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Measuring Caenorhabditis elegans Sleep during the Transition to Adulthood Using a Microfluidics-based System

Featured protocol,  Authors: Huiyan Huang
Huiyan HuangAffiliation: Department of Neuroscience, Brown University, Providence, USA
Bio-protocol author page: a4251
Komudi Singh
Komudi SinghAffiliation: Department of Neuroscience, Brown University, Providence, USA
Present address: Laboratory of Mitochondrial Biology & Metabolism, National Institute of Health, Bethesda, USA
Bio-protocol author page: a4252
 and Anne C. Hart
Anne C. HartAffiliation: Department of Neuroscience, Brown University, Providence, USA
For correspondence: anne_hart@brown.edu
Bio-protocol author page: a4253
date: 3/20/2017, 124 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2174.

Brief version appeared in Sleep, Sep 2014
C. elegans sleep during development is regulated by genes and cellular mechanisms that are conserved across the animal kingdom (Singh et al., 2014; Trojanowski and Raizen, 2016). C. elegans developmental sleep is usually assessed during the transition to adulthood, a 2.6 h time interval called lethargus (Raizen et al., 2008; Singh et al., 2011). During lethargus, animals cycle between periods of immobility (sleep bouts) and periods of active locomotion (motion bouts). Sleep bouts resemble sleep in other species based on behavioral criteria, including cessation of feeding and locomotion, increased arousal threshold for response to sensory stimulation, rapid reversibility, and homeostatic response to sleep loss. Several assays have been developed to study sleep in C. elegans (Belfer et al., 2013; Bringmann, 2011; Nelson et al., 2013; Raizen et al., 2008). Here, we contribute a detailed protocol for assessment of C. elegans sleep during lethargus, which has been used successfully by many research groups, incorporating simple microfluidic chambers, a low cost camera with lighting system, and computational analysis based on image subtraction. We note that this system could be easily adapted to assess sleep in any small animal.

Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP)

Featured protocol,  Authors: Nikos Kourtis
Nikos KourtisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Pathology, NYU School of Medicine, New York, USA
Bio-protocol author page: a4141
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 3/5/2017, 260 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2156.

Brief version appeared in Nature, Oct 2012
Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals.

Polyethylene Glycol-mediated Transformation of Drechmeria coniospora

Featured protocol,  Authors: Le D. He
Le D. HeAffiliation: Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
Bio-protocol author page: a4195
 and Jonathan J. Ewbank
Jonathan J. EwbankAffiliation: Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
For correspondence: ewbank@ciml.univ-mrs.fr
Bio-protocol author page: a4196
date: 3/5/2017, 193 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2157.

Brief version appeared in PLoS Genet, May 2016
Drechmeria coniospora is a nematophagous fungus and potential biocontrol agent. It belongs to the Ascomycota. It is related to Hirsutella minnesotensis, another nematophagous fungus but, phylogenetically, it is currently closest to the truffle parasite Tolypocladium ophioglossoides. Together with its natural host, Caenorhabditis elegans, it is used to study host-pathogen interactions. Here, we report a polyethylene glycol-mediated transformation method (Turgeon et al., 2010; Ochman et al., 1988) for this fungus. The protocol can be used to generate both knock-in or knock-out strains (Lebrigand et al., 2016).

Measuring Caenorhabditis elegans Sleep during the Transition to Adulthood Using a Microfluidics-based System

Authors: Huiyan Huang
Huiyan HuangAffiliation: Department of Neuroscience, Brown University, Providence, USA
Bio-protocol author page: a4251
Komudi Singh
Komudi SinghAffiliation: Department of Neuroscience, Brown University, Providence, USA
Present address: Laboratory of Mitochondrial Biology & Metabolism, National Institute of Health, Bethesda, USA
Bio-protocol author page: a4252
 and Anne C. Hart
Anne C. HartAffiliation: Department of Neuroscience, Brown University, Providence, USA
For correspondence: anne_hart@brown.edu
Bio-protocol author page: a4253
date: 3/20/2017, 124 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2174.

[Abstract] C. elegans sleep during development is regulated by genes and cellular mechanisms that are conserved across the animal kingdom (Singh et al., 2014; Trojanowski and Raizen, 2016). C. elegans developmental sleep is usually assessed during the transition to adulthood, a 2.6 h time interval called lethargus ...

Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP)

Authors: Nikos Kourtis
Nikos KourtisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Pathology, NYU School of Medicine, New York, USA
Bio-protocol author page: a4141
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 3/5/2017, 260 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2156.

[Abstract] Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis ...

