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Bio-protocol

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https://www.readbyqxmd.com/read/28603749/isolation-and-cultivation-of-primary-brain-endothelial-cells-from-adult-mice
#1
Julian Christopher Assmann, Kristin Müller, Jan Wenzel, Thomas Walther, Josefine Brands, Peter Thornton, Stuart M Allan, Markus Schwaninger
Brain endothelial cells are the major building block of the blood-brain barrier. To study the role of brain endothelial cells in vitro, the isolation of primary cells is of critical value. Here, we describe a protocol in which vessel fragments are isolated from adult mice. After density centrifugation and mild digestion of the fragments, outgrowing endothelial cells are selected by puromycin treatment and grown to confluence within one week.
May 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28573168/analysis-of-in-vivo-interaction-between-rna-binding-proteins-and-their-rna-targets-by-uv-cross-linking-and-immunoprecipitation-clip-method
#2
Pamela Bielli, Claudio Sette
RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic PKM2 variant...
May 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28534038/analysis-of-mitochondrial-transfer-in-direct-co-cultures-of-human-monocyte-derived-macrophages-mdm-and-mesenchymal-stem-cells-msc
#3
Megan V Jackson, Anna D Krasnodembskaya
Mesenchymal stem/stromal cells (MSC) are adult stem cells which have been shown to improve survival, enhance bacterial clearance and alleviate inflammation in pre-clinical models of acute respiratory distress syndrome (ARDS) and sepsis. These diseases are characterised by uncontrolled inflammation often underpinned by bacterial infection. The mechanisms of MSC immunomodulatory effects are not fully understood yet. We sought to investigate MSC cell contact-dependent communication with alveolar macrophages (AM), professional phagocytes which play an important role in the lung inflammatory responses and anti-bacterial defence...
May 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28580376/reversible-cryo-arrests-of-living-cells-to-pause-molecular-movements-for-high-resolution-imaging
#4
Jan Huebinger, Martin E Masip, Jens Christmann, Frank Wehner, Philippe I H Bastiaens
Fluorescence live-cell imaging by single molecule localization microscopy (SMLM) or fluorescence lifetime imaging microscopy (FLIM) in principle allows for the spatio-temporal observation of molecular patterns in individual, living cells. However, the dynamics of molecules within cells hamper their precise observation. We present here a detailed protocol for consecutive cycles of reversible cryo-arrest of living cells on a microscope that allows for a precise determination of the evolution of molecular patterns within individual living cells...
April 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28480316/expression-and-purification-of-mini-g-proteins-from-escherichia-coli
#5
Byron Carpenter, Christopher G Tate
Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR-G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation...
April 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28459086/automated-tracking-of-root-for-confocal-time-lapse-imaging-of-cellular-processes
#6
Mehdi Doumane, Claire Lionnet, Vincent Bayle, Yvon Jaillais, Marie-Cécile Caillaud
Here we describe a protocol that enables to automatically perform time-lapse imaging of growing root tips for several hours. Plants roots expressing fluorescent proteins or stained with dyes are imaged while they grow using automatic movement of the microscope stage that compensates for root growth and allows to follow a given region of the root over time. The protocol makes possible the image acquisition of multiple growing root tips, therefore increasing the number of recorded mitotic events in a given experiment...
April 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28616445/nitroxide-labeling-of-proteins-and-the-determination-of-paramagnetic-relaxation-derived-distance-restraints-for-nmr-studies
#7
Megan Sjodt, Robert T Clubb
Site-specific attachment of paramagnetic spin labels to biomolecules causes distance-dependent line-broadening effects, which can be exploited to study the structure and dynamics of these molecules in solution. This protocol describes how to attach nitroxide spin labels to proteins and how to collect and analyze NMR data using these labeled samples. We also explain how to derive distance restraints for paramagnetic relaxation enhancement nuclear magnetic resonance (PRE-NMR) studies.
April 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28603753/secretion-of-adipsin-as-an-assay-to-measure-flux-from-the-endoplasmic-reticulum-er
#8
Alexandria Brumfield, Natasha Chaudhary, Timothy E McGraw
In this protocol we describe a quantitative biochemical assay to assess the efficiency of endoplasmic reticulum (ER) to Golgi protein transport in adipocytes (Bruno et al., 2016). The assay takes advantage of the fact that adipocytes secrete various bioactive proteins, known as adipokines. As a measure of ER to Golgi flux we determine the rate of bulk secretion of the adipokine adipsin post washout of Brefeldin A (BFA) treatment using immunoblotting. Because BFA treatment results in an accumulation of adipsin in the ER, the exit of adipsin from the ER upon BFA washout is synchronized across cells and experimental conditions...
April 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28405595/in-vivo-mitophagy-monitoring-in-caenorhabditis-elegans-to-determine-mitochondrial-homeostasis
#9
Konstantinos Palikaras, Nektarios Tavernarakis
Perturbation of mitochondrial function is a major hallmark of several pathological conditions and ageing, underlining the essential role of fine-tuned mitochondrial activity (Lopez-Otin et al., 2013). Mitochondrial selective autophagy, known as mitophagy, mediates the removal of dysfunctional and/or superfluous organelles, preserving cellular and organismal homeostasis (Palikaras and Tavernarakis, 2014; Pickrell and Youle, 2015; Scheibye-Knudsen et al., 2015). In this protocol, we describe a method for assessing mitophagy in the nematode Caenorhabditis elegans...
