Volume: 6 Issue: 16 —BIO-PROTOCOL

Biochemistry

Purification of Flagellin from Acidovorax avenae and Analysis of Plant Immune Responses Induced by the Purified Flagellin Authors: Hiroyuki Hirai, Takehito Furukawa, Yuya Katsuragi and Fang-Sik Che, date: 08/20/2016, view: 4745, Q&A: 0

Plants sense potential pathogens by recognizing conserved pathogen-associated molecular patterns (PAMPs) that cause PAMP-triggered immunity (PTI) including the generation of reactive oxygen species, callose deposition, and expression of several PTI-related genes. Acidovorax avenae is a Gam-negative bacterium that causes a seedling disease ...

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Determination of VPS34/PIK3C3 Activity in vitro Utilising ³²P-γATP Authors: Michael J. Munson and Ian G. Ganley, date: 08/20/2016, view: 5533, Q&A: 0

VPS34 is the only class III phosphatidylinositol-3-kinase (PI3K) in mammalian cells and produces the vast majority of cellular phosphatidylinositol-3-phosphate [PI(3)P]. PI(3)P is a key signalling lipid that plays many membrane trafficking roles in processes such as endocytosis and autophagy. VPS34 is a key cellular regulator, loss of function can ...

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Determination of the H⁺-ATP Synthase and Hydrolytic Activities Authors: Javier García-Bermúdez, Cristina Nuevo-Tapioles and José M. Cuezva, date: 08/20/2016, view: 4985, Q&A: 0

The H⁺-ATP synthase of the inner mitochondrial membrane utilizes the proton gradient generated by the respiratory chain to synthesize ATP. Under depolarizing conditions, it can function in reverse by hydrolyzing ATP to generate a proton gradient. The protocols presented here allow the facile determination of both the synthetic and ...

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Quantification of 2-Hydroxyglutarate Enantiomers by Liquid Chromatography-mass Spectrometry Authors: William M. Oldham and Joseph Loscalzo, date: 08/20/2016, view: 6400, Q&A: 0

Two enantiomers of 2-hydroxyglutarate (2HG), L (L2HG) and D (D2HG), are metabolites of unknown function in mammalian cells that were initially associated with separate and rare inborn errors of metabolism resulting in increased urinary excretion of 2HG linked to neurological deficits in children (Chalmers et al., 1980; Duran et al ...

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Cell Biology

Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX) Authors: Michael J. Munson and Ian G. Ganley, date: 08/20/2016, view: 4758, Q&A: 0

The lipid Phosphatidylinositol-3-phosphate [PtdIns3P or PI(3)P] plays many membrane trafficking roles and is primarily produced by the Class III PI3K, VPS34. Determining the level of cellular PI(3)P however can be complex. Extraction of cellular lipids by methanol/chloroform can struggle to separate and identify distinct phospholipid species. ...

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Immunology

Adoptive Transfer of Tumor Expanded Regulatory T Cells (Tregs) Authors: Felipe Vences-Catalán and Shoshana Levy, date: 08/20/2016, view: 6024, Q&A: 0

Regulatory T cells (Tregs), a subset of CD4⁺CD25⁺ T cells, infiltrate tumors and suppress antitumor activity of effector T and NK cells. Depletion of Tregs by anti CD25⁺ antibodies has been shown to reduce tumor growth and metastasis (Olkhanud et al., 2009). Conversely, adoptive transfer of Tregs induced ...

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Macrophage Polarization by Tumor-induced MDSCs Assay Authors: Felipe Vences-Catalán, Minu K. Srivastava and Shoshana Levy, date: 08/20/2016, view: 7472, Q&A: 0

Myeloid derived suppressor cells (MDSCs) are a subset of granulocytes (immature myeloid cells) that exploit a variety of mechanism to modulate the innate and adaptive immune system. MDSCs are present normally in the body, but their numbers increase during inflammation and in cancer, promoting an immunosuppressive microenvironment. In addition to ...