Polyethylene Glycol-mediated Transformation of Drechmeria coniospora

Authors: Le D. He
Le D. HeAffiliation: Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
Bio-protocol author page: a4195
 and Jonathan J. Ewbank
Jonathan J. EwbankAffiliation: Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
For correspondence: ewbank@ciml.univ-mrs.fr
Bio-protocol author page: a4196
date: 3/5/2017, 193 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2157.

[Abstract] Drechmeria coniospora is a nematophagous fungus and potential biocontrol agent. It belongs to the Ascomycota. It is related to Hirsutella minnesotensis, another nematophagous fungus but, phylogenetically, it is currently closest to the truffle parasite Tolypocladium ophioglossoides. Together with its ...

P-body and Stress Granule Quantification in Caenorhabditis elegans

Authors: Matthias Rieckher
Matthias RieckherAffiliation: Institute for Genome Stability in Ageing and Disease, Cologne Cluster of Excellence in Cellular Stress Responses in Aging-associated Diseases (CECAD) Research Center, Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
Bio-protocol author page: a4014
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 1/20/2017, 536 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2108.

[Abstract] Eukaryotic cells contain various types of cytoplasmic, non-membrane bound ribonucleoprotein (RNP) granules that consist of non-translating mRNAs and a versatile set of associated proteins. One prominent type of RNP granules are Processing bodies (P bodies), which majorly harbors translationally inactive ...

Assay to Access Anthelmintic Activity of Small Molecule Drugs Using Caenohabidtis elegans as a Model

Authors: Viviane Sant’Anna
Viviane Sant’AnnaAffiliation: Laboratório de Biologia de Helmintos Otto Wucherer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Cidade Universitária, Bloco G, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
Bio-protocol author page: a4018
Wanderley de Souza
Wanderley de SouzaAffiliation: Laboratório de Ultraestrutura Celular Herta Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Cidade Universitária, Bloco G, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
Bio-protocol author page: a4019
 and Rossiane C. Vommaro
Rossiane C. VommaroAffiliation: Laboratório de Ultraestrutura Celular Herta Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Cidade Universitária, Bloco G, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
For correspondence: vommaro@biof.ufrj.br
Bio-protocol author page: a4020
date: 1/20/2017, 450 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2113.

[Abstract] This protocol proposes to use the nematode Caenorhabditis elegans as a model to screen and study the anthelmintic activity of natural and synthetic compounds and to observe their effects on the morphology and the ultrastructure of the helminthes. Furthermore, C. elegans can be used to investigate the ...

Measuring Oxidative Stress in Caenorhabditis elegans: Paraquat and Juglone Sensitivity Assays

Authors: Megan M. Senchuk
Megan M. SenchukAffiliation: Laboratory of Aging and Neurodegenerative Disease (LAND), Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, USA
Bio-protocol author page: a3949
Dylan J. Dues
Dylan J. Dues Affiliation: Laboratory of Aging and Neurodegenerative Disease (LAND), Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, USA
Bio-protocol author page: a3950
 and Jeremy M. Van Raamsdonk
Jeremy M. Van RaamsdonkAffiliation: Laboratory of Aging and Neurodegenerative Disease (LAND), Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, USA
For correspondence: jeremy.vanraamsdonk@vai.org
Bio-protocol author page: a3951
date: 1/5/2017, 647 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2086.

[Abstract] Oxidative stress has been proposed to be one of the main causes of aging and has been implicated in the pathogenesis of many diseases. Sensitivity to oxidative stress can be measured by quantifying survival following exposure to a reactive oxygen species (ROS)-generating compound such as paraquat or ...

Measuring Oxygen Consumption Rate in Caenorhabditis elegans

Authors: Konstantinos Palikaras
Konstantinos PalikarasAffiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Bio-protocol author page: a3832
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 12/5/2016, 723 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2049.

[Abstract] The rate of oxygen consumption is a vital marker indicating cellular function during lifetime under normal or metabolically challenged conditions. It is used broadly to study mitochondrial function (Artal-Sanz and Tavernarakis, 2009; Palikaras et al., 2015; Ryu et al., 2016) or investigate factors mediating ...

Intracellular Assessment of ATP Levels in Caenorhabditis elegans

Authors: Konstantinos Palikaras
Konstantinos PalikarasAffiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Bio-protocol author page: a3832
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 12/5/2016, 697 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2048.

[Abstract] Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others ...

Artificial Optogenetic TRN Stimulation of C. elegans

Authors: Ithai Rabinowitch
Ithai RabinowitchAffiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, USA
For correspondence: ithairab@gmail.com
Bio-protocol author page: a3605
Millet Treinin
Millet TreininAffiliation: Department of Medical Neurobiology, Hadassah Medical School, Hebrew University of Jerusalem, Jerusalem, Israel
Bio-protocol author page: a3606
 and Jihong Bai
Jihong BaiAffiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, USA
Bio-protocol author page: a3607
date: 10/20/2016, 601 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1966.

