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Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in Drosophila

Featured protocol,  Authors: Ines Erdmann
Ines ErdmannAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Bio-protocol author page: a4841
Kathrin Marter
Kathrin MarterAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Affiliation 3: Center for Behavioral Brain Sciences, Magdeburg, Germany
Bio-protocol author page: a4842
Oliver Kobler
Oliver KoblerAffiliation: Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany
Bio-protocol author page: a4843
Sven Niehues
Sven NiehuesAffiliation 1: Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany
Affiliation 2: Faculty of Medicine, University of Münster, Münster, Germany
Bio-protocol author page: a4844
Julia Bussmann
Julia BussmannAffiliation 1: Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany
Affiliation 2: Faculty of Medicine, University of Münster, Münster, Germany
Bio-protocol author page: a4845
Anke Müller
Anke MüllerAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Bio-protocol author page: a4846
Julia Abele
Julia AbeleAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Bio-protocol author page: a4847
Erik Storkebaum
Erik StorkebaumAffiliation 1: Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany
Affiliation 2: Faculty of Medicine, University of Münster, Münster, Germany
Bio-protocol author page: a4848
Ulrich Thomas
Ulrich ThomasAffiliation: Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany
For correspondence: thomas@lin-magdeburg.de
Bio-protocol author page: a4849
 and Daniela C. Dieterich
Daniela C. DieterichAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Affiliation 3: Center for Behavioral Brain Sciences, Magdeburg, Germany
For correspondence: daniela.dieterich@med.ovgu.de
Bio-protocol author page: a4850
date: 7/20/2017, 12 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2397.

Brief version appeared in Nat Commun, Jul 2015
Advanced mass spectrometry technology has pushed proteomic analyses to the forefront of biological and biomedical research. Limitations of proteomic approaches now often remain with sample preparations rather than with the sensitivity of protein detection. However, deciphering proteomes and their context-dependent dynamics in subgroups of tissue-embedded cells still poses a challenge, which we meet with a detailed version of our recently established protocol for cell-selective and temporally controllable metabolic labeling of proteins in Drosophila. This method is based on targeted expression of a mutated variant of methionyl-tRNA-synthetase, MetRSL262G, which allows for charging methionine tRNAs with the non-canonical amino acid azidonorleucine (ANL) and, thus, for detectable ANL incorporation into nascent polypeptide chains.

Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice

Featured protocol,  Authors: Michael A. Schmid
Michael A. SchmidAffiliation: Rega Institute for Medical Research, Virology and Chemotherapy, Department of Immunology and Microbiology, University of Leuven, Leuven, Belgium
For correspondence: michael.alex.schmid@gmail.com
Bio-protocol author page: a4893
Elizabeth Kauffman
Elizabeth KauffmanAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4894
Anne Payne
Anne PayneAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4895
Eva Harris
Eva HarrisAffiliation: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, USA
Bio-protocol author page: a4896
 and Laura D. Kramer
Laura D. KramerAffiliation 1: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Affiliation 2: School of Public Health, State University of New York at Albany, Albany, New York, USA
For correspondence: laura.kramer@health.ny.gov
Bio-protocol author page: a4897
date: 7/20/2017, 29 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2407.

Brief version appeared in PLoS Pathog, Jun 2016
Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and chikungunya viruses, as well as Plasmodium parasites, among others. Here, we describe standardized methods to assess the impact of mosquito-derived factors on immune responses and pathogenesis in mouse models of infection. This protocol includes the generation of mosquito salivary gland extracts and intradermal inoculation of mouse ears. Ultimately, the information obtained from using these techniques can help reveal fundamental mechanisms of interaction between pathogens, mosquito vectors, and the mammalian host. In addition, this protocol can help establish improved infection challenge models for pre-clinical testing of vaccines or therapeutics that take into account the natural route of transmission via mosquitoes.

Plasmodium Sporozoite Motility on Flat Substrates

Featured protocol,  Authors: Henriette L Prinz
Henriette L PrinzAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4835
Julia M Sattler
Julia M SattlerAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4836
 and Friedrich Frischknecht
Friedrich FrischknechtAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4837
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2395.

