Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.
Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity
A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.
Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response
1Department of Biological Sciences, University of Alabama
Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.
1Center for Stroke Research Berlin (CSB), Charité - Universitätsmedizin Berlin, 2Klinik und Hochschulambulanz für Neurologie, Charité - Universitätsmedizin Berlin, 3Medical School of the Universität Hamburg, Universitätsklinikum Hamburg - Eppendorf, 4Berliner Feuerwehr, 5STEMO-Consortium
Identification of suspected stroke in the dispatch center of the Berlin Fire Brigade prompts the deployment of a CT-equipped ambulance. If ischemic stroke is confirmed and contraindications are excluded prehospital thrombolysis is applied.
1Center for Advanced Microstructures and Devices (CAMD), Louisiana State University, 2Center for Atomic-Level Catalyst Design, Cain Department of Chemical Engineering, Louisiana State University, 3Department of Biological and Agricultural Engineering, Louisiana State University, 4Argonne National Laboratory
Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.
1Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center
We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.
Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.
Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose
1Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology
The active bacterial community associated with the gut of Spodoptera littoralis, was determined by stable-isotope-probing (SIP) coupled to pyrosequencing. Using this methodology, identification of the metabolically active bacteria species within the community was done with high resolution and precision.
1Tri-Institutional Training Program in Laboratory Animal Medicine and Science Memorial Sloan-Kettering Cancer Center, Weill Cornell Medical College, The Rockefeller University, 2Comparative Bioscience Center, The Rockefeller University
The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.
Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.