1Department of Biology, KU Leuven
The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).
Published July 28, 2014. Keywords: Cellular Biology, G protein-coupled receptor (GPCR), calcium mobilization assay, reverse pharmacology, deorphanization, cellular expression system, HEK293T, Fluo-4, FlexStation
1European Laboratory for Non-Linear Spectroscopy, University of Florence, 2National Institute of Optics, National Research Council, 3Department of Physics and Astronomy, University of Florence, 4International Center for Computational Neurophotonics (ICON Foundation)
Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.
Published July 28, 2014. Keywords: Neuroscience, axonal labeling, neuronal tracing, in vivo imaging, two-photon microscopy, cerebellum, climbing fibers, laser axotomy, craniotomy
1Veterans Administration Medical Center, San Diego, 2Department of Neurosciences, University of California, San Diego
Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.
Published July 27, 2014. Keywords: Neuroscience, nervous system diseases, wounds and injuries, biological factors, therapeutics, surgical procedures, neural stem cells, transplantation, spinal cord injury, fibrin, growth factors
1Department of Engineering Sciences, Uppsala University, 2Gatan Inc., 3Department of Microbiology, Swedish University of Agricultural Sciences, 4Physics Department, University of Oslo
Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.
Published July 26, 2014. Keywords: Bioengineering, Cryoelectron Microscopy, Life Sciences (General), Cryo-microscopy, Focused ion beam, Sample preparation, TEM, FIB
1Department of Medical Biophysics, University of Toronto, 2Platform Biological Sciences, Sunnybrook Research Institute, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Physical Sciences, Sunnybrook Research Institute, 5Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 6Manitoba Institute of Cell Biology, University of Manitoba
We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.
Published July 26, 2014. Keywords: Bioengineering, Histology Volume Reconstruction, Transgenic Mouse Model, Image Registration, Digital Histology, Image Processing, Mouse Mammary Gland
1Chemical Sensing & Fuel Technology, Chemistry Division, U.S. Naval Research Laboratory, 2NOVA Research, Inc., 3Bio/Analytical Chemistry, Chemistry Division, U.S. Naval Research Laboratory, 4Navy Technology Center for Safety and Survivability, Chemistry Division, U.S. Naval Research Laboratory
Trace explosive vapors of TNT and RDX collected on sorbent-filled thermal desorption tubes were analyzed using a programmed temperature desorption system coupled to GC with an electron capture detector. The instrumental analysis is combined with direct liquid deposition method to reduce sample variability and account for instrumentation drift and losses.
Published July 25, 2014. Keywords: Chemistry, Gas Chromatography (GC), Electron Capture Detector, Explosives, Quantitation, Thermal Desorption, TNT, RDX
1Institute of Pharmacology and Toxicology and Bio-Imaging Center/Rudolf Virchow Center, DFG-Research Center for Experimental Biomedicine, University of Würzburg, Germany
This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.
Published July 25, 2014. Keywords: Bioengineering, pharmacology, microscopy, receptor, live-cell imaging, single-molecule, total internal reflection fluorescence, tracking, dimerization, protein-protein interactions
JoVE Immunology and Infection
1Department of Biomedicine, Immunoregulation, University of Basel and University Hospital Basel
Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.
Published July 25, 2014. Keywords: Immunology, Tail-skin transplantation, I-Abm12 mismatch, CD4+ T cell, ABM, Rejection, Tolerance
1NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health
Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.
Published July 24, 2014. Keywords: Stem Cell Biology, Pluripotent stem cells, human embryonic stem cells, induced pluripotent stem cells, cell culture, non-colony type monolayer, single cell, plating efficiency, Rho-associated kinase, Y-27632, transfection, transduction
1Laboratoire Matière et Systèmes Complexes, UFR de Physique, Université Paris Diderot, 2Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, INRA Centre de Versailles-Grignon
We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.
Published July 24, 2014. Keywords: Plant Biology, Tissue growth, Cell wall, Plant mechanics, Elasticity, Young’s modulus, Root, Apical meristem, Hypocotyl, Organ formation, Biomechanics, Morphogenesis