Brief version appeared in PLoS Pathog, Jul 2016
Plasmodium sporozoites are the infectious, highly motile forms of the malaria parasite transmitted by Anopheles mosquitoes. Sporozoite motility can be assessed following the dissection of Anopheles salivary glands and isolation of sporozoites in vitro.

Ciberial Muscle 9 (CM9) Electrophysiological Recordings in Adult Drosophila melanogaster

Featured protocol,  Authors: Benjamin A. Eaton
Benjamin A. EatonAffiliation 1: Department of Cellular and Integrative Physiology, UT Health San Antonio, San Antonio, TX, USA
Affiliation 2: The Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health San Antonio, San Antonio, TX, USA
Bio-protocol author page: a4872
 and Rebekah E. Mahoney
Rebekah E. MahoneyAffiliation 1: The Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health San Antonio, San Antonio, TX, USA
Affiliation 2: Department of Cell Systems and Anatomy, UT Health San Antonio, San Antonio, TX, USA
For correspondence: mahoneyr@uthscsa.edu
Bio-protocol author page: a4873
date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2401.

Brief version appeared in J Neurosci, Feb 2014
The complexity surrounding presynaptic recordings in mammals is a significant barrier to the study of presynaptic mechanisms during neurotransmission in the mammalian central nervous system (CNS). Here we describe an adult fly neuromuscular junction (NMJ), the ciberial muscle 9 (CM9) NMJ, which allows for the recording of both evoked (EPSPs) and spontaneous postsynaptic excitatory potentials (mEPSPs) at a mature glutamatergic synapse. Combined with CM9-specific genetic technologies, the CM9 NMJ provides a powerful experimental system to better understand the regulation of neurotransmitter release at a mature synapse.

Ex vivo Ooplasmic Extract from Developing Drosophila Oocytes for Quantitative TIRF Microscopy Analysis

Featured protocol,  Authors: Imre Gáspár
Imre GáspárAffiliation: European Molecular Biology Laboratory (EMBL), Developmental Biology Unit, Heidelberg, Meyerhofstrasse 1, D-69117, Germany
For correspondence: imre.gaspar@embl.de
Bio-protocol author page: a4784
 and Anne Ephrussi
Anne EphrussiAffiliation: European Molecular Biology Laboratory (EMBL), Developmental Biology Unit, Heidelberg, Meyerhofstrasse 1, D-69117, Germany
For correspondence: anne.ephrussi@embl.de
Bio-protocol author page: a4785
date: 7/5/2017, 179 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2380.

Brief version appeared in EMBO J, Feb 2017
Understanding the dynamic behavior and the continuously changing composition of macromolecular complexes, subcellular structures and organelles is one of areas of active research in both cell and developmental biology, as these changes directly relate to function and subsequently to the development and homeostasis of the organism. Here, we demonstrate the use of the developing Drosophila oocyte to study dynamics of messenger ribonucleoprotein complexes (mRNPs) with high spatiotemporal resolution. The combination of Drosophila genetics with total internal reflection (TIRF) microscopy, image processing and data analysis gives insight into mRNP motility and composition dynamics with unprecedented precision.

Live Imaging of Myogenesis in Indirect Flight Muscles in Drosophila

Featured protocol,  Author: Dagan Segal
Dagan SegalAffiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
For correspondence: dagansegal@gmail.com
Bio-protocol author page: a4782
date: 7/5/2017, 164 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2377.

Brief version appeared in Dev Cell, Aug 2016
The indirect flight muscles (IFMs) are the largest muscles in the fly, making up the bulk of the adult thorax. IFMs in Drosophila are generated during pupariation by fusion of hundreds of muscle precursor cells (myoblasts) with larval muscle templates (myotubes). Prominent features, including the large number of fusion events, the structural similarity to vertebrate muscles, and the amenability to the powerful genetic techniques of the Drosophila system make the IFMs an attractive system to study muscle cell fusion. Here we describe methods for live imaging of IFMs, both in intact pupae, and in isolated IFMs ex-vivo. The protocols elaborated upon here were used in the manuscript by (Segal et al., 2016).

