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Cancer Biology

Karyotype Analysis Authors:  Chun-Hung Chou and Muh-Hwa Yang, date: 05/20/2014, view: 10659, Q&A: 0
A chromosome is the structure that organizes DNA and protein in cells. It is a single piece of coiled DNA containing coding and non-coding sequences. Human cells have 23 pairs of chromosomes including 22 pairs of autosomes and one pair of sex chromosome, giving a total of 46 per cell. In tumor cells, chromosomal instability has been considered to ...
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Cell Biology

Isolation and 3-dimensional Culture of Primary Murine Intestinal Epithelial Cells Authors:  Agnieszka Pastuła and Michael Quante, date: 05/20/2014, view: 13591, Q&A: 3
The intestine, together with skin and blood, belongs to the organs with the highest cell turnover, which makes it a perfect model to study cellular processes, such as proliferation and differentiation. Epithelial cell turnover in intestine is possible due to the presence of intestinal stem cells, which are located at the bottom of the crypt. Here, ...
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Developmental Biology

Barrier Function Assay Authors:  Tia DiTommaso and Floris Foijer, date: 05/20/2014, view: 5191, Q&A: 0
This protocol serves to determine the integrity of the barrier function of (murine) epidermis. Defects in the barrier function lead to dehydration and infection in neonatal mice/humans. One possible way to assess epidermal barrier integrity is by a dye penetration assay as described hereunder. This assay should be done on unfixed, untreated ...
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Microbiology

Construction of Deletion-knockout Mutant Fowlpox Virus (FWPV) Authors:  Stephen M. Laidlaw and Michael A. Skinner, date: 05/20/2014, view: 4369, Q&A: 0
The construction of deletion-knockout poxviruses is a useful approach to determining the function of specific virus genes. This protocol is an adaptation of the transient dominant knockout selection protocol published by Falkner and Moss (1990) for use with vaccinia virus. The protocol makes use of the dominant selectable marker Escherichia coli ...
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ELISA on Virus-Infected Cells Authors:  Daniel L. Glauser and Philip G. Stevenson, date: 05/20/2014, view: 5110, Q&A: 0
The gammaherpesvirus murid herpesvirus 4 (MuHV-4) enters cells by endocytosis from the cell surface and fusion of the viral envelope with the membrane of late endosomes. The viral envelope glycoproteins undergo antigenic changes both upon virion endocytosis and upon fusion of the viral envelope with the endosomal membrane. These changes in virion ...
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IFN-α/β Detection Assay Using Sensor Cell Lines Authors:  Valeria Lulla, Andres Merits and Aleksei Lulla, date: 05/20/2014, view: 5148, Q&A: 0
Type I interferons (IFN-α/β) play an important role in host resistance to viral infections. Signaling through the JAK-STAT pathway, IFN-α/β stimulates response elements (ISRE) in the promoters of ISG to regulate their expression (reviewed in Reference 2). This method was adapted from InvivoGen to specifically detect and quantify IFN-α/β secreted ...
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Purification and TEM of Afp and Its Variants Authors:  Daria Rybakova, Alok K. Mitra and Mark R. H. Hurst, date: 05/20/2014, view: 5000, Q&A: 0
The Serratia entomophila antifeeding prophage (Afp), forms a phage-tail-like particle (tailocin) that causes cessation of feeding activity of the New Zealand grass grub, Costelytra zealandica. Here, we describe the more detailed purification protocol for Afp particles and its variants which is based on the procedure described in ...
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Purification of HCV-remodeled and Control ER Membranes Authors:  David Paul and Ralf Bartenschlager, date: 05/20/2014, view: 3915, Q&A: 0
As for all positive strand RNA viruses, hepatitis C virus (HCV) RNA replication is tightly associated with rearranged host cell membranes, termed viral replication factories. However, up to now little is known about both viral and cellular constituents of viral replication factories. Here, we describe a protocol to specifically isolate ...
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Molecular Biology

Identification of Proteins Interacting with Genomic Regions of Interest in vivo Using Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (enChIP) Authors:  Toshitsugu Fujita and Hodaka Fujii, date: 05/20/2014, view: 6991, Q&A: 0
Elucidation of molecular mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. To this end, it is useful to isolate the target regions retaining molecular interactions. We established locus-specific chromatin immunoprecipitation (ChIP) technologies consisting of ...
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Plant Science

Arabidopsis thaliana Embryo Sac Mitochondrial Membrane Potential Stain Authors:  María Victoria Martin, Diego Fernando Fiol, Eduardo Julián Zabaleta and Gabriela Carolina Pagnussat, date: 05/20/2014, view: 5640, Q&A: 0
The aim of this experiment is to study mitochondrial functional status in Arabidopsis embryo sacs using the membrane potential indicator JC-1. Changes in the membrane potential are presumed to be due to the opening of the mitochondrial permeability transition pore (MPTP), allowing passage of ions and small molecules. The resulting ...
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Immunofluorescence Labeling of Pollen Tubes Authors:  Hao Wang and Liwen Jiang, date: 05/20/2014, view: 5616, Q&A: 0
Pollen tube is regarded as an excellent single-cell model system in plant cell studies. This protocol describes the use of a rapid and reliable immunofluorescence labeling method for studying in situ localization of proteins in pollen tubes. The whole experiment contains two major steps: pollen tube in vitro germination, and ...
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