1The ithree Institute, University of Technology, Sydney
Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.
Published September 29, 2014. Keywords: Molecular Biology, super-resolution microscopy, fluorescence microscopy, OMX, 3D-SIM, Blaze, cell division, bacteria, Bacillus subtilis, Staphylococcus aureus, FtsZ, Z ring constriction
1Penn Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania
In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.
Published September 29, 2014. Keywords: Cellular Biology, melanocyte-like cells, heart, mouse, atrial myocytes, primary isolation
1Department of Physics, University of Illinois at Urbana-Champaign, 2Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, 3Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign
Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.
Published September 26, 2014. Keywords: Molecular Biology, FIONA, fluorescence imaging, nanometer precision, myosin walking, thick tissue
1Department of Biochemistry, Medical College of Wisconsin, 2Stanford Cardiovascular Institute, Stanford University School of Medicine, 3Department of Anesthesiology, Medical College of Wisconsin, 4Stem Cell and Regenerative Medicine Consortium, LKS Faculty of Medicine, Hong Kong University, 5Division of Cardiology, Johns Hopkins University School of Medicine, 6Cardiovascular Research Center, Biotechnology and Bioengineering Center, Medical College of Wisconsin
The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.
Published September 23, 2014. Keywords: Cellular Biology, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
1McAllister Heart Institute, University of North Carolina at Chapel Hill, 2New York-Presbyterian Hospital/Weill-Cornell Medical Center
The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.
Published September 23, 2014. Keywords: Developmental Biology, heart, valve, embryonic, mouse, development, protein, western blot
1Department of Biology, Indiana University
The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.
Published September 20, 2014. Keywords: Cellular Biology, Drosophila, imaginal discs, eye, retina, dissection, developmental biology
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