Behavioral and Functional Assays for Investigating Mechanisms of Noxious Cold Detection and Multimodal Sensory Processing in Drosophila Larvae

Featured protocol,  Authors: Atit A. Patel
Atit A. PatelAffiliation: Neuroscience Institute, Georgia State University, Atlanta, GA, USA
Bio-protocol author page: a4794
 and Daniel N. Cox
Daniel N. CoxAffiliation: Neuroscience Institute, Georgia State University, Atlanta, GA, USA
For correspondence: dcox18@gsu.edu
Bio-protocol author page: a4795
date: 7/5/2017, 197 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2388.

Brief version appeared in Curr Biol, Dec 2016
To investigate cellular, molecular and behavioral mechanisms of noxious cold detection, we developed cold plate behavioral assays and quantitative means for evaluating the predominant noxious cold-evoked contraction behavior. To characterize neural activity in response to noxious cold, we implemented a GCaMP6-based calcium imaging assay enabling in vivo studies of intracellular calcium dynamics in intact Drosophila larvae. We identified Drosophila class III multidendritic (md) sensory neurons as multimodal sensors of innocuous mechanical and noxious cold stimuli and to dissect the mechanistic bases of multimodal sensory processing we developed two independent functional assays. First, we developed an optogenetic dose response assay to assess whether levels of neural activation contributes to the multimodal aspects of cold sensitive sensory neurons. Second, we utilized CaMPARI, a photo-switchable calcium integrator that stably converts fluorescence from green to red in presence of high intracellular calcium and photo-converting light, to assess in vivo functional differences in neural activation levels between innocuous mechanical and noxious cold stimuli. These novel assays enable investigations of behavioral and functional roles of peripheral sensory neurons and multimodal sensory processing in Drosophila larvae.

Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in Drosophila

Authors: Ines Erdmann
Ines ErdmannAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Bio-protocol author page: a4841
Kathrin Marter
Kathrin MarterAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Affiliation 3: Center for Behavioral Brain Sciences, Magdeburg, Germany
Bio-protocol author page: a4842
Oliver Kobler
Oliver KoblerAffiliation: Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany
Bio-protocol author page: a4843
Sven Niehues
Sven NiehuesAffiliation 1: Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany
Affiliation 2: Faculty of Medicine, University of Münster, Münster, Germany
Bio-protocol author page: a4844
Julia Bussmann
Julia BussmannAffiliation 1: Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany
Affiliation 2: Faculty of Medicine, University of Münster, Münster, Germany
Bio-protocol author page: a4845
Anke Müller
Anke MüllerAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Bio-protocol author page: a4846
Julia Abele
Julia AbeleAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Bio-protocol author page: a4847
Erik Storkebaum
Erik StorkebaumAffiliation 1: Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany
Affiliation 2: Faculty of Medicine, University of Münster, Münster, Germany
Bio-protocol author page: a4848
Ulrich Thomas
Ulrich ThomasAffiliation: Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany
For correspondence: thomas@lin-magdeburg.de
Bio-protocol author page: a4849
 and Daniela C. Dieterich
Daniela C. DieterichAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Affiliation 3: Center for Behavioral Brain Sciences, Magdeburg, Germany
For correspondence: daniela.dieterich@med.ovgu.de
Bio-protocol author page: a4850
date: 7/20/2017, 12 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2397.

[Abstract] Advanced mass spectrometry technology has pushed proteomic analyses to the forefront of biological and biomedical research. Limitations of proteomic approaches now often remain with sample preparations rather than with the sensitivity of protein detection. However, deciphering proteomes and their context-dependent ...

Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice

Authors: Michael A. Schmid
Michael A. SchmidAffiliation: Rega Institute for Medical Research, Virology and Chemotherapy, Department of Immunology and Microbiology, University of Leuven, Leuven, Belgium
For correspondence: michael.alex.schmid@gmail.com
Bio-protocol author page: a4893
Elizabeth Kauffman
Elizabeth KauffmanAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4894
Anne Payne
Anne PayneAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4895
Eva Harris
Eva HarrisAffiliation: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, USA
Bio-protocol author page: a4896
 and Laura D. Kramer
Laura D. KramerAffiliation 1: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Affiliation 2: School of Public Health, State University of New York at Albany, Albany, New York, USA
For correspondence: laura.kramer@health.ny.gov
Bio-protocol author page: a4897
date: 7/20/2017, 29 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2407.

[Abstract] Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese ...

Plasmodium Sporozoite Motility on Flat Substrates

Authors: Henriette L Prinz
Henriette L PrinzAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4835
Julia M Sattler
Julia M SattlerAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4836
 and Friedrich Frischknecht
Friedrich FrischknechtAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4837
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2395.

[Abstract] Plasmodium sporozoites are the infectious, highly motile forms of the malaria parasite transmitted by Anopheles mosquitoes. Sporozoite motility can be assessed following the dissection of Anopheles salivary glands and isolation of sporozoites in vitro. ...

Ciberial Muscle 9 (CM9) Electrophysiological Recordings in Adult Drosophila melanogaster

Authors: Benjamin A. Eaton
Benjamin A. EatonAffiliation 1: Department of Cellular and Integrative Physiology, UT Health San Antonio, San Antonio, TX, USA
Affiliation 2: The Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health San Antonio, San Antonio, TX, USA
Bio-protocol author page: a4872
 and Rebekah E. Mahoney
Rebekah E. MahoneyAffiliation 1: The Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health San Antonio, San Antonio, TX, USA
Affiliation 2: Department of Cell Systems and Anatomy, UT Health San Antonio, San Antonio, TX, USA
For correspondence: mahoneyr@uthscsa.edu
Bio-protocol author page: a4873
date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2401.

[Abstract] The complexity surrounding presynaptic recordings in mammals is a significant barrier to the study of presynaptic mechanisms during neurotransmission in the mammalian central nervous system (CNS). Here we describe an adult fly neuromuscular junction (NMJ), the ciberial muscle 9 (CM9) NMJ, which allows ...

Ex vivo Ooplasmic Extract from Developing Drosophila Oocytes for Quantitative TIRF Microscopy Analysis

Authors: Imre Gáspár
Imre GáspárAffiliation: European Molecular Biology Laboratory (EMBL), Developmental Biology Unit, Heidelberg, Meyerhofstrasse 1, D-69117, Germany
For correspondence: imre.gaspar@embl.de
Bio-protocol author page: a4784
 and Anne Ephrussi
Anne EphrussiAffiliation: European Molecular Biology Laboratory (EMBL), Developmental Biology Unit, Heidelberg, Meyerhofstrasse 1, D-69117, Germany
For correspondence: anne.ephrussi@embl.de
Bio-protocol author page: a4785
date: 7/5/2017, 179 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2380.

[Abstract] Understanding the dynamic behavior and the continuously changing composition of macromolecular complexes, subcellular structures and organelles is one of areas of active research in both cell and developmental biology, as these changes directly relate to function and subsequently to the development ...

Live Imaging of Myogenesis in Indirect Flight Muscles in Drosophila

Author: Dagan Segal
Dagan SegalAffiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
For correspondence: dagansegal@gmail.com
Bio-protocol author page: a4782
date: 7/5/2017, 164 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2377.

[Abstract] The indirect flight muscles (IFMs) are the largest muscles in the fly, making up the bulk of the adult thorax. IFMs in Drosophila are generated during pupariation by fusion of hundreds of muscle precursor cells (myoblasts) with larval muscle templates (myotubes). Prominent features, including the large ...

Behavioral and Functional Assays for Investigating Mechanisms of Noxious Cold Detection and Multimodal Sensory Processing in Drosophila Larvae

Authors: Atit A. Patel
Atit A. PatelAffiliation: Neuroscience Institute, Georgia State University, Atlanta, GA, USA
Bio-protocol author page: a4794
 and Daniel N. Cox
Daniel N. CoxAffiliation: Neuroscience Institute, Georgia State University, Atlanta, GA, USA
For correspondence: dcox18@gsu.edu
Bio-protocol author page: a4795
date: 7/5/2017, 197 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2388.

