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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.

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 (Raizen et al., 2008; Singh et al., 2011). During ...

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 elegans animals....

Transplantation of Mesenchymal Cells Including the Blastema in Regenerating Zebrafish Fin

Authors: Eri Shibata
Eri ShibataAffiliation: School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
Bio-protocol author page: a4049
Kazunori Ando
Kazunori AndoAffiliation: School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
Bio-protocol author page: a4050
 and Atsushi Kawakami
Atsushi KawakamiAffiliation: School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
For correspondence: atkawaka@bio.titech.ac.jp
Bio-protocol author page: a4051
date: 1/20/2017, 369 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2109.

[Abstract] Regeneration of fish fins and urodele limbs occurs via formation of the blastema, which is a mass of mesenchymal cells formed at the amputated site and is essential for regeneration. The blastema transplantation, a novel technique developed in our previous studies (Shibata et al., 2016; Yoshinari et al., 2012) is a useful approach for tracking and ...

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 mRNAs and an array of proteins mediating mRNA ...

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 the switch from oxidative phosphorylation ...

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 (Khakh and Burnstock, 2009). Therefore, ATP levels ...

Vascular Smooth Muscle Cell Isolation and Culture from Mouse Aorta

Authors: Callie S. Kwartler
Callie S. KwartlerAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3821
Ping Zhou
Ping ZhouAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3822
Shao-Qing Kuang
Shao-Qing KuangAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3823
Xue-Yan Duan
Xue-Yan DuanAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3824
Limin Gong
Limin GongAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
Bio-protocol author page: a3825
 and Dianna M. Milewicz
Dianna M. MilewiczAffiliation: Department of Internal Medicine, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas, USA
For correspondence: Dianna.M.Milewicz@uth.tmc.edu
Bio-protocol author page: a3827
date: 12/5/2016, 700 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2045.

[Abstract] Vascular smooth muscle cells (SMC) in the ascending thoracic aorta arise from neural crest cells, whereas SMCs in the descending aorta are derived from the presomitic mesoderm. SMCs play important roles in cardiovascular development and aortic aneurysm formation. This protocol describes the detailed process for explanting ascending and descending SMCs ...

Visualising Differential Growth of Arabidopsis Epidermal Pavement Cells Using Thin Plate Spline Analysis

Authors: William Jonathan Armour
William Jonathan ArmourAffiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
For correspondence: armour.william@gmail.com
Bio-protocol author page: a3748
Deborah Anne Barton
Deborah Anne BartonAffiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
Bio-protocol author page: a3749
 and Robyn Lynette Overall
Robyn Lynette OverallAffiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
Bio-protocol author page: a3750
date: 11/20/2016, 705 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2022.

[Abstract] Epidermal pavement cells in Arabidopsis leaves and cotyledons develop from relatively simple shapes to form complex cells that have multiple undulations of varying sizes. Analyzing the growth of individual parts of the cell wall boundaries over time is essential to understanding how pavement cells develop their complex shapes. Thin plate spline analysis ...

Isolation, Culture, and Staining of Single Myofibers

Authors: Yann Simon Gallot
Yann Simon GallotAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a3535
Sajedah M. Hindi
Sajedah M. HindiAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
Bio-protocol author page: a3536
Aman K. Mann
Aman K. MannAffiliation: duPont Manual High Schoo, Louisville, USA
Bio-protocol author page: a3538
 and Ashok Kumar
Ashok KumarAffiliation: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, USA
For correspondence: ashok.kumar@louisville.edu
Bio-protocol author page: a3537
date: 10/5/2016, 862 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1942.

[Abstract] Adult skeletal muscle regeneration is orchestrated by a specialized population of adult stem cells called satellite cells, which are localized between the basal lamina and the plasma membrane of myofibers. The process of satellite cell-activation, proliferation, and subsequent differentiation that occurs during muscle regeneration can be recapitulated ...

Analysis of Enteric Neural Crest Cell Migration Using Heterotopic Grafts of Embryonic Guts

Authors: Rodolphe Soret
Rodolphe SoretAffiliation: Molecular Genetics of Development Laboratory, Department of Biological Sciences; BioMed Research Center, Faculty of Sciences, University of Quebec at Montreal (UQAM), Montreal, PQ, Canada
Bio-protocol author page: a3490
 and Nicolas Pilon
Nicolas PilonAffiliation: Molecular Genetics of Development Laboratory, Department of Biological Sciences; BioMed Research Center, Faculty of Sciences, University of Quebec at Montreal (UQAM), Montreal, PQ, Canada
For correspondence: pilon.nicolas@uqam.ca
Bio-protocol author page: a3491
date: 9/5/2016, 696 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1924.

