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Cancer Biology

Vaccine-induced Cytokine Production Detected by Luminex Multiplex Analysis Authors:  Rachel Perret, Sophie R. Sierro, Natalia K. Botelho, Stephanie Corgnac, Alena Donda and Pedro Romero, date: 08/20/2014, view: 4465, Q&A: 0
Different vaccine and adjuvant combinations are known to rapidly induce antigen presenting cell (APC) maturation and pro-inflammatory cytokine and production, which in turn play an important role in the priming of antigen-specific T cells. Measuring cytokine production systemically in the serum fails to detect localized responses in the lymph ...
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Analysis of Tumor-infiltrating Lymphocytes Following CD45 Enrichment Authors:  Rachel Perret, Sophie R. Sierro, Natalia K. Botelho, Stephanie Corgnac, Alena Donda and Pedro Romero, date: 08/20/2014, view: 11539, Q&A: 0
Measuring antigen-specific T cell responses in the blood and lymphoid organs of vaccinated mice can give us a useful indication of the potency of a vaccine formulation. Unfortunately, systemic or even localized lymphoid T cell responses are not always predictive of the ability of a vaccine to induce tumor protection. Measuring the antigen-specific ...
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Immunology

Enzymatic Reactions and Detection of C3 Cleavage Fragments Authors:  Claudia Kemper and Martin Kolev, date: 08/20/2014, view: 4983, Q&A: 0
The complement component C3 is the major effector molecule of the complement system. C3 circulates in the blood and interstitial fluids as pro-enzyme and is activated by enzymatic cleavage into a C3a portion, a classic anaphylatoxin that functions as chemoattractant and immune cell activator, and the C3b portion, the body’s most potent opsonin. C3 ...
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Signaling Assays for Detection of Human G-protein-coupled Receptors in Yeast Authors:  Yasuyuki Nakamura, Jun Ishii and Akihiko Kondo, date: 08/20/2014, view: 5294, Q&A: 0
G-protein-coupled receptors (GPCRs) are the largest group of cell-surface proteins and are major molecular targets for drug development. The protocol described herein is for the detection of human GPCR signaling in the yeast Saccharomyces cerevisiae. Using Zoanthus sp. green fluorescent protein (ZsGreen) as the reporter, ...
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Microbiology

In vitro DNA Polymerization Activity Assay Using Cell-free Extracts Authors:  Anurag K. Sinha and Malay K. Ray, date: 08/20/2014, view: 4151, Q&A: 0
This protocol has been designed to measure the in-vitro DNA polymerization activity in crude cell extracts of the Antarctic bacterium Pseudomonas syrinagae Lz4W. This bacterium can grow at 4 °C with optimum growth rate at 22 °C. The slow growth rate of the bacterium observed at low temperature (4 °C) compared to higher ...
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Endolysin Expression, Purification and Activity Determination by Zymography Authors:  Alexander B. Westbye, Paul C. Fogg and John Thomas Beatty, date: 08/20/2014, view: 7251, Q&A: 0
Endolysins are peptidoglycan-degrading (muralytic) enzymes produced by many bacteriophages for cell lysis of the host bacterium. The enzymatic activity of muralytic enzymes can be assayed qualitatively using a zymogram containing incorporated peptidoglycan. This protocol describes the expression of a recombinant 6x His-tagged endolysin using an ...
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VZV Replication Assays Authors:  Samantha J. Griffiths and Jürgen Haas, date: 08/20/2014, view: 5587, Q&A: 0
Varicella zoster virus (VZV) is a human herpesvirus which causes Varicella (chickenpox) upon primary infection and Zoster (shingles) following reactivation from latency (von Bokay, 1909). Whilst VZV is extensively studied, inherent features of VZV replication, such as cell-association of virus particles during in vitro culture and a ...
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HIV-1 Fusion Assay Authors:  Marielle Cavrois, Jason Neidleman and Warner C. Greene, date: 08/20/2014, view: 7769, Q&A: 0
The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase–Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a ...
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Neuroscience

Elevated Plus Maze Test to Assess Anxiety-like Behavior in the Mouse Authors:  Luciana M. Leo and Fabricio A. Pamplona, date: 08/20/2014, view: 12525, Q&A: 0
The elevated plus maze task is a simple method to assess anxiety-like behaviors in rodents. This version describes the procedure used in mice. However, the protocol may also be applied to rats, considering a proportionally larger apparatus (arms: 10 x 50 cm; height: 55 cm). Briefly, the test is performed on a plus-shaped apparatus with two open ...
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Plant Science

Assays of Polyphenol Oxidase Activity in Walnut Leaf Tissue Authors:  Ross Gertzen and Matthew A. Escobar, date: 08/20/2014, view: 9938, Q&A: 0
Polyphenol oxidase (PPO) is an enzyme that catalyzes the hydroxylation of monophenols into ortho-diphenols (cresolase activity) and the oxidation of o-diphenols into quinones (catecholase activity) (Figure 1). These quinones spontaneously polymerize to form dark-colored phytomelanins, most often seen in the browning of damaged plant ...
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SGR-based Reporter to Assay Plant Transcription Factor-promoter Interactions Authors:  Shisong Ma and Savithramma Dinesh-Kumar, date: 08/20/2014, view: 5096, Q&A: 0
We developed an in vivo method to assay plant transcription factor (TF)–promoter interactions using the transient expression system in Nicotiana benthamiana (N. benthamiana) plants. The system uses the Arabidopsis stay green (SGR) gene as a reporter. Induction of SGR expression in N. ...
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Salinity Assay in Tomato Authors:  Begoña Renau-Morata, Manuel Sánchez-Perales, Joaquín Medina, Rosa Victoria Molina, Rocío Corrales, Laura Carrillo, Pedro Fernández-Nohales, Jorge Marqués, Stephan Pollmann, Jesús Vicente-Carbajosa, Antonio Granell and Sergio G. Nebauer, date: 08/20/2014, view: 7884, Q&A: 1
Tomato is one of the most important horticultural crops worldwide, and is cultivated in semi-arid regions in which soil and groundwater salinity is an increasing limitation to yield. The assessment of the responses of new cultivars to salt and the comparisons among cultivars and wild species are of great interest in tomato breeding. This assay ...
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Salinity Assay in Arabidopsis Authors:  Alba Rocío Corrales, Laura Carrillo, Sergio G. Nebauer, Begoña Renau-Morata, Manuel Sánchez-Perales, Pedro Fernández-Nohales, Jorge Marqués, Antonio Granell, Stephan Pollmann, Jesús Vicente-Carbajosa, Rosa Victoria Molina and Joaquín Medina, date: 08/20/2014, view: 8899, Q&A: 0
Salinity is an important environmental constraint to crop productivity in arid and semi-arid regions of the world. The evaluation of the responses to salinity of different Arabidopsis ecotypes or transgenic lines is important to identify and investigate the role of different key genes. These new characterized genes involved in the ...
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Systems Biology

siRNA Screening for Genes Involved in HSV-1 Replication Authors:  Samantha J. Griffiths and Jürgen Haas, date: 08/20/2014, view: 4941, Q&A: 0
Small interfering RNAs (siRNAs) are small (typically 18-24 nucleotides) RNA molecules capable of silencing gene expression post-transcriptionally and as such, they provide a simple method by which the role of individual genes in complex cellular systems can be easily assessed. As siRNAs are easy to use experimentally, and can be designed to target ...
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