Volume: 6 Issue: 10 —BIO-PROTOCOL

Cell Biology

Isolating Liver Mitochondria by Differential Centrifugation Authors: Ignacio Amigo, Javier Traba and Carlos B. Rueda, date: 05/20/2016, view: 14337, Q&A: 0

In addition to methods aimed at the study of mitochondrial function in-situ, a full understanding of mitochondrial function requires their purification from cells or tissues under specific physiological or pathological conditions. This protocol illustrates a sequential procedure to obtain functional mitochondria with high yield from mice liver ...

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Liquid Luminescent DNA-precipitation Assay Authors: Frédérique Végran, Mélanie Bruchard, Valentin Derangère and François Ghiringhelli, date: 05/20/2016, view: 6401, Q&A: 0

Working on transcription factors requires studying interactions between protein and DNA. After identification of putative binding-sequences and motifs, Electrophoretic Mobility Shift Assay (EMSA) experiment is classically used to determine specific interactions of proteins and nucleic acids. This lengthy process is rather heavy-handed because of ...

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Immunology

Proximity Ligation Assay (PLA) Protocol Using Duolink^® for T Cells Authors: Valentin Derangère, Mélanie Bruchard, Frédérique Végran and François Ghiringhelli, date: 05/20/2016, view: 14040, Q&A: 0

Protein-protein interaction experiments, such as co-immunoprecipitation (IP) assays, classically require tremendous amount of cells. This becomes a problem when your work focuses on rare cell populations (e.g., lymphocyte subtypes). O-link Bioscience has developed Proximity Ligation Assay (PLA) reagents and procedures to alleviate and ...

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Reconstitution of Lymphopaenic Mice with Regulatory and Conventional T cell Subsets Authors: Holly A. Bolton and Barbara Fazekas de St Groth, date: 05/20/2016, view: 6262, Q&A: 0

Transfer of mature T cells into immunodeficient mice results in sub-optimal reconstitution of the peripheral T cell pool. Under lymphopenic conditions, dendritic cells are released from tonic control by regulatory T cells (Tregs), and consequently drive activation and proliferation of low affinity T cells specific for endogenous antigens. This ...

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Study of Epithelium Barrier Functions by Real-time TER Measurement Authors: Jingjing Meng and Sabita Roy, date: 05/20/2016, view: 6123, Q&A: 0

Transepithelial Electrical Resistance (TER) measurement is a reliable and efficient method to quantify the permeability of barrier forming cells such as epithelial cells. Measuring the permeability of the epithelial cells will help the researchers to investigate the barrier function of epithelium in various infectious and inflammatory diseases. ...

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Extraction and Quantification of Sphingosine 1-Phosphate (S1P) Authors: Christina-Maria Reimann and Markus H. Gräler, date: 05/20/2016, view: 7085, Q&A: 0

Sphingosine 1-phosphate (S1P) is a lipid metabolite and signaling molecule involved in many different physiological processes including lymphocyte circulation, T cell differentiation, antigen presentation, and maintenance of the vascular endothelial barrier. S1P is a ligand of five different G protein-coupled cell surface receptors designated S1P ...

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Microbiology

Induction, Isolation and Counting of Akinetes in Aphanizomenon ovalisporum Authors: Assaf Sukenik, Ruth N. Kaplan-Levi, Yehudit Viner-Mozzini, Achsa Lupu and Dotan Sela, date: 05/20/2016, view: 6178, Q&A: 0

Akinetes are spore-like resting (dormant) cells formed by strains of filamentous cyanobacteria for surviving long periods of unfavorable conditions. During deprivation for potassium, vegetative photosynthetic cells along the filaments of the cyanobacterium Aphanizomenon ovalisporum (A. ovalisporum) (strain ILC-164) differentiate ...

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Nitrite Reduction Assay for Whole Pseudomonas Cells Authors: Michael P. Thorgersen and Michael W. Adams, date: 05/20/2016, view: 9028, Q&A: 1

The second step of the dissimilatory denitrification pathway in which nitrite (NO₂^-) is converted to nitric oxide (NO) is catalyzed by the enzyme nitrite reductase. Two distinct enzymes are found in nature that catalyze this reaction, and they contain different metal sites, either iron (Fe), in the form of heme, or copper ...