April 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28405594/screening-for-novel-endogenous-inflammatory-stimuli-using-the-secreted-embryonic-alkaline-phosphatase-nf-%C3%AE%C2%BAb-reporter-assay
#10
Lorena Zuliani-Alvarez, Anna M Piccinini, Kim S Midwood
An immune response can be activated by pathogenic stimuli, as well as endogenous danger signals, triggering the activation of pattern recognition receptors and initiating signalling cascades that lead to inflammation. This method uses THP1-Blue™ cells, a human monocytic cell line which contains an embryonic alkaline phosphatase reporter gene allowing the detection of NF-κB-induced transcriptional activation. We validated this protocol by assessing NF-κB activation after stimulation of toll-like receptor 4 (TLR4) by two different agonists: lipopolysaccharide (LPS), derived from the cell wall of Gram negative bacteria, and tenascin-C, an extracellular matrix protein whose expression is induced upon tissue injury...
April 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28612042/determination-of-adeno-associated-virus-rep-dna-binding-using-fluorescence-anisotropy
#11
Francisco Zarate-Perez, Vishaka Santosh, Martino Bardelli, Leticia Agundez, R Michael Linden, Els Henckaerts, Carlos R Escalante
Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the integration site AAVS1 using fluorescent anisotropy. This protocol can be used to measure the binding constants of any DNA binding protein provided the substrate DNA is fluorescently labeled...
March 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28603752/polysome-analysis
#12
Dipak Kumar Poria, Partho Sarothi Ray
Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By this protocol, cell lysates are fractionated by sucrose density gradient ultracentrifugation. Free mRNA fraction and various ribosomal fractions, such as 40S, 60S, monosomes and polysomes are collected by fractionation. Association of particular mRNAs with these fractions is detected by reverse transcription - PCR to investigate the translational state of the mRNA...
March 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28580375/rna-protein-uv-crosslinking-assay
#13
Dipak Kumar Poria, Partho Sarothi Ray
RNA-protein interactions play a crucial role in every aspect of RNA metabolism, and also plays a major role in post-transcriptional gene regulation. RNA-binding proteins have been implicated in viral gene expression (Ray and Das, 2002) and microRNA-mediated gene regulation (Poria et al., 2016). Here we have described the protocol which (1) covalently links transiently interacting RNA-protein complexes by UV crosslinking, (2) removes the unprotected RNA by RNase digestion and (3) detects the RNA-protein complexes by SDS-PAGE analysis...
March 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28573169/surface-inoculation-and-quantification-of-pseudomonas-syringae-population-in-the-arabidopsis-leaf-apoplast
#14
Cristián Jacob, Shweta Panchal, Maeli Melotto
Bacterial pathogens must enter the plant tissue in order to cause a successful infection. Foliar bacterial pathogens that are not able to directly penetrate the plant epidermis rely on wounds or natural openings to internalize leaves. This protocol describes a procedure to estimate the population size of Pseudomonas syringae in the leaf apoplast after surface inoculation of Arabidopsis rosettes.
March 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28516126/an-hplc-based-method-to-quantify-coronatine-production-by-bacteria
#15
Shweta Panchal, Zachary S Breitbach, Maeli Melotto
Coronatine is a polyketide phytotoxin produced by several pathovars of the plant pathogenic bacterium Pseudomonas syringae. It is one of the most important virulence factors determining the success of bacterial pathogenesis in the plant at both epiphytic and endophytic stages of the disease cycle. This protocol describes an optimized procedure to culture bacterial cells for coronatine production and to quantify the amount of coronatine secreted in the culture medium using an HPLC-based method.
March 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28382319/ca-2-measurements-in-mammalian-cells-with-aequorin-based-probes
#16
Anna Tosatto, Rosario Rizzuto, Cristina Mammucari
Aequorin is a Ca(2+) sensitive photoprotein suitable to measure intracellular Ca(2+) transients in mammalian cells. Thanks to recombinant cDNAs expression, aequorin can be specifically targeted to various subcellular compartments, thus allowing an accurate measurement of Ca(2+) uptake and release of different intracellular organelles. Here, we describe how to use this probe to measure cytosolic Ca(2+) levels and mitochondrial Ca(2+) uptake in mammalian cells.
March 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28286807/protein-synthesis-rate-assessment-by-fluorescence-recovery-after-photobleaching-frap
#17
Nikos Kourtis, Nektarios Tavernarakis
Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals.
March 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28603751/production-of-guide-rnas-in-vitro-and-in-vivo-for-crispr-using-ribozymes-and-rna-polymerase-ii-promoters
#18
Tao Zhang, Yangbin Gao, Rongchen Wang, Yunde Zhao
CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method also makes it feasible to conduct tissue/cell specific gene editing.
February 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28603750/quantitative-analysis-of-exosome-secretion-rates-of-single-cells
#19
Yu-Jui Chiu, Wei Cai, Tiffany Lee, Julia Kraimer, Yu-Hwa Lo
To study the inhomogeneity within a cell population including exosomes properties such as exosome secretion rate of cells and surface markers carried by exosomes, we need to quantify and characterize those exosomes secreted by each individual cell. Here we develop a method to collect and analyze exosomes secreted by an array of single cells using antibody-modified glass slides that are position-registered to each single cell. After each collection, anti-body conjugated quantum dots are used to label exosomes to allow counting and analysis of exosome surface proteins...
February 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28255574/automatic-quantification-of-the-number-of-intracellular-compartments-in-arabidopsis-thaliana-root-cells
#20
Vincent Bayle, Matthieu Pierre Platre, Yvon Jaillais
In the era of quantitative biology, it is increasingly required to quantify confocal microscopy images. If possible, quantification should be performed in an automatic way, in order to avoid bias from the experimenter, to allow the quantification of a large number of samples, and to increase reproducibility between laboratories. In this protocol, we describe procedures for automatic counting of the number of intracellular compartments in Arabidopsis root cells, which can be used for example to study endocytosis or secretory trafficking pathways and to compare membrane organization between different genotypes or treatments...
February 20, 2017: Bio-protocol
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