[Abstract] Optogenetics is a powerful tool for manipulating neuronal activity with high temporal and spatial precision. In the nematode C. elegans optogentics is especially useful and easy to apply. This is because C. elegans is translucent, so its neurons are highly accessible to optic stimulation. In addition, ...

Evaluation of Burkholderia cepacia Complex Bacteria Pathogenicity Using Caenorhabditis elegans

Authors: Pietro Tedesco
Pietro TedescoAffiliation: Institute of Protein Biochemistry, National Research Council, Naples, Italy
Bio-protocol author page: a3599
Elia Di Schiavi
Elia Di SchiaviAffiliation: Institute of Bioscience and Bioresources, National Research Council, Naples, Italy
Bio-protocol author page: a3600
Fortunato Palma Esposito
Fortunato Palma EspositoAffiliation: Institute of Protein Biochemistry, National Research Council, Naples, Italy
Bio-protocol author page: a3601
 and Donatella de Pascale
Donatella de PascaleAffiliation: Institute of Protein Biochemistry, National Research Council, Naples, Italy
For correspondence: d.depascale@ibp.cnr.it
Bio-protocol author page: a3602
date: 10/20/2016, 488 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1964.

[Abstract] This protocol describes two biological assays to evaluate pathogenicity of Burkholderia cepacia complex (Bcc) strains against the nematode Caenorhabditis elegans. Specifically, these two assays allow one to identify if the under-investigated Bcc strains are able to kill the nematodes by intestinal colonization ...
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[Bio101] Common Worm Media and Buffers

Author: Fanglian He date: 4/5/2011, 22561 views, 10 Q&A
DOI: https://doi.org/10.21769/BioProtoc.55.

[Abstract] Here are recipes of some media and solutions often used in C. elegans research....

[Bio101] Lifespan Assay

Author: Fanglian He date: 4/5/2011, 14001 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.57.

[Abstract] This assay is used to address aging-related questions in worms....

[Bio101] Total RNA Extraction from C. elegans

Author: Fanglian He date: 3/20/2011, 13858 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.47.

[Abstract] This protocol describes total RNA extraction from worms with or without using commercial RNA extraction kits....

[Bio101] Making Males of C. elegans

Author: Fanglian He date: 4/20/2011, 13128 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.58.

[Abstract] Getting males from a hermaphrodite population. This is a modified version of protocol originally written by Michael Koelle at Yale University....

[Bio101] RNA Interference (RNAi) by Bacterial Feeding

Author: Fanglian He date: 4/20/2011, 12957 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.59.

[Abstract] There are 3 ways to perform RNAi in worms: microinjection, soaking and feeding. In the feeding protocol, RNAi is induced by cultivating worms on bacteria expressing gene-specific dsRNA. dsRNA is expressed in E. coli and ingested by worms. This protocol describes the feeding protocol to induce RNAi....

[Bio101] Synchronization of Worm

Author: Fanglian He date: 4/5/2011, 12609 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.56.

[Abstract] Attaining a large synchronized population of worms is desirable for use in some assays in order to eliminate variation in results due to age differences. Two ways to get synchronized worms include egg preparation via bleaching and egg lay. The former in general yields more progeny than the latter, however, ...

PCR-RFLP Genotyping of Point Mutations in Caenorhabditis elegans

Author: Peichuan Zhang
Peichuan ZhangAffiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, USA
For correspondence: peichuan.zhang@ucsf.edu
Bio-protocol author page: a11
date: 3/20/2012, 11983 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.128.

[Abstract] This protocol describes the basic principle of PCR/restriction digest genotyping of point mutations in worms, based on the principle of Restriction Fragment Length Polymorphism (RFLP) analysis. This type of genotyping is, particularly, useful when phenotypic analysis of animals carrying point mutations ...

[Bio101] Oil Red O Staining of Fixed Worm

Author: Fanglian He date: 7/5/2012, 11601 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.230.

[Abstract] Oil red O staining is used to assess major fat stores in C. elegans. This protocol is adapted from the Ashrafi Lab at the University of California-San Francisco....

[Bio101] DAPI Nuclear Staining of Live Worm

Author: Fanglian He date: 6/5/2011, 10075 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.77.

[Abstract] Adapted from the Villenueve Lab at Stanford University. This is a very simple method using ethanol fixation, but works very well....

[Bio101] Single Worm PCR

Author: Fanglian He date: 4/20/2011, 9439 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.60.

[Abstract] This protocol is used for genotyping single worms....
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