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Bio-protocol

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https://www.readbyqxmd.com/read/28286807/protein-synthesis-rate-assessment-by-fluorescence-recovery-after-photobleaching-frap
#1
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
#2
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/28286806/assessment-of-cellular-redox-state-using-nad-p-h-fluorescence-intensity-and-lifetime
#3
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
#4
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/28191488/measurement-of-mechanical-tension-at-cell-cell-junctions-using-two-photon-laser-ablation
#5
Xuan Liang, Magdalene Michael, Guillermo A Gomez
The cortical actomyosin cytoskeleton is found in all non-muscle cells where a key function is to control mechanical force (Salbreux et al., 2012). When coupled to E-cadherin cell-cell adhesion, cortical actomyosin generates junctional tension that influences many aspects of tissue function, organization and morphogenesis (Lecuit and Yap, 2015). Uncovering the molecular mechanisms underlying the generation of junctional tension requires tools for measuring it in live cells with a high spatio-temporal resolution...
December 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28251171/analysis-of-myosin-ii-minifilament-orientation-at-epithelial-zonula-adherens
#6
Magdalene Michael, Xuan Liang, Guillermo A Gomez
Non-muscle myosin II (NMII) form bipolar filaments, which bind F-actin to exert cellular contractility during physiological processes (Vicente-Manzanares et al., 2009). Using a combinatorial approach to fluorescently label both N- and C-termini of the NMII heavy chain, recent works have demonstrated the ability to visualize NMII bipolar filaments at various subcellular localizations (Ebrahim et al., 2013; Beach et al., 2014). At the zonula adherens (ZA) of epithelia, NMII minifilaments bind the circumferential actin bundles in a pseudo-sarcomeric manner (Ebrahim et al...
December 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28239623/isolation-of-latex-bead-phagosomes-from-dictyostelium-for-in-vitro-functional-assays
#7
Ashwin D'Souza, Paulomi Sanghavi, Ashim Rai, Divya Pathak, Roop Mallik
We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined periods inside cells before extraction for in vitro motility. These assays allow us to probe how lipids on the phagosome membrane recruit and organize motors, and also measure the motion and force generation resulting from underlying lipid-motor interactions...
December 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28239622/measuring-oxygen-consumption-rate-in-caenorhabditis-elegans
#8
Konstantinos Palikaras, Nektarios Tavernarakis
The rate of oxygen consumption is a vital marker indicating cellular function during lifetime under normal or metabolically challenged conditions. It is used broadly to study mitochondrial function (Artal-Sanz and Tavernarakis, 2009; Palikaras et al., 2015; Ryu et al., 2016) or investigate factors mediating the switch from oxidative phosphorylation to aerobic glycolysis (Chen et al., 2015; Vander Heiden et al., 2009). In this protocol, we describe a method for the determination of oxygen consumption rates in the nematode Caenorhabditis elegans...
December 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28194429/intracellular-assessment-of-atp-levels-in-caenorhabditis-elegans
#9
Konstantinos Palikaras, Nektarios Tavernarakis
Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP levels can serve as a metabolic gauge for cellular homeostasis and survival (Artal-Sanz and Tavernarakis, 2009; Gomes et al., 2011; Palikaras et al., 2015). In this protocol, we describe a method for the determination of intracellular ATP levels using a bioluminescence approach in the nematode Caenorhabditis elegans...
December 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28018942/murine-leukemia-virus-mlv-based-coronavirus-spike-pseudotyped-particle-production-and-infection
#10
Jean Kaoru Millet, Gary R Whittaker
Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors...
December 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28280752/determination-of-h2o2-generation-by-phpa-assay
#11
Jennifer L Larson-Casey, A Brent Carter
The production of reactive oxygen species, including H2O2, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H2O2 release from cells (1-5). This assay takes advantage of H2O2-mediated oxidation of horseradish peroxidase (HRP) to Complex I, which, in turn, oxidizes p-hydroxyphenylacetic acid (pHPA) to a stable, fluorescent pHPA dimer (2,2'-dihydroxy-biphenyl-5,5' diacetate [(pHPA)2])...
November 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28239621/assay-to-evaluate-bal-fluid-regulation-of-fibroblast-%C3%AE-sma-expression
#12
Jennifer L Larson-Casey, A Brent Carter
Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol allows evaluating the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. Fibroblast differentiation was measured by the expression of α-smooth muscle actin (α-SMA). BACKGROUND: Alveolar macrophages play an integral role in pulmonary fibrosis development by increasing the expression of TGF-β1 (He et al...