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Chromium-51 (⁵¹Cr) Release Assay to Assess Human T Cells for Functional Avidity and Tumor Cell Recognition Authors: Petra Baumgaertner, Daniel E. Speiser, Pedro Romero, Nathalie Rufer and Michael Hebeisen, date: 08/20/2016, view: 10192, Q&A: 0

Cytotoxic CD8⁺ T cells are able to specifically recognize and kill target cells through specific interaction between their T cell receptors (TCRs) and small immunogenic peptides (antigens) presented by major histocompatibility complex (MHC) molecules. The antigen recognition capacity and in vitro lytic activity of ...

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Microbiology

A Modified Chromogenic Assay for Determination of the Ratio of Free Intracellular NAD⁺/NADH in Streptococcus mutans Authors: Jonathon L. Baker, Roberta C. Faustoferri and Robert G. Quivey, Jr, date: 08/20/2016, view: 7259, Q&A: 2

Nicotinamide adenine dinucleotide is a coenzyme present in all kingdoms of life and exists in two forms: oxidized (NAD⁺) and reduced (NADH). NAD(H) is involved in a multitude of essential metabolic redox reactions, providing oxidizing or reducing equivalents. The ratio of free intracellular NAD⁺/NADH is fundamentally ...

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Neuroscience

A Controlled Cortical Impact Mouse Model for Mild Traumatic Brain Injury Authors: Heidi Y Febinger, Hannah E Thomasy and Carmelina Gemma, date: 08/20/2016, view: 7548, Q&A: 0

Traumatic brain injury (TBI) affects millions of people worldwide; however, the immediate impact of TBI and the secondary injury mechanisms are still not fully understood. TBI can cause devastating neuromotor deficits in both acute and chronic stages. Time course studies utilizing animal models of focal TBI have provided essential insight into TBI ...

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Primary Explosive Blast-induced Traumatic Brain Injury Model in PC12 Cell Culture Authors: Thuvan Piehler, Nicole Zander and Richard Benjamin, date: 08/20/2016, view: 4137, Q&A: 0

While it is understood that structural damage occurs at the cellular level from the traumatic brain injury event, the effect on functional activity remains largely unknown. Simplified models such as in vitro models of primary explosive blast are critically needed to deconvolute mechanisms of cellular damage. This protocol details an in ...

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Plant Science

Determination of Recombinant Mannitol-1-phosphatase Activity from Ectocarpus sp. Authors: Agnès Groisillier and Thierry Tonon, date: 08/20/2016, view: 4829, Q&A: 0

Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly, but not exclusively, in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of ...

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¹⁵N-nitrate Uptake Activity and Root-to-shoot Transport Assay in Rice Authors: Yongqiang Liu, Bin Hu and Chengcai Chu, date: 08/20/2016, view: 6390, Q&A: 0

¹⁵N is a nonradioactive heavy isotope of nitrogen, widely used for biochemical and physiological research in plants. For instance, ¹⁵N-KNO₃ was used as the nitrogen source in plants in order to investigate nitrate uptake activity and transport from roots to shoots (Lin et al., 2008). Here, we describe a ...

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Experimental Pipeline for SNP and SSR Discovery and Genotyping Analysis of Mango (Mangifera indica L.) Authors: Michal Sharabi-Schwager, Mor Rubinstein, Mazal Ish shalom, Ravit Eshed, Ada Rozen, Amir Sherman, Yuval Cohen and Ron Ophir, date: 08/20/2016, view: 6242, Q&A: 0

Establishing a reservoir of polymorphic markers is an important key for marker-assisted breeding. Many crops are still lack of such genomic infrastructure. Single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) are useful as markers because they are widespread over the genome and many technologies were developed for high ...

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Stem Cell

Bone Marrow-derived Endothelial Progenitor Cell Intercellular Adhesion Assay Authors: Yubo Tang, Angela Jacobi, Corina Vater, Xuenong Zou and Maik Stiehler, date: 08/20/2016, view: 6080, Q&A: 0

Cell-cell adhesion ensures tight contacts between neighbouring cells, which is necessary for cell segregation, as well as for the morphological and functional differentiation of different tissues. Evidently there are cell-cell recognition systems that make cells of the same type preferentially adherent to one another. Homotypic cell adhesion is ...

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Past Issues in 2016

Volume: 6 Issue: 16

Biochemistry

Cell Biology

Immunology

Microbiology

Neuroscience

Plant Science

Stem Cell