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Past Issues in 2013

Volume: 3 Issue: 22

Cell Biology

Harvest and Culture of Mouse Peritoneal Macrophages Authors: Mingfang Lu and Alan W. Varley, date: 11/20/2013, view: 33529, Q&A: 1

Peritoneal macrophages are used as primary macrophages in lots of studies, mainly because they are easy to obtain. Injection of thioglycollate broth i.p. induces inflammatory responses and elicits large numbers of macrophages. This protocol can be used for harvesting resident or thioglycollate-elicited peritoneal cells. Peritoneal macrophages are ...

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Immunology

Gastric Aspiration Models Authors: Bruce A. Davidson and Ravi Alluri, date: 11/20/2013, view: 16896, Q&A: 0

The procedures described below are for producing gastric aspiration pneumonitis in mice with alterations for rats and rabbits described parenthetically. We use 4 different injury vehicles delivered intratracheally to investigate the inflammatory responses to gastric aspiration:
1) Normal saline (NS) as the injury vehicle control ...

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Immunocytochemical Detection of Recombinant Biomphalysin on Schistosoma mansoni Sporocysts Authors: David Duval, Richard Galinier, Julien Portela, Guillaume Mitta and Benjamin Gourbal, date: 11/20/2013, view: 7439, Q&A: 0

Schistosomiasis, or bilharzia, is a tropical disease caused by worms of the genus Schistosoma which infect about 200 million people. The life cycle of the parasite requires Biomphalaria, a specific genus of freshwater snails, as intermediate. Using an interactome approach employing B. glabrata plasma and S. mansoni ...

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Assessment of Human Dendritic Cell Antigen Uptake by Flow Cytometry Authors: Ana Luque, Sonia Cárdenas-Brito, Rut Olivar and Josep M. Aran, date: 11/20/2013, view: 14410, Q&A: 0

Antigen uptake by dendritic cells is the first key step towards induction of antigen-specific T-cell responses. This flow cytometry-based protocol describes the analysis of dendritic cell uptake of soluble antigens through two different mechanisms: non-specific macropinocytosis (using Lucifer Yelloy CH), and receptor-mediated endocytosis (using DQ ...

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Microbiology

Separation and Detection of Phosphorylated and Nonphosphorylated BvgA, a Bordetella pertussis Response Regulator, in vivo and in vitro Authors: Qing Chen, Alice Boulanger, Deborah M. Hinton and Scott Stibitz, date: 11/20/2013, view: 9332, Q&A: 0

Protein phosphorylation plays a central role in signal transduction in bacteria. However, separation and detection of the phosphorylated protein from its nonphosphorylated form remain challenging. Here we describe a method to detect phosphorylation of the Bordetella pertussis response regulator BvgA, which is phosphorylated at an ...

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Transport Assays in Aspergillus nidulans Authors: Emilia Krypotou and George Diallinas, date: 11/20/2013, view: 6933, Q&A: 0

Transport assays allow the direct kinetic analysis of a specific transporter by measuring apparent K_m and V_max values, and permit the characterization of substrate specificity profiles through competition assays. In this protocol, we describe a rapid and easy method for performing uptake assays in the model ...

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Shigella IpaD and IpaB Surface Localizations Authors: Lionel Schiavolin, Alaeddine Meghraoui and Abdelmounnaim Allaoui, date: 11/20/2013, view: 8778, Q&A: 1

Shigella uses a type III secretion system to invade host cell and to cause disease. Secretion control and insertion of a translocation pore into cell membrane are critical steps for pathogenesis and are tightly linked to the formation of the needle tip complex formed by the IpaB and IpaD proteins (Veenendaal et al., 2007). ...

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Assay to Evaluate Vascular Permeability Induction in Mice Authors: Henry Puerta-Guardo, Arturo Raya-Sandino, Lorenza González-Mariscal, Victor H. Rosales, José Ayala-Dávila, Bibiana Chávez-Mungía, Daniel Martínez-Fong, Fernando Medina, Juan E. Ludert and Rosa María del Angel, date: 11/20/2013, view: 19743, Q&A: 0

Dengue virus infection usually courses as a benign self-limited fever, called dengue fever. However, on occasions it can progress to a life-threatening complication known as severe dengue (SD). A hallmark of SD is a sharp increase in vascular permeability. Secondary infections are considered a risk factor to develop SD, presumably through a ...

