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Developmental Biology

Physical Removal of the Midbody Remnant from Polarised Epithelial Cells Using Take-Up by Suction Pressure (TUSP)

Authors: Miguel Bernabé-Rubio
Miguel Bernabé-RubioAffiliation: Department of Cell Biology and Immunology, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid, Spain
Bio-protocol author page: a4399
David C. Gershlick
David C. GershlickAffiliation: Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, USA
Bio-protocol author page: a4400
 and Miguel A. Alonso
Miguel A. AlonsoAffiliation: Department of Cell Biology and Immunology, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid, Spain
For correspondence: maalonso@cbm.csic.es
Bio-protocol author page: a4401
date: 4/20/2017, 249 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2244.

[Abstract] In polarised epithelial cells the midbody forms at the apical cell surface during cytokinesis. Once severed, the midbody is inherited by one of the daughter cells remaining tethered to the apical plasma membrane where it participates in non-cytokinetic processes, such as primary ciliogenesis. Here, we describe a novel method to physically remove the ...

In vivo Mitophagy Monitoring in Caenorhabditis elegans to Determine Mitochondrial Homeostasis

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: 4/5/2017, 294 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2215.

[Abstract] Perturbation of mitochondrial function is a major hallmark of several pathological conditions and ageing, underlining the essential role of fine-tuned mitochondrial activity (Lopez-Otin et al., 2013). Mitochondrial selective autophagy, known as mitophagy, mediates the removal of dysfunctional and/or superfluous organelles, preserving cellular and organismal ...

In vivo Live Imaging of Calcium Waves and Other Cellular Processes during Fertilization in Caenorhabditis elegans

Authors: Jun Takayama
Jun TakayamaAffiliation: RIKEN Quantitative Biology Center, Laboratory for Developmental Dynamics, Kobe, Japan
Bio-protocol author page: a4310
Masashi Fujita
Masashi FujitaAffiliation: RIKEN Quantitative Biology Center, Laboratory for Developmental Dynamics, Kobe, Japan
Bio-protocol author page: a4311
 and Shuichi Onami
Shuichi OnamiAffiliation: RIKEN Quantitative Biology Center, Laboratory for Developmental Dynamics, Kobe, Japan
For correspondence: sonami@riken.jp
Bio-protocol author page: a4312
date: 4/5/2017, 416 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2205.

[Abstract] Fertilization calcium waves are a conserved trigger for animal development; however, genetic analysis of these waves has been limited due to the difficulty of imaging in vivo fertilization. Here we describe a protocol to image calcium dynamics during in vivo fertilization in the genetic animal model Caenorhabditis elegans. This protocol consists of ...

Assessment of Murine Retinal Function by Electroretinography

Authors: Gillie Benchorin
Gillie BenchorinAffiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, United States
Bio-protocol author page: a4327
Melissa A. Calton
Melissa A. CaltonAffiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, United States
Bio-protocol author page: a4328
Marielle O. Beaulieu
Marielle O. BeaulieuAffiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, United States
Bio-protocol author page: a4329
 and Douglas Vollrath
Douglas VollrathAffiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, United States
For correspondence: vollrath@stanford.edu
Bio-protocol author page: a4330
date: 4/5/2017, 274 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2218.

[Abstract] The electroretinogram (ERG) is a sensitive and noninvasive method for testing retinal function. In this protocol, we describe a method for performing ERGs in mice. Contact lenses on the mouse cornea measure the electrical response to a light stimulus of photoreceptors and downstream retinal cells, and the collected data are analyzed to evaluate retinal ...

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, 404 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, 571 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, 649 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, 916 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, 1162 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, 1032 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 ...
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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, 20133 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, 16160 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, 14561 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, 13664 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....

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, 11294 views, 1 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 ...

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, 10992 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 ...

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, 10436 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, 9506 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, 8757 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, 8481 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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