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Bio-protocol

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https://www.readbyqxmd.com/read/28405595/in-vivo-mitophagy-monitoring-in-caenorhabditis-elegans-to-determine-mitochondrial-homeostasis
#1
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
#2
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/28382319/ca-2-measurements-in-mammalian-cells-with-aequorin-based-probes
#3
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
#4
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
#5
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/28367479/heterochronic-pellet-assay-to-test-cell-cell-communication-in-the-mouse-retina
#6
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/28286806/assessment-of-cellular-redox-state-using-nad-p-h-fluorescence-intensity-and-lifetime
#7
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
#8
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/28413812/quantitative-3d-time-lapse-imaging-of-muscle-progenitors-in-skeletal-muscle-of-live-mice
#9
Micah T Webster, Tyler Harvey, Chen-Ming Fan
For non-optically clear mammalian tissues, it is now possible to use multi-photon microscopy to penetrate deep into the tissue and obtain detailed single cell images in a live animal, i.e., intravital imaging. This technique is in principle applicable to any fluorescently marked cell, and we have employed it to observe stem cells during the regenerative process. Stem cell-mediated skeletal muscle regeneration in the mouse model has been classically studied at specific time points by sacrificing the animal and harvesting the muscle tissue for downstream analyses...
December 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28352647/single-step-marker-switching-in-schizosaccharomyces-pombe-using-a-lithium-acetate-transformation-protocol
#10
Simon D Brown, Alexander Lorenz
The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4(+) and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4(+) and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains...
December 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28191488/measurement-of-mechanical-tension-at-cell-cell-junctions-using-two-photon-laser-ablation
#11
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
#12
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
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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