[Abstract] Hirschsprung disease (HSCR), also named aganglionic megacolon, is a severe congenital malformation characterized by a lack of enteric nervous system (ENS) in the terminal regions of the bowel (Bergeron et al., 2013). As the ENS notably regulates motility in the whole gastrointestinal track, the segment without neurons remains tonically contracted, ...
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[Bio101] In vivo BrdU Incorporation and Detection in Mouse

Author: Zheng Liu
Zheng LiuAffiliation: The Feinstein Institute for Medical Research, Manhasset, NY, USA
For correspondence: zl2119@caa.columbia.edu
Bio-protocol author page: a12
Anne Davidson Lab, date: 6/5/2011, 19455 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.81.

[Abstract] BrdU (Bromodeoxyuridine or 5-bromo-2’-deoxyuridine) is a synthetic nucleoside that is incorporated into DNA by proliferating cells. This protocol is to be used to incorporate and detect BrdU in murine plasma cells. The plasma cells described in this protocol are formed spontaneously in autoimmune mice ...

Alcian Blue – Alizarin Red Staining of Mouse Skeleton

Author: Peichuan Zhang
Peichuan ZhangAffiliation 1: Department of Biology, The Pennsylvania State University, University Park, PA, USA
Affiliation 2: Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA
For correspondence: peichuan.zhang@ucsf.edu
Bio-protocol author page: a11
date: 4/20/2012, 15676 views, 9 Q&A
DOI: https://doi.org/10.21769/BioProtoc.162.

[Abstract] Our lab has used the Alcian blue – Alizarin red staining method with certain modifications to characterize skeleton deformities in mice lacking Pek/Perk, encoding a translational control eIF2alpha kinase....

[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] 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....

c-Fos and Arc Immunohistochemistry on Rat Cerebellum

Author: Soyun Kim
Soyun KimAffiliation: Neuroscience Program, University of Southern California, Los Angeles, USA
For correspondence: soyunkimucsd@gmail.com
Bio-protocol author page: a45
date: 5/20/2012, 10612 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.191.

[Abstract] This protocol aims to introduce methods for sacrificing rats by transcardial perfusion and extracting the brain, and introduce methods for staining the rat brain tissue with c-Fos and Arc antibodies. Please note the expression of the proteins is very sensitive to behavioral paradigm that triggers neural ...

Mouse Cochlear Whole Mount Immunofluorescence

Authors: Omar Akil
Omar AkilAffiliation: Department Of Otolaryngology-HNS, University of California, San Francisco, USA
For correspondence: oakil@ohns.ucsf.edu
Bio-protocol author page: a238
 and Lawrence R. Lustig
Lawrence R. LustigAffiliation: Department Of Otolaryngology-HNS, University of California, San Francisco, USA
Bio-protocol author page: a239
date: 3/5/2013, 10607 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.332.

[Abstract] This protocol comprises the entire process of immunofluorescence staining on mouse cochlea whole mount, starting from tissue preparation to the mounting of the tissue. This technique provides “three-dimensional” views of the stained components in order to determine the localization of a protein of interest ...

Phalloidin Staining and Immunohistochemistry of Zebrafish Embryos

Authors: Michelle F. Goody
Michelle F. Goody Affiliation: Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, USA
Bio-protocol author page: a619
 and Clarissa A. Henry
Clarissa A. HenryAffiliation: School of Biology and Ecology, University of Maine, Orono, USA
For correspondence: clarissa.henry@umit.maine.edu
Bio-protocol author page: a620
date: 6/5/2013, 9795 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.786.

[Abstract] Fluorescent conjugated Phalloidin is a stain that allows for visualization of F-actin. In immunohistochemistry, primary antibodies and fluorescent conjugated secondary antibodies can be used to visualize subcellular localization and relative amounts of proteins of interest. Here is a protocol for Phalloidin ...

[Bio101] Cell Culture Transfection for Production and Purification of Wnt Ligands

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 1/20/2012, 9165 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.176.

[Abstract] Wnt ligand proteins are extremely difficult to purify and enrich in vitro. This protocol uses Wnt11r protein as an example to illustrate how to use 293T cells to produce secreted Wnt11r and collect it in vitro for further biochemical experiments....

[Bio101] Mouse Embryonic Stem Cell Maintenance for Differentiation

Author: Hogune Im
Hogune ImAffiliation: Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin Medical Schoo, Madison, USA
For correspondence: hoguneim@stanford.edu
Bio-protocol author page: a18
date: 10/20/2011, 8384 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.145.

[Abstract] Embryonic stem cells are derived from inner cell mass of an embryo that can differentiate into every cell type in the body. Clinically, cultured red blood cell supply is of great interest. However, some of the hurdles need to be overcome. This protocol describes a protocol to maintain mouse stem cells ...

[Bio101] Culturing of C57BL/6 Mouse Embryonic Stem (ES) Cell Line

Author: Yuqiong Pan date: 10/5/2011, 8155 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.142.

[Abstract] Using ATCC ES C57BL/6 as an example, it is shown here how to culture mouse embryonic stem (ES) cell line. The clonal embryonic stem cell line #693 ES C57BL/6 was derived from a strain C57BL/6J (B6) mouse blastocyst [PubMed: 11730008]. The ES cells were shown to populate the germ line of two host blastocyst ...
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