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Bio-protocol

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https://www.readbyqxmd.com/read/28191488/measurement-of-mechanical-tension-at-cell-cell-junctions-using-two-photon-laser-ablation
#1
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/28194429/intracellular-assessment-of-atp-levels-in-caenorhabditis-elegans
#2
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
#3
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/28203614/transfer-of-large-contiguous-dna-fragments-onto-a-low-copy-plasmid-or-into-the-bacterial-chromosome
#4
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
#5
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
#6
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
#7
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
#8
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
#9
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
https://www.readbyqxmd.com/read/27840835/generation-of-mitochondrial-nuclear-exchange-mice-via-pronuclear-transfer
#10
Robert A Kesterson, Larry W Johnson, Laura J Lambert, Jay L Vivian, Danny R Welch, Scott W Ballinger
The mitochondrial paradigm for common disease proposes that mitochondrial DNA (mtDNA) sequence variation can contribute to disease susceptibility and progression. To test this concept, we developed the Mitochondrial-nuclear eXchange (MNX) model, in which isolated embryonic pronuclei from one strain of species are implanted into an enucleated embryo of a different strain of the same species (e.g., C57BL/6 and C3H/HeN, Mus musculus), generating a re-constructed zygote harboring nuclear and mitochondrial genomes from different strains...
October 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27785458/aniline-blue-and-calcofluor-white-staining-of-callose-and-cellulose-in-the-streptophyte-green-algae-zygnema-and-klebsormidium
#11
Klaus Herburger, Andreas Holzinger
Plant including green algal cells are surrounded by a cell wall, which is a diverse composite of complex polysaccharides and crucial for their function and survival. Here we describe two simple protocols to visualize callose (β-1→3-glucan) and cellulose (β-1→4-glucan) and related polysaccharides in the cell walls of streptophyte green algae by using standard dyes and epifluorescence microscopy.
October 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27819014/isolation-culture-and-staining-of-single-myofibers
#12
Yann Simon Gallot, Sajedah M Hindi, Aman K Mann, Ashok Kumar
Adult skeletal muscle regeneration is orchestrated by a specialized population of adult stem cells called satellite cells, which are localized between the basal lamina and the plasma membrane of myofibers. The process of satellite cell-activation, proliferation, and subsequent differentiation that occurs during muscle regeneration can be recapitulated ex vivo by isolation of single myofibers from skeletal muscles and culturing them under suspension conditions. Here, we describe an improved protocol to evaluate ex vivo satellite cells activation through isolation of single myofibers from extensor digitorum longus (EDL) muscle of mice and culturing and staining of myofiber-associated satellite cells with the markers of self-renewal, proliferation, and differentiation...
October 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28191487/preparation-of-protein-containing-extracts-from-microbiota-rich-intestinal-contents
#13
Patricia Dillenburg-Pilla, Carlos R Zárate-Bladés, Phyllis B Silver, Reiko Horai, Rachel R Caspi
The contribution of microbiota in regulating multiple physiological and pathological host responses has been studied intensively in recent years. Evidence suggests that commensal microbiota can directly modulate different populations of cells of the immune system (e.g., Ivanov et al., 2008; Atarashi et al., 2011). Recently, we showed that protein extracts from gut commensal microbiota can activate retina-specific T cells, allowing these autoreactive T cells to then break through the blood-retinal barrier and trigger autoimmune uveitis in the recipient (Horai et al...
September 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27642615/analysis-of-enteric-neural-crest-cell-migration-using-heterotopic-grafts-of-embryonic-guts
#14
Rodolphe Soret, Nicolas Pilon
Hirschsprung disease (HSCR), also named aganglionic megacolon, is a severe congenital malformation characterized by a lack of enteric nervous system (ENS) in the terminal regions of the bowel (Bergeron et al., 2013). As the ENS notably regulates motility in the whole gastrointestinal track, the segment without neurons remains tonically contracted, resulting in functional intestinal obstruction and accumulation of fecal material (megacolon). HSCR occurs when enteric neural progenitors of vagal neural crest origin fail to fully colonize the developing intestines...
September 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28127575/determination-of-vps34-pik3c3-activity-in-vitro-utilising-32-p-%C3%AE-atp
#15
Michael J Munson, Ian G Ganley
VPS34 is the only class III phosphatidylinositol-3-kinase (PI3K) in mammalian cells and produces the vast majority of cellular phosphatidylinositol-3-phosphate [PI(3)P]. PI(3)P is a key signalling lipid that plays many membrane trafficking roles in processes such as endocytosis and autophagy. VPS34 is a key cellular regulator, loss of function can have catastrophic effects and is embryonic lethal (Zhou et al., 2011). The levels of cellular PI(3)P can be determined by fluorescent staining techniques and can be used to monitor effects upon VPS34 activity, however it is important to verify that any changes are mediated by VPS34, particularly as alternate pathways of PI(3)P production are possible such as via class II PI3Ks (Devereaux et al...
August 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28127574/determination-of-cellular-phosphatidylinositol-3-phosphate-pi3p-levels-using-a-fluorescently-labelled-selective-pi3p-binding-domain-px
#16
Michael J Munson, Ian G Ganley
The lipid Phosphatidylinositol-3-phosphate [PtdIns3P or PI(3)P] plays many membrane trafficking roles and is primarily produced by the Class III PI3K, VPS34. Determining the level of cellular PI(3)P however can be complex. Extraction of cellular lipids by methanol/chloroform can struggle to separate and identify distinct phospholipid species. Alternately mass spectrometry may be utilised but this requires significant set up of specialised equipment and time to utilise. Use of a PI(3)P-binding-specific recombinant protein domain is a quick method for ascertaining cellular PI(3)P levels and can also allow visualisation of sub-cellular localisation...
August 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28093578/mouse-liver-mitochondria-isolation-size-fractionation-and-real-time-momp-measurement
#17
Thibaud T Renault, Mark P A Luna-Vargas, Jerry E Chipuk
The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators...
August 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28042595/expression-purification-and-crystallization-of-the-herpesvirus-nuclear-egress-complex-nec
#18
Janna M Bigalke, Ekaterina E Heldwein
The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pure NEC from two different viruses in a prokaryotic expression system, which enabled us to crystallize these viral protein complexes and determine their structures...
July 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27366759/aorta-atherosclerosis-lesion-analysis-in-hyperlipidemic-mice
#19
Sarajo Mohanta, Changjun Yin, Christian Weber, Desheng Hu, Andreas Jr Habenicht
Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries. Apolipoprotein E-deficient (ApoE(-/-)) mice are used as experimental models to study human atherosclerosis. ApoE(-/-) mice are constitutively hyperlipidemic and develop intima plaques that resemble human plaques. Various issues including experimental design for lesion analysis, dietary conditions, isolation of the aorta, staining methods, morphometry, group size, age, the location within the arterial tree, and statistical analyses are important parameters that need to be addressed to obtain robust data...
June 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27335895/preparation-of-single-cell-suspensions-from-mouse-aorta
#20
Desheng Hu, Changjun Yin, Sarajo K Mohanta, Christian Weber, Andreas J R Habenicht
Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by lipid deposition, plaque formation, and immune cell infiltration. Innate and adaptive immune cells infiltrate the artery during development of the disease. Moreover, advanced disease leads to formation of artery tertiary lymphoid organs in the adventitia (Grabner et al., 2009; Hu et al., 2015). Various and diverse types of immune cells have been identified in the aorta adventitia vs atherosclerotic plaques (Elewa et al., 2016; Galkina et al...
June 5, 2016: Bio-protocol
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