zahn and Javier M. Di Noia, date: 02/05/2016, view: 7019, Q&A: 0

The enzyme Activation induced deaminase (AID) underpins antibody affinity maturation and isotype switching through its mutagenic activity of deaminating deoxycytidine to deoxyuridine in DNA. Subsequent processing of the deoxyuridine initiates the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. ...

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Microbiology

Calculation of Microorganism Lag Times as a Measure of Adaptative Capability between Different Growth Conditions Author: Brice Enjalbert, date: 02/05/2016, view: 5282, Q&A: 0

This protocol has been designed as a simple and efficient way to investigate microorganism adaptive capabilities (Enjalbert et al., 2015). It is performed using switch experiments in which cells are initially grown in the first condition (primary cultures), then rapidly switched to the second condition (secondary culture) without ...

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Design and Functional Analysis of Fluorescent Nitrate and Peptide Transporter Activity Sensors in Yeast Cultures Authors: Cheng-Hsun Ho and Wolf B. Frommer, date: 02/05/2016, view: 8046, Q&A: 0

This protocol describes the methods used to engineer and deploy genetically encoded fluorescence activity reporters for nitrate and peptide transporter activity in yeast cells. Fusion of the dual-affinity nitrate transceptor CHL1/AtNRT1.1/AtNPF6.3 or four different peptide transporters (AtPTR1, 2, 4, and 5) from Arabidopsis to a pair of ...

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Neuroscience

Craniotomy for Cortical Voltage-sensitive Dye Imaging in Mice Authors: Takayuki Suzuki and Masanori Murayama, date: 02/05/2016, view: 11539, Q&A: 0

Cortico-cortical interactions play crucial roles in various brain functions. Here, we present a detailed surgical procedure for cortical voltage-sensitive dye (VSD) imaging that allows monitoring of spatiotemporal dynamics in cortical activity in living mice. Cortical neurons in the upper layers (layer 1-3) are stained with a VSD, and an image ...

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Plant Science

Measurement of PI4P Levels in Intact Chloroplasts Isolated from Arabidopsis thaliana Authors: Kumiko Okazaki, Shin-ya Miyagishima and Hajime Wada, date: 02/05/2016, view: 7523, Q&A: 1

Phosphatidylinositol 4-phosphate (PI4P), a major species of phosphoinositides, modulates many fundamental cellular processes. We have recently revealed that PI4P plays an important role in chloroplast division as a negative regulator. Despite its importance in chloroplasts, the content of PI4P in chloroplasts is very low and it is difficult to ...

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Preparation of Chloroplast Lipid Membrane and Lipid-protein Interaction Assay Authors: Kumiko Okazaki, Shin-ya Miyagishima and Hajime Wada, date: 02/05/2016, view: 7446, Q&A: 0

Lipid-Protein interaction assay is a method to search lipids, which are bound with proteins in vitro. Since membranes that are spotted with chloroplast lipids such as monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG) are not commercially available, we extracted these lipids ...

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Quantification of Ethylene Production in Tomato Leaves Infected by Xanthomonas euvesicatoria Authors: Jung-Gun Kim, William Stork and Mary Beth Mudgett, date: 02/05/2016, view: 8575, Q&A: 0

Ethylene is a gaseous plant hormone controlling fruit ripening, flower opening, leaf senescence as well as abscission, and disease symptom development. Ethylene plays a critical role in the bacterial pathogen Xanthomonas euvesicatoria (X. euvesicatoria)-elicited symptom development in tomato. This protocol describes the ...

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Structured Illumination Microscopy (SIM) and Photoactivated Localization Microscopy (PALM) to Analyze the Abundance and Distribution of RNA Polymerase II Molecules on Flow-sorted Arabidopsis Nuclei Authors: Klaus Weisshart, Jörg Fuchs and Veit Schubert, date: 02/05/2016, view: 10156, Q&A: 0

RNA polymerase II (RNAPII) is the enzyme transcribing most of the eukaryotic protein-coding genes. Analysing the distribution and quantification of RNAPII can help understanding its function in interphase nuclei. Although several investigations in mammals indicate the organization of RNAPII in so-called ‘transcription factories’ (Jackson et al. ...

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Stem Cell

Transfection of Embryoid Bodies with miRNA Precursors to Induce Cardiac Differentiation Authors: Federica Pisano and Massimiliano Gnecchi, date: 02/05/2016, view: 7099, Q&A: 1

In recent years, the utilization of stem cell therapy to regenerate cardiac tissue has been proposed as a possible strategy to treat cardiac damage (Gnecchi et al., 2012, Aguirre et al., 2013; Sanganalmath and Bolli, 2013). Although encouraging results have been obtained in experimental models, the efficiency of cardiac ...

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