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Cancer Biology

Protocol for T-cell Adhesion Strength on Tumor Cells under Flow Conditions Authors:  Marie Boutet, Katarzyna Franciszkiewicz, Audrey Le Floc’h and Fathia Mami-Chouaib, date: 10/20/2013, view: 4101, Q&A: 0
This method allows evaluating the relative adhesion strength between T lymphocytes and specific adherent target cells using a shear force in flow chambers. It is based on the measure of the resistance of conjugates formed between T cells and adherent tumor cells to shear stress in a microfluidic system. For this purpose, T cells, stained with a ...
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Soft Agar Anchorage-independent Assay Authors:  Li-Ting Wang and Shih-Hsien Hsu, date: 10/20/2013, view: 6379, Q&A: 2
Chronic inflammation drives initiation of hepatocellular carcinoma (HCC), but the underlying mechanisms linking inflammation and tumor formation remain obscure. In this study, soft agar anchorage-independent assay were used to determine tumor transform activity of hepatoma cells with ISX over expression or knockdown in vitro.
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Xenograft Tumor Growth Assay Authors:  Li-Ting Wang and Shih-Hsien Hsu, date: 10/20/2013, view: 5512, Q&A: 0
Chronic inflammation drives initiation of hepatocellular carcinoma (HCC), but the underlying mechanisms linking inflammation and tumor formation remain obscure. In this study, Xenograft tumor assay was used to determine the tumorigenic activity of hepatoma cells with ISX over expression on nude mice in vivo.
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Cell Biology

Colon Tissue Immunoelectron Microscopy Authors:  Megumi Iwano, Akio Tsuru and Kenji Kohno, date: 10/20/2013, view: 3055, Q&A: 0
The method described here is intended to study intracellular localization of proteins in colon cells. This protocol was used to localize IRE1β in the endoplasmic reticulum membrane. We used anti-IRE1β antibody raised in guinea pig for this purpose. We also studied the location of BiP (also known as GRP78), with the antibody raised in rabbit. Both ...
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Bimolecular Fluorescence Complementation (BiFC) Assay for Direct Visualization of Protein-Protein Interaction in vivo Authors:  Hsien-Tsung Lai and Cheng-Ming Chiang, date: 10/20/2013, view: 8832, Q&A: 0
Bimolecular Fluorescence Complementation (BiFC) assay is a method used to directly visualize protein-protein interaction in vivo using live-cell imaging or fixed cells. This protocol described here is based on our recent paper describing the functional association of human chromatin adaptor and transcription cofactor Brd4 with p53 tumor ...
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Measurement of Junctional Protein Dynamics Using Fluorescence Recovery After Photobleaching (FRAP) Authors:  Rashmi Priya and Guillermo A. Gomez, date: 10/20/2013, view: 6090, Q&A: 0
Fluorescence Recovery After Photobleaching (FRAP) (Lippincott-Schwartz et al., 2003; Reits and Neefjes, 2001) was employed to determine dynamic properties of proteins localized at the ephitelial zonula adherens (ZA) (Kovacs et al., 2011; Otani et al., 2006). The proteins of interest were expressed in cells using a ...
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Hypertrophy Analysis and Quantification in Embryonic Cardiomyocites Authors:  Guillermo Luxán and José Luis de la Pompa, date: 10/20/2013, view: 3940, Q&A: 0
Myocardial growth goes from proliferation to hypertrophy during development. The measurement of the relative cell area provides information of cardiomyocyte hypertrophy, which is ideal for studying myocardial development.
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Isolation of Whole Mononuclear Cells from Peripheral Blood Authors:  Beatriz Martínez-Poveda, Guillermo Luxán and José Luis de la Pompa, date: 10/20/2013, view: 3109, Q&A: 0
Whole mononuclear cells from peripheral blood are an easy to obtain and useful population of cells where protein and expression patterns of genes can be studied in patients.
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Fluorescence Recovery After Photobleaching (FRAP) in the Fission Yeast Nucleus Authors:  Petrina Delivani, Mariola R. Chacón, Britta Schroth-Diez and Iva M. Tolić-Nørrelykke, date: 10/20/2013, view: 4778, Q&A: 0
We use fluorescence recovery after photobleaching (FRAP) to calculate the diffusion coefficient of GFP in the nucleoplasm of fission yeast. The FRAP method can be generally used to measure the mobility of proteins inside the cell or its organelles.
In our experiment we only measured the diffusion of GFP inside the nucleoplasm of fission ...
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Immunology