[Abstract] To investigate cellular, molecular and behavioral mechanisms of noxious cold detection, we developed cold plate behavioral assays and quantitative means for evaluating the predominant noxious cold-evoked contraction behavior. To characterize neural activity in response to noxious cold, we implemented ...

Validating Candidate Congenital Heart Disease Genes in Drosophila

Authors: Jun-yi Zhu*
Jun-yi ZhuAffiliation: Center for Cancer and Immunology Research, Children’s National Medical Center, 111 Michigan Ave. NW, Washington, DC, USA
Bio-protocol author page: a4710
Yulong Fu*
Yulong FuAffiliation: Center for Cancer and Immunology Research, Children’s National Medical Center, 111 Michigan Ave. NW, Washington, DC, USA
Bio-protocol author page: a4711
Adam Richman
Adam RichmanAffiliation: Center for Cancer and Immunology Research, Children’s National Medical Center, 111 Michigan Ave. NW, Washington, DC, USA
Bio-protocol author page: a4712
 and Zhe Han
Zhe HanAffiliation 1: Center for Cancer and Immunology Research, Children’s National Medical Center, 111 Michigan Ave. NW, Washington, DC, USA
Affiliation 2: Department of Pediatrics, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
For correspondence: zhan@childrensnational.org
Bio-protocol author page: a4713
 (*contributed equally to this work) date: 6/20/2017, 227 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2350.

[Abstract] Genomic sequencing efforts can implicate large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system to validate candidate gene association with pathology is therefore useful. We present such a system employing Drosophila to validate candidate ...

Chromatin Immunoprecipitation Experiments from Whole Drosophila Embryos or Larval Imaginal Discs

Authors: Vincent Loubiere
Vincent LoubiereAffiliation 1: Institute of Human Genetics, UMR9002 CNRS-UM, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, France
Affiliation 2: University of Montpellier, 163 Rue Auguste Broussonnet, 34090 Montpellier, France
Bio-protocol author page: a4619
Anna Delest
Anna DelestAffiliation: Centre de Recherche en Cancérologie de Lyon, INSERM U1052, 151 cours Albert Thomas, 69003 Lyon, France
Bio-protocol author page: a4620
Bernd Schuettengruber
Bernd SchuettengruberAffiliation: Institute of Human Genetics, UMR9002 CNRS-UM, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, France
Bio-protocol author page: a4621
Anne-Marie Martinez
Anne-Marie MartinezAffiliation 1: Institute of Human Genetics, UMR9002 CNRS-UM, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, France
Affiliation 2: University of Montpellier, 163 Rue Auguste Broussonnet, 34090 Montpellier, France
For correspondence: anne-marie.martinez@igh.cnrs.fr
Bio-protocol author page: a4622
 and Giacomo Cavalli
Giacomo CavalliAffiliation: Institute of Human Genetics, UMR9002 CNRS-UM, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, France
For correspondence: giacomo.cavalli@igh.cnrs.fr
Bio-protocol author page: a4623
date: 6/5/2017, 335 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2327.

[Abstract] Chromatin Immunoprecipitation coupled either to qPCR (qChIP) or high-throughput sequencing (ChIP-Seq) has been extensively used in the last decades to identify the DNA binding sites of transcription factors or the localization of various histone marks along the genome. The ChIP experiment generally ...

Isolation and Infection of Drosophila Primary Hemocytes

Authors: Charles Tracy
Charles TracyAffiliation: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
Bio-protocol author page: a4450
 and Helmut Krämer
Helmut KrämerAffiliation 1: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
Affiliation 2: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
Affiliation 3: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
For correspondence: helmut.kramer@utsouthwestern.edu
Bio-protocol author page: a4451
date: 6/5/2017, 355 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2300.

