Featured protocol,
Authors: Collin Yvès Ewald 
Collin Yvès EwaldAffiliation 1: Department of Health Sciences and Technology, Eidgenössische Technische Hochschule (ETH) Zürich, Schwerzenbach-Zürich, Switzerland
Affiliation 2: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
Affiliation 3: Harvard Stem Cell Institute, Harvard University, Boston, Massachusetts, USA
Affiliation 4: Joslin Diabetes Center, Research Division, Boston, Massachusetts, USA
For correspondence: collin-ewald@ethz.chBio-protocol author page: a4749 John M. HourihanAffiliation 1: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
Affiliation 2: Harvard Stem Cell Institute, Harvard University, Boston, Massachusetts, USA
Affiliation 3: Joslin Diabetes Center, Research Division, Boston, Massachusetts, USA
Bio-protocol author page: a4750 T. Keith BlackwellAffiliation 1: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
Affiliation 2: Harvard Stem Cell Institute, Harvard University, Boston, Massachusetts, USA
Affiliation 3: Joslin Diabetes Center, Research Division, Boston, Massachusetts, USA
Bio-protocol author page: a4751
DOI: https://doi.org/10.21769/BioProtoc.2365.
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| Brief version appeared in Elife, Jan 2017 |
Cells and organisms face constant exposure to reactive oxygen species (ROS), either from the environment or as a by-product from internal metabolic processes. To prevent cellular damage from ROS, cells have evolved detoxification mechanisms. The activation of these detoxification mechanisms and their downstream responses represent an overlapping defense response that can be tailored to different sources of ROS to adequately adapt and protect cells. In this protocol, we describe how to measure the sensitivity to oxidative stress from two different sources, arsenite and tBHP, using the nematode
C. elegans.
Featured protocol,
Authors: Facundo Rodriguez Ayala Facundo Rodriguez AyalaAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
For correspondence: facundoayala.foncyt@gmail.comBio-protocol author page: a4690 Sebastián CogliatiAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4691 Carlos BaumanAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4692 Cecilia LeñiniAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4693 Marco BartoliniAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4694 Juan Manuel VillalbaAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4695 Federico ArgañarazAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4696 Roberto GrauAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4697
DOI: https://doi.org/10.21769/BioProtoc.2345.
 |
| Brief version appeared in Nat Commun, Jan 2017 |
Determining an accurate count of intestinal bacteria from
Caenorhabditis elegans is one critical way to assess colonization proficiency by a given bacteria. This can be accomplished by culturing appropriate dilutions of worm gut bacteria on selective or differential agarized media. Because of the high concentration of bacteria in gut worm, dilution is necessary before plating onto growth media. Serial dilutions can reduce the concentration of the original intestinal sample to levels low enough for single colonies to be grown on media plates, allowing for the calculation of the initial counts of bacteria in the intestinal sample.
Featured protocol,
Authors: ChangHwan LeeChangHwan LeeAffiliation 1: Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA
Affiliation 2: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Bio-protocol author page: a4726 Hannah S. SeidelAffiliation 1: Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA
Affiliation 2: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Present address: Department of Biology, Eastern Michigan University, Ypsilanti, Michigan, USA
Bio-protocol author page: a4727 Tina R. LynchAffiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Bio-protocol author page: a4728 Erika B. SorensenAffiliation 1: Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA
Affiliation 2: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Present address: Department of Biology, Wabash College, Crawfordsville, Indiana, USA
Bio-protocol author page: a4729 Sarah L. CrittendenAffiliation: Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA
Bio-protocol author page: a4730 Judith KimbleAffiliation 1: Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA
Affiliation 2: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
For correspondence: jekimble@wisc.eduBio-protocol author page: a4731
DOI: https://doi.org/10.21769/BioProtoc.2357.
 |
| Brief version appeared in Elife, Oct 2016 |
Single-molecule RNA fluorescence
in situ hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA (Raj
et al., 2008; Lee
et al., 2016a). We adapted this technique to visualize RNAs in the
C. elegans whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm (Lee
et al., 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for
C. elegans extruded gonads.