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Bio-protocol

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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
#1
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/28480316/expression-and-purification-of-mini-g-proteins-from-escherichia-coli
#2
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
#3
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/28405595/in-vivo-mitophagy-monitoring-in-caenorhabditis-elegans-to-determine-mitochondrial-homeostasis
#4
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
#5
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/28516126/an-hplc-based-method-to-quantify-coronatine-production-by-bacteria
#6
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
#7
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
#8
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/28255574/automatic-quantification-of-the-number-of-intracellular-compartments-in-arabidopsis-thaliana-root-cells
#9
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
https://www.readbyqxmd.com/read/28523286/synthetic-lethality-screens-using-rnai-in-combination-with-crispr-based-knockout-in-drosophila-cells
#10
Benjamin E Housden, Hilary E Nicholson, Norbert Perrimon
A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi...
February 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28516125/determination-of-the-in-vitro-sporulation-frequency-of-clostridium-difficile
#11
Adrianne N Edwards, Shonna M McBride
The anaerobic, gastrointestinal pathogen, Clostridium difficile, persists within the environment and spreads from host-to-host via its infectious form, the spore. To effectively study spore formation, the physical differentiation of vegetative cells from spores is required to determine the proportion of spores within a population of C. difficile. This protocol describes a method to accurately enumerate both viable vegetative cells and spores separately and subsequently calculate a sporulation frequency of a mixed C...
February 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28516124/bioinformatic-analysis-for-profiling-drug-induced-chromatin-modification-landscapes-in-mouse-brain-using-chlp-seq-data
#12
Yong-Hwee Eddie Loh, Jian Feng, Eric Nestler, Li Shen
Chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) is a powerful technology to profile genome-wide chromatin modification patterns and is increasingly being used to study the molecular mechanisms of brain diseases such as drug addiction. This protocol discusses the typical procedures involved in ChIP-seq data generation, bioinformatic analysis, and interpretation of results, using a chronic cocaine treatment study as a template. We describe an experimental design that induces significant chromatin modifications in mouse brain, and the use of ChIP-seq to derive novel information about the chromatin regulatory mechanisms involved...
February 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28516123/polysome-fractionation-to-analyze-mrna-distribution-profiles
#13
Amaresh C Panda, Jennifer L Martindale, Myriam Gorospe
Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al...
February 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28367479/heterochronic-pellet-assay-to-test-cell-cell-communication-in-the-mouse-retina
#14
Nobuhiko Tachibana, Dawn Zinyk, Randy Ringuette, Valerie Wallace, Carol Schuurmans
All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells...
February 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28516122/two-electrode-voltage-clamp-recordings-in-xenopus-laevis-oocytes-reconstitution-of-abscisic-acid-activation-of-slac1-anion-channel-via-pyl9-aba-receptor
#15
Cun Wang, Jingbo Zhang, Julian I Schroeder
Two-Electrode Voltage-Clamp (TEVC) recording in Xenopus laevis oocytes provides a powerful method to investigate the functions and regulation of ion channel proteins. This approach provides a well-known tool to characterize ion channels or transporters expressed in Xenopus laevis oocytes. The plasma membrane of the oocyte is impaled by two microelectrodes, one for voltage sensing and the other one for current injection. Here we list a protocol that allows robust reconstitution of multi-component signaling pathways...
January 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28503634/an-acute-mouse-spinal-cord-slice-preparation-for-studying-glial-activation-ex-vivo
#16
Juan Mauricio Garré, Guang Yang, Feliksas F Bukauskas, Michael V L Bennett
Pathological conditions such as amyotrophic lateral sclerosis, spinal cord injury and chronic pain are characterized by activation of astrocytes and microglia in spinal cord and have been modeled in rodents. In vivo imaging at cellular level in these animal models is limited due to the spinal cord's highly myelinated funiculi. The preparation of acute slices may offer an alternative and valuable strategy to collect structural and functional information in vitro from dorsal, lateral and ventral regions of spinal cord...
January 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28286806/assessment-of-cellular-redox-state-using-nad-p-h-fluorescence-intensity-and-lifetime
#17
Thomas S Blacker, Tunde Berecz, Michael R Duchen, Gyorgy Szabadkai
NADH and NADPH are redox cofactors, primarily involved in catabolic and anabolic metabolic processes respectively. In addition, NADPH plays an important role in cellular antioxidant defence. In live cells and tissues, the intensity of their spectrally-identical autofluorescence, termed NAD(P)H, can be used to probe the mitochondrial redox state, while their distinct enzyme-binding characteristics can be used to separate their relative contributions to the total NAD(P)H intensity using fluorescence lifetime imaging microscopy (FLIM)...
January 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28239624/p-body-and-stress-granule-quantification-in-caenorhabditis-elegans
#18
Matthias Rieckher, Nektarios Tavernarakis
Eukaryotic cells contain various types of cytoplasmic, non-membrane bound ribonucleoprotein (RNP) granules that consist of non-translating mRNAs and a versatile set of associated proteins. One prominent type of RNP granules are Processing bodies (P bodies), which majorly harbors translationally inactive mRNAs and an array of proteins mediating mRNA degradation, translational repression and cellular mRNA transport (Sheth and Parker, 2003). Another type of RNP granules, the stress granules (SGs), majorly contain mRNAs associated with translation initiation factors and are formed upon stress-induced translational stalling (Kedersha et al...
January 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28516121/simultaneous-intranasal-intravascular-antibody-labeling-of-cd4-t-cells-in-mouse-lungs
#19
Yanqun Wang, Jing Sun, Rudragouda Channappanavar, Jingxian Zhao, Stanley Perlman, Jincun Zhao
CD4(+) T cell responses have been shown to be protective in many respiratory virus infections. In the respiratory tract, CD4(+) T cells include cells in the airway and parenchyma and cells adhering to the pulmonary vasculature. Here we discuss in detail the methods that are useful for characterizing CD4(+) T cells in different anatomic locations in mouse lungs.
January 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28516119/affinity-pulldown-of-biotinylated-rna-for-detection-of-protein-rna-complexes
#20
Amaresh C Panda, Jennifer L Martindale, Myriam Gorospe
RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al., 2008). There are several methodologies available today to identify RNAs bound to specific RBPs; some detect only recombinant molecules in vitro, others detect recombinant and endogenous molecules, while others detect only endogenous molecules...
December 20, 2016: Bio-protocol
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