[Abstract] Phagocytosis of invading pathogens and their subsequent clearance in lysosomes is important for organismal fitness. We have devised the following protocol to extract phagocytic hemocytes from wild-type and mutant Drosophila larvae and infect the isolated hemocytes with GFP-labeled E. coli to measure ...
1 2 3 

[Bio101] Transfection of S2 Cell with DNA Using CellFectin Reagent

Author: Darran G. Cronshaw
Darran G. CronshawAffiliation: Center for Autoimmune and Musculoskeletal Diseases, Feinstein Institute for Medical Research, Manhasset, USA
For correspondence:
Bio-protocol author page: a41
date: 3/5/2012, 11265 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.190.

[Abstract] This method provides a step-by-step guide to transfecting Drosophila S2 cells with the pRmHA-3 (or similar) vector with insert of choice (in this case SDF-1β-FLAG) and generating a stable cell line. This cell line is then capable of producing the protein of interest under inducible conditions by addition ...

Tandem Affinity Purification in Drosophila Heads and Ovaries

Authors: Anita Pepper
Anita PepperAffiliation: Department of Genetics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a75
Balpreet Bhogal
Balpreet BhogalAffiliation: Department of Genetics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a76
 and Thomas Jongens
Thomas JongensAffiliation: Department of Genetics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA
For correspondence: jongens@mail.med.upenn.edu
Bio-protocol author page: a77
date: 8/5/2012, 7037 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.245.

[Abstract] Tandem affinity purification (TAP) (Pugi et al.,2001; Rigaut et al., 1999) is a method that uses a tagging approach of a target protein of interest for a two-step purification scheme in order to pull down protein complexes under native conditions and expression levels. The TAP tag consists of three ...

FLP/FRT Induction of Mitotic Recombination in Drosophila Germline

Authors: Pedro Prudêncio
Pedro PrudêncioAffiliation 1: Regenerative Medicine Program, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, Campus de Gambelas, Faro, Portugal
Affiliation 2: IBB-Institute for Biotechnology and Bioengineering, CBME-Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, Campus de Gambelas, Faro, Portugal
Affiliation 3: Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, Oeiras, Portugal
Bio-protocol author page: a2168
 and Leonardo G. Guilgur
Leonardo G. GuilgurAffiliation 1: Regenerative Medicine Program, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, Campus de Gambelas, Faro, Portugal
Affiliation 2: IBB-Institute for Biotechnology and Bioengineering, CBME-Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, Campus de Gambelas, Faro, Portugal
Affiliation 3: Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, Oeiras, Portugal
For correspondence: lguilgur@igc.gulbenkian.pt
Bio-protocol author page: a2169
date: 5/5/2015, 6767 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1458.

[Abstract] The FLP/FRT system is a site-directed recombination technology based on the targeting of a recombination enzyme (flipase - FLP) to specific DNA regions designated as flipase recognition target (FRT) sites. Initially identified in Saccharomyces cerevisiae, the yeast FLP-enzyme and its FRT recombination ...

Protein Extraction from Drosophila Embryos and Ovaries

Authors: Pedro Prudêncio
Pedro PrudêncioAffiliation 1: Regenerative Medicine Program, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, Campus de Gambelas, Faro, Portugal
Affiliation 2: IBB-Institute for Biotechnology and Bioengineering, CBME-Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, Campus de Gambelas, Faro, Portugal
Affiliation 3: Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, Oeiras, Portugal
Bio-protocol author page: a2168
 and Leonardo G. Guilgur
Leonardo G. GuilgurAffiliation 1: Regenerative Medicine Program, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, Campus de Gambelas, Faro, Portugal
Affiliation 2: IBB-Institute for Biotechnology and Bioengineering, CBME-Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, Campus de Gambelas, Faro, Portugal
Affiliation 3: Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, Oeiras, Portugal
For correspondence: lguilgur@igc.gulbenkian.pt
Bio-protocol author page: a2169
date: 5/5/2015, 5007 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1459.

[Abstract] Here we provide the description of protocols to efficiently obtain protein extracts from embryos and ovaries of Drosophila melanogaster. These protocols are routinely applied in our laboratory and are based on two techniques: either embryos or ovaries are homogenized using a pestle and then the soluble ...

