• Volume 9, 2019
  • Volume 8, 2018
  • Volume 7, 2017
  • Volume 6, 2016
  • Volume 5, 2015
  • Volume 4, 2014
  • Volume 3, 2013
  • Volume 2, 2012
  • Volume 1, 2011

Biochemistry

Detection of Wnt5 in Media Conditioned by Mouse Embryonic Fibroblast Authors:  Juan Flores and Nan Gao, date: 10/20/2016, view: 4836, Q&A: 0
This protocol describes the procedure of visualizing secreted Wnt5 protein in serum free media via western blotting. This procedure can also be used to visualize other secreted proteins larger than 10,000 daltons. The work presented in this paper visualizes Wnt5 secreted by mouse embryonic fibroblast (MEF), but can be adapted to other cell lines ...
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Cancer Biology

The Chick Embryo Chorioallantoic Membrane as an in vivo Model to Study Metastasis Authors:  Piero Crespo and Berta Casar, date: 10/20/2016, view: 11704, Q&A: 0
Metastasis is a complex process that includes several steps: neoplastic progression, angiogenesis, cell migration and invasion, intravasation into nearby blood vessels, survival in the circulatory system, extravasation followed by homing into distant tissues, the formation of micrometastases, and finally the growth into macroscopic secondary ...
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Cell Surface Protein Detection to Assess Receptor Internalization Authors:  Magdalena Czarnecka and Joanna Kitlinska, date: 10/20/2016, view: 5702, Q&A: 0
The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our ...
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In vivo Imaging of Tumor and Immune Cell Interactions in the Lung Authors:  Richard N. Hanna, Grzegorz Chodaczek and Catherine C. Hedrick, date: 10/20/2016, view: 5171, Q&A: 0
Immunotherapy has demonstrated great therapeutic potential by activating the immune system to fight cancer. However, little is known about the specific dynamics of interactions that occur between tumor and immune cells. In this protocol we describe a novel method to visualize the interaction of tumor and immune cells in the lung of live mice, ...
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Quantification of Tumor Material Uptake Authors:  Richard N. Hanna and Catherine C. Hedrick, date: 10/20/2016, view: 5149, Q&A: 0
Extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris may help cancers invade new organs. Enhancing the removal of extracellular tumor material by immune cells represents a novel immunotherapy approach for preventing cancer metastasis. This protocol quantifies the uptake and removal of extracellular tumor ...
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Detection of Anaphase Bridge Formation by Immunofluorescence Microscopy in Mammalian Cells Authors:  Thomas Aschacher and Florian Enzmann, date: 10/20/2016, view: 6641, Q&A: 0
The aim of this protocol is to provide a comprehensive description of the materials, equipment and reproducible methods to detect and analyze anaphase bridges in immunofluorescence microscopy using DAPI to detect cells that failed to completely segregate during mitosis. It describes the process of cell preparation, staining and microscopic ...
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Cell Biology

A 3D Culture System of Human Immortalized Myometrial Cells Authors:  Minnie Malik, Joy Britten and William H. Catherino, date: 10/20/2016, view: 4881, Q&A: 0
Myometrium forms the middle layer of the uterus and is mainly composed of the smooth muscle cells. The cells in vitro are usually grown in a single layer (2-dimensional; 2D) format, whereas in vivo cells are structured in an extracellular matrix scaffolding that allows the cells to communicate and respond to environmental cues. ...
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Immunology

Lipid Extraction from HeLa Cells, Quantification of Lipids, Formation of Large Unilamellar Vesicles (LUVs) by Extrusion and in vitro Protein-lipid Binding Assays, Analysis of the Incubation Product by Transmission Electron Microscopy (TEM) and by Flotation across a Discontinuous Sucrose Gradient Authors:  Amina Bittame, Jodie Lopez, Gregory Effantin, Nicolas Blanchard, Marie-France Cesbron-Delauw, Jean Gagnon and Corinne Mercier, date: 10/20/2016, view: 8291, Q&A: 0
Dissecting the interactions established between proteins and membranes in a given type of cells is not an easy task. Using a cell-free system of large unilamellar vesicles (LUVs) to analyze these interactions may help decipher these interactions and identify potential membrane deformations induced by the proteins incubated with these LUVs. This ...
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Ear Inflammation and Whole-mount Ear Staining Authors:  Jia Tong Loh, Merry Gunawan and I-hsin Su, date: 10/20/2016, view: 5895, Q&A: 0
The recruitment of circulating neutrophils from the bloodstream to the site of inflammation represents one of the earliest events during an innate immune response. During this response, neutrophils tether and roll along the vessel walls before transmigrating across the endothelium into the interstitial space to exert their functions. Here, we ...
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Assessment of TCR-induced Sumoylation of PKC-θ Authors:  Xudong Wang, Zhilong Chen, Qilong Wang and Yingqiu Li, date: 10/20/2016, view: 3661, Q&A: 0
Sumoylation controls many cellular processes. Protein kinase C-θ (PKC-θ), a member of the Ca2+-independent PKC subfamily of kinases, serves as a regulator of T cell activation by mediating the T cell antigen receptor (TCR)- and coreceptor CD28-induced activation of the transcription factors NF-κB and AP-1 and, to a lesser extent, NFAT, ...
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PKC-θ in vitro Kinase Activity Assay Authors:  Xudong Wang and Yingqiu Li, date: 10/20/2016, view: 5163, Q&A: 0
Protein kinase C-θ (PKC-θ), a member of the Ca2+-independent PKC subfamily of kinases, serves as a regulator of T cell activation by mediating the T cell antigen receptor (TCR)- and coreceptor CD28-induced activation of the transcription factors NF-κB and AP-1 and, to a lesser extent, NFAT, and, subsequently, interleukin 2 (IL-2) ...
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Molecular Biology