November 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28239620/analysis-of-phagosomal-antigen-degradation-by-flow-organellocytometry
#13
Eik Hoffmann, Anne-Marie Pauwels, Andrés Alloatti, Fiorella Kotsias, Sebastian Amigorena
Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome maturation kinetics differ between cell types and cell activation states. This protocol allows to quantify phagosome maturation kinetics on a single organelle level in different types of phagocytes using flow cytometry...
November 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28239619/evaluation-of-cross-presentation-in-bone-marrow-derived-dendritic-cells-in-vitro-and-splenic-dendritic-cells-ex-vivo-using-antigen-coated-beads
#14
Andrés Alloatti, Fiorella Kotsias, Eik Hoffmann, Sebastian Amigorena
Antigen presentation by MHC class I molecules, also referred to as cross-presentation, elicits cytotoxic immune responses. In particular, dendritic cells (DC) are the most proficient cross-presenting cells, since they have developed unique means to control phagocytic and degradative pathways. This protocol allows the evaluation of antigen cross-presentation both in vitro (by using bone marrow-derived DC) and ex vivo (by purifying CD8(+) DC from spleen after incorporation of particulate antigen) using ovalbumin (OVA)-coupled particles...
November 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28203614/transfer-of-large-contiguous-dna-fragments-onto-a-low-copy-plasmid-or-into-the-bacterial-chromosome
#15
Analise Z Reeves, Cammie F Lesser
Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E...
November 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28184379/establishment-of-patient-derived-xenografts-in-mice
#16
Dongkyoo Park, Dongsheng Wang, Guo Chen, Xingming Deng
Patient-derived xenograft (PDX) models for cancer research have recently attracted considerable attention in both the academy and industry (Hidalgo et al., 2014; Wilding and Bodmer, 2014). PDX models have been developed from different tumor types including lung cancer to improve the drug development process. These models are used for pre-clinical drug evaluation and can be used for the predictive results of clinical outcomes because they conserve original tumor characteristics such as heterogeneity, complexity and molecular diversity (Kopetz et al...
November 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28180137/isolation-of-highly-pure-primary-mouse-alveolar-epithelial-type-ii-cells-by-flow-cytometric-cell-sorting
#17
Meenal Sinha, Clifford A Lowell
In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 × 10(6) ATII cells per mouse lung. The cell preps are highly pure and viable and can be used for genomic or proteomic analyses or cultured ex vivo to understand their roles in various biological processes...
November 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27928550/ionization-properties-of-phospholipids-determined-by-zeta-potential-measurements
#18
Murugappan Sathappa, Nathan N Alder
Biological membranes are vital for diverse cellular functions such as maintaining cell and organelle structure, selective permeability, active transport, and signaling. The surface charge of the membrane bilayer plays a critical role in these myriad processes. For most biomembranes, the surface charge of anionic phospholipids contributes to the negative surface charge density within the interfacial region of the bilayer. To quantify surface charge, it is essential to understand the proton dissociation behavior of the titratable headgroups within such lipids...
November 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28180136/sequencing-of-ebola-virus-genomes-using-nanopore-technology
#19
Thomas Hoenen
Sequencing of virus genomes during disease outbreaks can provide valuable information for diagnostics, epidemiology, and evaluation of potential countermeasures. However, particularly in remote areas logistical and technical challenges can be significant. Nanopore sequencing provides an alternative to classical Sanger and next-generation sequencing methods, and was successfully used under outbreak conditions (Hoenen et al., 2016; Quick et al., 2016). Here we describe a protocol used for sequencing of Ebola virus under outbreak conditions using Nanopore technology, which we successfully implemented at the CDC/NIH diagnostic laboratory (de Wit et al...
November 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28180135/identification-of-methylated-deoxyadenosines-in-genomic-dna-by-da-6m-dna-immunoprecipitation
#20
Magdalena J Koziol, Charles R Bradshaw, George E Allen, Ana S H Costa, Christian Frezza
dA(6m) DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N(6)-methyldeoxyadenosine (dA(6m)). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start sites and excluded from exons, suggesting a role in transcriptional regulation (Koziol et al., 2015). Importantly, its existence suggests that DNA might be more diverse than previously believed, as further DNA modifications might exist in eukaryotic DNA (Koziol et al...
November 5, 2016: Bio-protocol
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