1Center for Innovative Cancer Research, Ottawa Hospital Research Institute, 2Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 3Faculty of Medicine, University of Ottawa
This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.
Published September 19, 2014. Keywords: Virology, titration, virus, plaque assay, high-throughput, transgene, luciferase, automated, cytotoxicity assay, Vesicular Stomatitis Virus, Herpes Simplex virus, Vaccinia virus, Adeno-Associated virus
1Department of Biochemistry, Université de Montréal, CRCHUM, 2Department of Neurosciences, Université de Montréal, CRCHUM
Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.
Published September 18, 2014. Keywords: Cellular Biology, Mitochondria, flow cytometry, organelle isolation, immunolabeling, spinal cord, TMRM
1Department of Pathology, University of Cambridge
The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.
Published September 18, 2014. Keywords: Cellular Biology, innate immunity, DNA, double stranded DNA (dsDNA), concatemer, signaling, transfection, stimulation, ligation
1Department of Structural Biology, Max Planck Institute of Biophysics
We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.
Published September 14, 2014. Keywords: Structural Biology, electron microscopy, electron cryo-tomography, mitochondria, ultrastructure, membrane structure, membrane protein complexes, ATP synthase, energy conversion, bioenergetics
1Department of Biosciences, University of Exeter
Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.
Published September 13, 2014. Keywords: Biophysics, differential scanning fluorimetry, dissociation constant, protein-ligand interactions, StepOne, cooperativity, WcbI.
1Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, 2Department of Biological Chemistry and the Developmental Biology Center, University of California, Irvine
Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.
Published September 11, 2014. Keywords: Molecular Biology, Hydra, transgenic, microinjection, gene overexpression, gene knockdown
1Fachbereich Biologie, Entwicklungsbiologie, Philipps-Universität Marburg, 2Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg
This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.
Published September 11, 2014. Keywords: Developmental Biology, Ex vivo culture, testis, male germ-line cells, Drosophila, imaging, pharmacological assay
1Department of Pediatrics, Center for Environmental Medicine, Asthma, and Lung Biology, The University of North Carolina at Chapel Hill
Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.
Published September 11, 2014. Keywords: Biophysics, Freeze-fracture; Freeze-etch; Membranes; Intercellular junctions; Materials science; Nanotechnology; Electron microscopy
1Department of Biology, Boston College
The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.
Published September 7, 2014. Keywords: Molecular Biology, plusTipTracker, microtubule plus-end-tracking proteins, EB1, growth cone, Xenopus laevis, live cell imaging analysis, microtubule dynamics
1Department of Medicine, Vanderbilt University, 2Department of Cell and Developmental Biology, Vanderbilt University
Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.
Published September 7, 2014. Keywords: Cellular Biology, cardiomyocyte isolation, Langendorff, contractility, calcium transients