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Neuroscience

Cell Cycle Analysis in the Vertebrate Brain Using Immunolabeled Fresh Cell Nuclei Authors: Noelia Lopez-Sanchez and Jose M. Frade, date: 11/20/2013, view: 14503, Q&A: 0

Flow cytometry, a standard technique used for quantitative analysis of isolated cells, is routinely employed by immunologists and oncologists to study DNA content, protein expression, and other functional parameters in blood and tumor cells. Unfortunately, the use of this technique by neurobiologists has been hampered by the complexity of the ...

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Plant Science

Mapping and Analysis of Illumina Reads for Transcriptome of Medicago Truncatula During the Early Organogenesis of the Nodule Authors: Alexandre Boscari, Alberto Ferrarini, Jennifer del Giudice, Luca Venturini, Massimo Delledone and Alain Puppo, date: 11/20/2013, view: 9120, Q&A: 0

Medicago truncatula serves as a model plant for legume genetics and genomics. We used RNA-Seq to characterize the transcriptome during the early organogenesis of the nodule and during its functioning. We generated approximately 135.5 million high-quality 36-bp reads, which were then aligned with the M. truncatula genome sequence ...

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Analysis of RNA-protein Interactions Using Electrophoretic Mobility Shift Assay (Gel Shift Assay) Authors: Saiprasad Goud Palusa and Anireddy S. N. Reddy, date: 11/20/2013, view: 25349, Q&A: 5

RNA binding proteins (RBPs) play a crucial role in regulating gene expression at the post-transcriptional level at multiple steps including pre-mRNA splicing, polyadenylation, mRNA stability, mRNA localization and translation. RBPs regulate these processes primarily by binding to specific sequence elements in nascent or mature transcripts. There ...

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Cotton Ovules Culture and Analysis Authors: Jiafu Tan, Fenglin Deng, Wenxin Tang, Jie Han, Guo Kai, Lili Tu and Xianlong Zhang, date: 11/20/2013, view: 8764, Q&A: 0

The cotton ovules culture was innovated by Beasley and Ting (1973), and named after them. It is a convenient system to analyze the effect of chemical or environmental treatment on fiber development directly on ovules. This protocol was generated according to previous published papers and our practical experience.

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Shikimate Hydroxycinnamoyl Transferase (HCT) Activity Assays in Populus nigra Authors: Igor Cesarino, Ruben Vanholme, Geert Goeminne, Bartel Vanholme and Wout Boerjan, date: 11/20/2013, view: 11853, Q&A: 0

Lignin is a complex phenolic polymer deposited in secondarily-thickened plant cell walls. The polymer is mainly derived from the three primary monolignols: p-coumaryl, coniferyl and sinapyl alcohol which give rise to p-hydroxyphenyl, guaiacyl and syringyl units (H, G and S units, respectively) when coupled into the polymer. The building ...

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Bimolecular Fluorescence Complementation (BIFC) Protocol for Rice Protoplast Transformation Authors: Kun Wang, Ying Liu and Shaoqing Li, date: 11/20/2013, view: 11963, Q&A: 0

After the plant cells are removed the cell walls by digestive enzyme, the plant protoplasts still have good cell activity. The protoplasts can be used to transiently express proteins of target genes in living plant cells through polyethylene glycol (PEG) mediated transformation. The purpose of this method is to employ the rice protoplasts and ...

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Binding Assay of Cytosolic Proteins to the Cytoskeleton Authors: Stefano Del Duca and Giampiero Cai, date: 11/20/2013, view: 10191, Q&A: 2

Many cellular proteins interact with the cytoskeleton (both actin filaments and microtubules), either dynamically or permanently. This interaction is required during different aspects of the cell life, for example during the process of cell division. In addition, many enzymes interact transiently with actin filaments and microtubules in order to ...

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