In vitro T Cell–DC and T Cell–T Cell Clustering Assays Author:  Audrey Gérard, date: 10/20/2013, view: 5559, Q&A: 0
To get activated, T cells need to find their cognate antigen at the surface of an antigen-presenting cell (APC). Recognition of cognate antigen in the context of the MHC (Major histocompatibility complex) by the TCR (T-Cell Receptor) results in long lasting interactions between T cells and APCs. Subsequently, T cells form homotypic interactions ...
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Microbiology

Surface Polysaccharide Extraction and Quantification Authors:  Cedric Arthur Brimacombe and John Thomas Beatty, date: 10/20/2013, view: 7113, Q&A: 0
Gram-negative bacterial cells possess two membranes - the inner cytoplasmic membrane and the outer membrane. The two membranes are distinct in their composition; the inner membrane is composed of a phospholipid bilayer, whereas the outer membrane (OM) is composed of an asymmetrical bilayer, with the outer leaflet containing lipopolysaccharide ...
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Immunoblot Analysis of Histone H4 Acetylation and Histone H2A Phosphorylation in Candida albicans Authors:  Michael Tscherner and Karl Kuchler, date: 10/20/2013, view: 4498, Q&A: 0
Posttranslational modifications of histones are required for different processes including transcription, replication and DNA damage repair. This protocol describes the preparation of a whole-cell extracts for the fungal pathogen Candida albicans. Furthermore, the extract is used to detect lysine acetylation of histone H4 as well as ...
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Cell Fractionation and Quantitative Analysis of HIV-1 Reverse Transcription in Target Cells Authors:  Vaibhav B Shah and Christopher Aiken, date: 10/20/2013, view: 5417, Q&A: 0
This is a protocol to detect HIV-1 reverse transcription products in cytoplasmic and nuclear fractions of cells infected with VSV-G-pseudotyped envelope-defective HIV-1. This protocol can also be extended to HIV-1 with regular envelope.
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Neuroscience

[3H]-Spiperone Competition Binding to Dopamine D2, D3 and D4 Receptors Authors:  Jan-Peter van Wieringen and Martin C. Michel, date: 10/20/2013, view: 3830, Q&A: 0
This protocol is intended for use in 96 well plates (1,200 μl wells) but it can similarly be applied to standard test tubes (Levant, 2007). D2, D3, and D4 dopamine receptors are members of the D2-like class of dopamine receptors. They can be studied using the radioligand [3H]-spiperone, which ...
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[3H]-Spiperone Saturation Binding to Dopamine D2, D3 and D4 Receptors Authors:  Jan-Peter van Wieringen and Martin C. Michel, date: 10/20/2013, view: 4349, Q&A: 0
This protocol is intended for use in 96 well plates (1,200 μl wells) but it can similarly be applied to standard test tubes (Levant, 2007). D2, D3, and D4 dopamine receptors are members of the D2-like class of dopamine receptors. They can be studied using the radioligand [3H]-spiperone, which ...
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Plant Science

A High Resolution Short Interfering RNA (siRNA) Detection Method from Virus-infected Plants Authors:  Vinay Panwar and Guus Bakkeren, date: 10/20/2013, view: 5679, Q&A: 0
Plant viruses are strong inducers as well as targets of RNA silencing. In plants RNA silencing acts as a natural defense mechanism against viral infection and is associated with accumulation of virus-specific small interfering RNAs (siRNAs). The continuing discoveries, increasing awareness and interest in the regulatory roles of non-coding small ...
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Trehalase Activity in Arabidopsis thaliana Optimized for 96-well Plates Authors:  Hilde Van Houtte and Patrick Van Dijck, date: 10/20/2013, view: 4825, Q&A: 0
Trehalose is a nonreducing disaccharide. It is a common sugar in bacteria, fungi and yeast, where it functions as a carbon source and stress protectant. In contrast, plants, although encoding large trehalose biosynthesis gene families, contain only small amounts of trehalose. The intermediate compound of trehalose, trehalose-6-phosphate (T6P), is ...
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Stem Cell

Immunolabelling of Thin Slices of Mouse Descending Colon and Jejunum Authors:  Julien Bellis, Isabelle Duluc, Jean-Noël Freund and Jan R. De Mey, date: 10/20/2013, view: 4464, Q&A: 0
This protocol describes a method for efficient immunolabelling of thin tissue slices containing a few rows of intact intestinal crypts, which yields large numbers of them being oriented favorably for recording stacks of optical sections aligned with the crypt long axis (Bellis et al., 2012). The latter can then be used for cell positional ...
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