1Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, 2Department of Neurobiology and Anatomy, Eccles Institute of Human Genetics, University of Utah
Cilia-generated fluid flow in Kupffer’s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.
Published March 31, 2013. Keywords: Developmental Biology, Genetics, Cellular Biology, Neurobiology, Neuroscience, Molecular Biology, Bioengineering, Biophysics, Anatomy, Physiology, Cilia, Zebrafish, Danio rerio, Gene Knockdown Techniques, Left-right asymmetry, cilia, Kupffer's Vesicle, morpholinos, microinjection, animal model
1Pharmacology and Chemical Biology, University of Pittsburgh Drug Discovery Institute, 2Department of Microbiology and Molecular Genetics, University of Pittsburgh, 3Department of Pharmaceutical Sciences, University of Pittsburgh, 4Department of Chemistry, University of Pittsburgh
We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.
Published June 24, 2010. Keywords: Cellular Biology, Zebrafish, Chemical Screens, Cognition Network Technology, Fibroblast Growth Factor, (E)-2-benzylidene-3-(cyclohexylamino)-2, 3-dihydro-1H-inden-1-one (BCI), Tg(dusp6:d2EGFP)
JoVE Immunology and Infection
1Lurie Family Imaging Center, Dana-Farber Cancer Institute, Harvard Medical School, 2Division of Pediatric Hematology/Oncology/Stem Cell Transplantation, Columbia University Medical Center
We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.
Published August 16, 2013. Keywords: Immunology, Infection, Medicine, Cellular Biology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Cancer Biology, Stem Cell Biology, Inflammation, Phagocytes, Phagocyte, Superoxides, Molecular Imaging, chemiluminescence, in vivo imaging, superoxide, bioluminescence, chronic inflammation, acute inflammation, phagocytes, cells, imaging, animal model
1Institute of Anatomy, Technische Universität Dresden
Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.
Published October 26, 2010. Keywords: Neuroscience, neurogical disorders, Parkinson's disease, animal model, mouse, rotenone, gavage, image analysis
1Division of Nutritional Sciences, Cornell University, 2Field of Biochemistry, and Molecular Cell Biology, Cornell University
Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.
Published May 27, 2014. Keywords: Chemistry, high-resolution mass spectrometry, metabolomics, positive/negative switching, low mass calibration, Orbitrap
1Laboratory of Structural Biology, NIEHS, National Institutes of Health
Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.
Published August 19, 2013. Keywords: Chemistry, Biochemistry, Genetics, Molecular Biology, Microbiology, Structural Biology, Chemical Biology, Eukaryota, Amino Acids, Peptides, and Proteins, Nucleic Acids, Nucleotides, and Nucleosides, Enzymes and Coenzymes, Life Sciences (General), enzymology, rapid quench-flow, active site titration, steady-state, pre-steady-state, single-turnover, kinetics, base excision repair, DNA glycosylase, 8-oxo-7, 8-dihydroguanine, 8-oxoG, sequencing
JoVE Clinical and Translational Medicine
1Cancer Research Center, Sheba Medical Center, 2The Neufeld Cardiac Research Institute, Tel-Aviv University, 3Department of Physiology and Pharmacology, Tel-Aviv University, 4Imaging Unit, Sackler Faculty of Medicine, Tel-Aviv University, 5Biotechnology and Cell Signaling, Ecole Superieure de Biotechnologie Strasbourg, 6Department of Human Molecular Genetics and Biochemistry, Tel-Aviv University
The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.
Published August 21, 2013. Keywords: Cancer Biology, Medicine, Cellular Biology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Genetics, Neoplastic Processes, Pharmacologic Actions, Live confocal imaging, Extra-centrosomes clustering/de-clustering, Mitotic Catastrophe cell death, PJ-34, myocardial infarction, microscopy, imaging
1Unit on Neural Circuits and Adaptive Behaviors, Genes Cognition and Psychosis Program, National Institute of Mental Health, 2Department of Neuroscience, Brown University - National Institutes of Health Graduate Partnership Program, 3Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, 4Champalimaud Neuroscience Programme, Champalimaud Center for the Unknown
Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.
Published January 5, 2013. Keywords: Neuroscience, Medicine, Anatomy, Neurobiology, Surgery, Cerebral Cortex, Frontal Cortex, Stereotaxic Techniques, Molecular Imaging, Neuronal Plasticity, Neurosciences, In Vivo Imaging, Two-photon Microscopy, Experience-dependent Gene Expression, Arc-GFP Mice, Cranial Window, in situ hybridization, immunohistochemistry, animal model
1Department of Biochemistry, University of Geneva
We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.
Published November 5, 2013. Keywords: Microbiology, Biology (general), Biochemistry, Reactive oxygen species, Superoxide, Hydrogen peroxide, OxyBurst Green, Carboxylated beads, Dihydroethidium, Amplex UltraRed, Phagocytosis, Dictyostelium discoideum
1The Davis Heart and Lung Research Institute, The Ohio State University, 2Department of Pharmacology, College of Medicine, The Ohio State University
Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.
Published August 18, 2012. Keywords: Molecular Biology, Cellular Biology, Physics, Biophysics, spin trap, eNOS, ROS, superoxide, NO, EPR