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Respiratory Syncytial Virus Infection in Mice and Detection of Viral Genomes in the Lung Using RT-qPCR Authors: Yan Sun and Carolina B. López, date: 05/20/2016, view: 7401, Q&A: 0

Respiratory syncytial virus (RSV) is a single-stranded negative sense RNA virus that belongs to the paramyxovirus family. RSV infections lead to a variety of clinical outcomes ranging from a mild “cold-like disease” to death. Infection is usually more severe in infants and the elderly. RSV is associated with the development and ...

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Preparation of Respiratory Syncytial Virus with High or Low Content of Defective Viral Particles and Their Purification from Viral Stocks Authors: Yan Sun and Carolina B. López, date: 05/20/2016, view: 13182, Q&A: 0

Respiratory syncytial virus (RSV) belongs to the paramyxovirus family that includes many clinically relevant viruses, such as the human metapneumovirus and measles. RSV infection can cause severe disease in infants, the elderly, and some immunocompromised adults. During RSV replication, a series of truncated forms of the viral genome is ...

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Molecular Biology

Affymetrix Genome-wide Human SNP Assay Author: Alexis Bosman, date: 05/20/2016, view: 6041, Q&A: 0

To assess genomic variation, it is possible to identify the single nucleotide polymorphisms (SNP) which an individual carries. Using the Affymetrix Genome-wide Human SNP Assay, it is possible to assess 906,600 SNPs on a single array. This protocol, the next iteration of the GeneChip Mapping 500K array set, is based directly on the manufacturers’ ...

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Preparation of Knockdown Transformants of Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex Authors: Jun Abe, Naoko Hirano, Ayumi Komiya, Naho Kanda, Anri Fujiwara, Sachie Hori, Yuki Tsuchikane and Hiroyuki Sekimoto, date: 05/20/2016, view: 6713, Q&A: 0

To prepare the knockdown transformants of the Closterium peracerosum-strigosum-littorale (C. psl.) complex, particle bombardment was applied with a newly constructed vector (pSA0104) with an endogenous constitutive promoter fused to a DNA fragment corresponding to an antisense strand of a target gene. Using a hygromycin ...

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Neuroscience

Biotinylation and Purification of Plasma Membrane-associated Proteins from Rodent Cultured Neurons Authors: Margarida Caldeira, Joana S. Ferreira, Ana Luísa Carvalho and Carlos B. Duarte, date: 05/20/2016, view: 8040, Q&A: 0

This protocol aims at the biotin labeling and affinity purification of plasma membrane proteins from cultured neurons. Protein biotinylation consists in the covalent attachment of biotin to proteins. Biotin is a membrane unpermeable molecule with a small size (MW 244.31 g/mol) and therefore does not interfere with the normal function of proteins. ...

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Isolating Brain Mitochondria by Differential Centrifugation Authors: Ignacio Amigo, Javier Traba and Carlos B. Rueda, date: 05/20/2016, view: 12878, Q&A: 3

In addition to methods aimed at the study of mitochondrial function in-situ, a full understanding of mitochondrial function requires their purification from cells or tissues under specific physiological or pathological conditions. This protocol illustrates a sequential procedure to obtain functional mitochondria with high yield from mice ...

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Running Reward Conditioned Place Preference Task Authors: Maria Fernanda A. Fernandes and Stephanie Fulton, date: 05/20/2016, view: 10026, Q&A: 0

The conditioned place preference (CPP) test is a standard pre-clinical behavioral tool used to study the motivational effects of drugs and non-drug treatments in experimental animals. The basic characteristic of this task involves the association of a particular environment and contextual cues with a reward stimulus, followed by the association of ...

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Past Issues in 2016

Volume: 6 Issue: 10

Cell Biology

Immunology

Microbiology

Molecular Biology

Neuroscience