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Cancer Biology

Isolation and Separation of Epithelial CD34+ Cancer Stem Cells from Tgfbr2-deficient Squamous Cell Carcinoma Authors:  Heather A. McCauley and Géraldine Guasch, date: 09/05/2017, view: 2121, Q&A: 0
Most epithelial tumors have been shown to contain cancer stem cells that are potentially the driving force in tumor progression and metastasis (Kreso and Dick, 2014; Nassar and Blanpain, 2016). To study these cells in depth, cell isolation strategies relying on cell surface markers or fluorescent reporters are essential, and the isolation ...
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GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling Authors:  Julia Petschnigg and Robin Ketteler, date: 09/05/2017, view: 1944, Q&A: 0
High-content screening is a useful tool to understand complex cellular processes and to identify genes, proteins or small molecule compounds that modulate such pathways. High-content assays monitor the function of a protein or pathway by visualizing a change in an image-based readout, such as a change in the localization of a reporter protein. ...
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Protocol for Establishing a Multiplex Image-based Autophagy RNAi Screen in Cell Cultures Authors:  Jennifer Jung and Christian Behrends, date: 09/05/2017, view: 2140, Q&A: 0
Autophagy is a recycling pathway, in which intracellular cargoes including protein aggregates and bacteria are engulfed by autophagosomes and subsequently degraded after fusion with lysosomes. Dysregulation of this process is involved in several human diseases such as cancer or neurodegeneration. Hence, advancing our understanding of how autophagy ...
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Cell Biology

Peroxisome Motility Measurement and Quantification Assay Authors:  Jeremy Metz, Inês G. Castro and Michael Schrader, date: 09/05/2017, view: 1983, Q&A: 0
Organelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation ...
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Staining of Membrane Receptors with Fluorescently-labeled DNA Aptamers for Super-resolution Imaging Authors:  Maria Angela Gomes de Castro, Claudia Höbartner and Felipe Opazo, date: 09/05/2017, view: 2040, Q&A: 0
One of the most prominent applications of fluorescent super-resolution microscopy is the study of nanodomain arrangements of receptors and the endocytic pathway. Staining methods are becoming crucial for answering questions on the nanoscale, therefore, the use of small and monovalent affinity probes is of great interest in super-resolution ...
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Developmental Biology

Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells Authors:  Huy Nguyen Duc and Xiaojun Ren , date: 09/05/2017, view: 2402, Q&A: 0
HaloTag has been widely used to label proteins in vitro and in vivo (Los et al., 2008). In this protocol, we describe labelling HaloTag-Cbx fusion proteins by HaloTag ligands for live-cell single-molecule imaging (Zhen et al., 2016).
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Isolation of Mouse Cardiac Neural Crest Cells and Their Differentiation into Smooth Muscle Cells Authors:  Xia Wang and Sophie Astrof, date: 09/05/2017, view: 2108, Q&A: 0
Cardiac neural crest cells (CNCCs) originate at the dorsal edge of the neural tube between the otic pit and the caudal edge of the 3rd somite, and migrate into the pharyngeal arches and the heart. We have shown that fibronectin (Fn1) plays an important role in the development of the CNCC by regulating the differentiation of CNCCs into ...
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Immunology

In vivo Priming of T Cells with in vitro Pulsed Dendritic Cells: Popliteal Lymph Node Assay Authors:  Songjie Cai, Masayuki Fujino, Lina Lu and Xiao-Kang Li, date: 09/05/2017, view: 2554, Q&A: 0
One-way mixed lymphocyte reaction (MLR) is a classic tool to measure how T cells react to external stimuli. However, MLR is an in vitro reaction system, which shows different response intensity compared with in vivo trails sometimes due to the lack of cytokines, tissue matrix and other immune response associated factors. The ...
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Microbiology

Sterol Analysis in Kluyveromyces lactis Authors:  Yvetta Gbelska, Nora Toth Hervay, Marcela Morvova and Alexandra Konecna, date: 09/05/2017, view: 1432, Q&A: 0
Sterols are essential lipids of most eukaryotic cells with multiple functions (structural, regulatory and developmental). Sterol profile of yeast cells is often determined during the studies of ergosterol synthesis mutants used to uncover a number of functions for various sterols in yeast cells. Molecular studies of ergosterol biosynthesis have ...
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Determination of Local pH Differences within Living Salmonella Cells by High-resolution pH Imaging Based on pH-sensitive GFP Derivative, pHluorin(M153R) Authors:  Yusuke V. Morimoto, Nobunori Kami-ike, Keiichi Namba and Tohru Minamino, date: 09/05/2017, view: 1649, Q&A: 0
The bacterial flagellar type III protein export apparatus is composed of a transmembrane export gate complex and a cytoplasmic ATPase complex. The export apparatus requires ATP hydrolysis and the proton motive force across the cytoplasmic membrane to unfold and transport flagellar component proteins for the construction of the bacterial flagellum ...
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Rearing of Culex spp. and Aedes spp. Mosquitoes Authors:  Elizabeth Kauffman, Anne Payne, Mary A. Franke, Michael A. Schmid, Eva Harris and Laura D. Kramer, date: 09/05/2017, view: 3141, Q&A: 0
Mosquito-transmitted pathogens cause major public health problems and contribute substantially to the global burden of disease. Aedes mosquitoes transmit dengue, Zika, yellow fever, and Chikungunya viruses; Culex mosquitoes transmit West Nile, Japanese encephalitis, and Saint Louis encephalitis viruses, among others. Experiments ...
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Multicolor Stimulated Emission Depletion (STED) Microscopy to Generate High-resolution Images of Respiratory Syncytial Virus Particles and Infected Cells Authors:  Masfique Mehedi, Margery Smelkinson, Juraj Kabat, Sundar Ganesan, Peter L. Collins and Ursula J. Buchholz, date: 09/05/2017, view: 2008, Q&A: 0
Human respiratory syncytial virus (RSV) infection in human lung epithelial A549 cells induces filopodia, cellular protrusions consisting of F-actin, that extend to neighboring uninfected cells (Mehedi et al., 2016). High-resolution imaging via stimulated emission depletion (STED) microscopy revealed filamentous RSV particles along these ...
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Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells Authors:  Akira Karasawa and Toshimitsu Kawate, date: 09/05/2017, view: 2828, Q&A: 0
The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms ...
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Detection of Reactive Oxygen Species (ROS) in Cyanobacteria Using the Oxidant-sensing Probe 2’,7’-Dichlorodihydrofluorescein Diacetate (DCFH-DA) Authors:  Rajneesh, Jainendra Pathak, Ananya Chatterjee, Shailendra P. Singh and Rajeshwar P. Sinha, date: 09/05/2017, view: 3680, Q&A: 0
Reactive oxygen species (ROS) are cell signaling molecules synthesized inside the cells as a response to routine metabolic processes. In stress conditions such as ultraviolet radiation (UVR), ROS concentration increases several folds in the cells that become toxic for the cell survival. Here we present the method for in vivo detection of ...
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Molecular Biology