Generation of Mitochondrial-nuclear eXchange Mice via Pronuclear Transfer Authors:  Robert A. Kesterson, Larry W. Johnson, Laura J. Lambert, Jay L. Vivian, Danny R. Welch and Scott W. Ballinger, date: 10/20/2016, view: 5983, Q&A: 0
The mitochondrial paradigm for common disease proposes that mitochondrial DNA (mtDNA) sequence variation can contribute to disease susceptibility and progression. To test this concept, we developed the Mitochondrial-nuclear eXchange (MNX) model, in which isolated embryonic pronuclei from one strain of species are implanted into an enucleated ...
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Neuroscience

Evaluation of Burkholderia cepacia Complex Bacteria Pathogenicity Using Caenorhabditis elegans Authors:  Pietro Tedesco, Elia Di Schiavi, Fortunato Palma Esposito and Donatella de Pascale, date: 10/20/2016, view: 3875, Q&A: 0
This protocol describes two biological assays to evaluate pathogenicity of Burkholderia cepacia complex (Bcc) strains against the nematode Caenorhabditis elegans. Specifically, these two assays allow one to identify if the under-investigated Bcc strains are able to kill the nematodes by intestinal colonization (slow killing ...
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Artificial Optogenetic TRN Stimulation of C. elegans Authors:  Ithai Rabinowitch, Millet Treinin and Jihong Bai, date: 10/20/2016, view: 4708, Q&A: 0
Optogenetics is a powerful tool for manipulating neuronal activity with high temporal and spatial precision. In the nematode C. elegans optogentics is especially useful and easy to apply. This is because C. elegans is translucent, so its neurons are highly accessible to optic stimulation. In addition, many of its neurons can be ...
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Plant Science

Mungbean Yellow Mosaic India Virus (MYMIV)-infection, Small RNA Library Construction and Deep Sequencing for MicroRNA Identification in Vigna mungo Authors:  Anirban Kundu, Sujay Paul, Amita Pal and Genotypic Technology, date: 10/20/2016, view: 6239, Q&A: 0
This protocol describes small RNA library preparation from Vigna mungo total RNA followed by deep sequencing and analysis for microRNA identification.​
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Heterologous Expression and Purification of Catalytic Domain of CESA1 from Arabidopsis thaliana Authors:  Venu Gopal Vandavasi and Hugh O’ Neill, date: 10/20/2016, view: 4183, Q&A: 0
Heterologous expression of plant cellulose synthase (CESA) and its purification has remained a challenge for decades impeding detailed biophysical, biochemical and structural characterization of this key enzyme. An in-depth knowledge of structure and function of CESA proteins would enable us to better understand the hierarchical structure of the ...
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Aniline Blue and Calcofluor White Staining of Callose and Cellulose in the Streptophyte Green Algae Zygnema and Klebsormidium Authors:  Klaus Herburger and Andreas Holzinger, date: 10/20/2016, view: 7335, Q&A: 0
Plant including green algal cells are surrounded by a cell wall, which is a diverse composite of complex polysaccharides and crucial for their function and survival. Here we describe two simple protocols to visualize callose (1→3-β-D-glucose) and cellulose (1→4-β-D-glucose) and related polysaccharides in the cell walls of streptophyte green algae. ...
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Determination of Molecular Structures of Condensed Tannins from Plant Tissues Using HPLC-UV Combined with Thiolysis and MALDI-TOF Mass Spectrometry Authors:  Sauro Bianchi, Ivana Kroslakova and Ingo Mayer, date: 10/20/2016, view: 6492, Q&A: 0
Condensed tannins extracted from plant tissues are suitable substitutes for phenolic resins. Their molecular structure, which might influence their chemical reactivity, can be assessed by the use of both HPLC-UV after acid thiolysis and MALDI-TOF mass spectrometry. Thiolysis of plant extracts in acidic methanol with cysteamine hydrochloride ...
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Rapid Determination of Cellulose, Neutral Sugars, and Uronic Acids from Plant Cell Walls by One-step Two-step Hydrolysis and HPAEC-PAD Authors:  Trevor Yeats, Tamara Vellosillo, Nadav Sorek, Ana B. Ibáñez and Stefan Bauer, date: 10/20/2016, view: 8855, Q&A: 1
The plant cell wall is primarily composed of the polysaccharides cellulose, hemicellulose and pectin. The structural and compositional complexity of these components are important for determining cell wall function during plant growth. Moreover, cell wall structure defines a number of functional properties of plant-derived biomass, such as ...
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Stem Cell

Experimental Liver Fibrosis and Intrasplenic Transplantation of CD45+ Bone Marrow Cells Authors:  Prakash Baligar, Sebanta Pokhrel and Asok Mukhopadhyay, date: 10/20/2016, view: 6575, Q&A: 1
Liver fibrosis results from the excessive collagen deposition (collagen scar) by activated hepatic stellate cells (HpSCs), leading to the inhibition of normal liver regeneration and function. Fibrogenesis is a complex mechanism involving both the synthesis and degradation of matrix proteins by different cell types, mainly macrophages in the liver. ...
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