Using CRISPR-ERA Webserver for sgRNA Design Authors:  Honglei Liu, Xiaowo Wang and Lei S. Qi, date: 09/05/2017, view: 2835, Q&A: 0
The CRISPR-Cas9 system is emerging as a powerful technology for gene editing (modifying the genome sequence) and gene regulation (without modifying the genome sequence). Designing sgRNAs for specific genes or regions of interest is indispensable to CRISPR-based applications. CRISPR-ERA (http://crispr-era.stanford.edu/ ...
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Neuroscience

FM1-43 Photoconversion and Electron Microscopy Analysis at the Drosophila Neuromuscular Junction Authors:  Nadezhda S. Sabeva and Maria Bykhovskaia, date: 09/05/2017, view: 2127, Q&A: 0
We developed a protocol for photoconversion of endocytic marker FM1-43 followed by electron microscopy analysis of synaptic boutons at the Drosophila neuromuscular junction. This protocol allows detection of stained synaptic vesicle even when release rates are very low, such as during the spontaneous release mode. The preparations are ...
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Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation Authors:  Steven Boeynaems, Mathias De Decker, Peter Tompa and Ludo Van Den Bosch, date: 09/05/2017, view: 3374, Q&A: 0
Studying liquid-liquid phase separation (LLPS) of proteins provides key insights into the biogenesis of membraneless organelles and pathological protein aggregation in disease. We have established a protocol for inducing the phase separation of arginine-rich peptides, which allows for studying their molecular determinants and dynamics (Boeynaems ...
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Predator Odor-induced Freezing Test for Mice Author:  Shintaro Otsuka, date: 09/05/2017, view: 1841, Q&A: 0
The innate fear response is an emotional response that does not require any previously acquired conditioning. One of the standard methods to analyze the innate fear response is a 2,4,5-trimethylthiazoline (TMT)-induced freezing test. TMT is an odor originally isolated from anal secretion of the red fox. Acute TMT exposure has been shown to induce ...
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Isolation of Rodent Brain Vessels Authors:  Cristina Carvalho and Paula I. Moreira, date: 09/05/2017, view: 1902, Q&A: 0
The prevalence of neurodegenerative diseases is increasing worldwide. Cerebrovascular disorders and/or conditions known to affect brain vasculature, such as diabetes, are well-known risk factors for neurodegenerative diseases. Thus, the evaluation of the brain vasculature is of great importance to better understand the mechanisms underlying brain ...
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Plant Science

Ubiquitin Proteasome Activity Measurement in Total Plant Extracts Authors:  Suayib Üstün and Frederik Börnke, date: 09/05/2017, view: 2147, Q&A: 0
The fine-tuned balance of protein level, conformation and location within the cell is vital for the dynamic changes required for a cell to respond to a given stimulus. This requires the regulated turnover of damaged or short-lived proteins through the ubiquitin proteasome system (UPS). Thus, the protease activity of the proteasome is adjusted to ...
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Isolation and Quantification of Plant Extracellular Vesicles Authors:  Brian D. Rutter, Katie L. Rutter and Roger W. Innes, date: 09/05/2017, view: 3674, Q&A: 3
Extracellular vesicles (EVs) play an important role in intercellular communication by transporting proteins and RNA. While plant cells secrete EVs, they have only recently been isolated and questions regarding their biogenesis, release, uptake and function remain unanswered. Here, we present a detailed protocol for isolating EVs from the ...
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Extraction and Molybdenum Blue-based Quantification of Total Phosphate and Polyphosphate in Parachlorella Authors:  Shuhei Ota and Shigeyuki Kawano, date: 09/05/2017, view: 2187, Q&A: 0
Inorganic phosphorus is a non-renewable resource and an essential element for life on Earth. Organisms such as algae, protists, and animals can store phosphate (Pi) through uptake of Pi as polyphosphate (poly-P), which is a linear polymer of orthophosphate residues linked by high-energy phosphoanhydride bonds. Here, we describe procedures for ...
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Construction of a Single Transcriptional Unit for Expression of Cas9 and Single-guide RNAs for Genome Editing in Plants Authors:  Xu Tang, Zhaohui Zhong, Xuelian Zheng and Yong Zhang, date: 09/05/2017, view: 3178, Q&A: 0
The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein9 (Cas9) is a simple and efficient tool for genome editing in many organisms including plant and crop species. The sgRNAs of the CRISPR/Cas9 system are typically expressed from RNA polymerase III promoters, such as U6 and U3. In many transformation events, ...
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