Fluorescence in situ Hybridization to the Polytene Chromosomes of Anopheles Mosquitoes

Authors: Ai Xia
Ai XiaAffiliation: Department of Entomology, Nanjing Agricultural University, Nanjing, China
Bio-protocol author page: a757
Ashley Peery
Ashley PeeryAffiliation: Department of Entomology, Virginia Tech, Blacksburg, USA
Bio-protocol author page: a758
Maryam Kamali
Maryam KamaliAffiliation: Department of Entomology, Virginia Tech, Blacksburg, USA
Bio-protocol author page: a759
Jiangtao Liang
Jiangtao LiangAffiliation: Department of Entomology, Nanjing Agricultural University, Nanjing, China
Bio-protocol author page: a760
Maria V. Sharakhova
Maria V. SharakhovaAffiliation: Department of Entomology, Virginia Tech, Blacksburg, USA
Bio-protocol author page: a761
 and Igor V. Sharakhov
Igor V. SharakhovAffiliation: Department of Entomology, Virginia Tech, Blacksburg, USA
For correspondence: igor@vt.edu
Bio-protocol author page: a427
date: 8/20/2013, 4897 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.860.

[Abstract] Fluorescence in situ hybridization (FISH) is a method that uses a fluorescently labeled DNA probe for mapping the position of a genetic element on chromosomes. A DNA probe is prepared by incorporating Cy-3 or Cy-5 labeled nucleotides into DNA by nick-translation or a random primed labeling method. This ...

Electrical Penetration Graph Recording (Whitefly)

Authors: Baiming Liu
Baiming LiuAffiliation: Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
Bio-protocol author page: a1202
Xuguo Zhou
Xuguo ZhouAffiliation 1: Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
Affiliation 2: Department of Entomology, University of Kentucky, Lexington, USA
For correspondence: xuguozhou@uky.edu
Bio-protocol author page: a1203
 and Youjun Zhang
Youjun ZhangAffiliation: Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
For correspondence: zhangyoujun@caas.cn
Bio-protocol author page: a1204
date: 3/5/2014, 4529 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1063.

[Abstract] Electrical penetration graph (EPG) was first developed by Mclean and Kinsey (1964) and later modified by Tjallingii (1978 and 1985). The EPG system turns a phloem-sucking insects and its host plant into part of an electrical circuit that is completed when the insect’s mouthparts penetrate the plant. ...

LC3B Labeling on Terrestrial Isopod Adipocytes

Authors: Christine Braquart-Varnier
Christine Braquart-VarnierAffiliation: Ecologie et Biologie des Interactions, UMR 7267 CNRS, Université de Poitiers, Poitiers Cedex, France
For correspondence: christine.braquart@univ-poitiers.fr
Bio-protocol author page: a625
Maryline Raimond
Maryline RaimondAffiliation: Ecologie et Biologie des Interactions, UMR 7267 CNRS, Université de Poitiers, Poitiers Cedex, France
Bio-protocol author page: a626
 and Mathieu Sicard
Mathieu SicardAffiliation: Ecologie et Biologie des Interactions, UMR 7267 CNRS, Université de Poitiers, Poitiers Cedex, France
Bio-protocol author page: a341
date: 6/20/2013, 4073 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.792.

[Abstract] The LC3B protein plays a critical role in autophagy. Normally, this protein resides in the cytosol, but following cleavage and lipidation with phosphatidylethanolamine, LC3B associates with the phagophore. This localization can be used as a general marker for autophagic membranes. To visualize the LC3B, ...

Measuring Anxiety-like Behavior in Crayfish by Using a Sub Aquatic Dark-light Plus Maze

Authors: Pascal Fossat
Pascal FossatAffiliation: Université de Bordeaux, Bordeaux, France
For correspondence: pascal.fossat@u-bordeaux.fr
Bio-protocol author page: a1985
Julien Bacqué-Cazenave
Julien Bacqué-CazenaveAffiliation: Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a1986
Jean-Paul Delbecque*
Jean-Paul DelbecqueAffiliation 1: Université de Bordeaux, Bordeaux, France
Affiliation 2: Centre National de la Recherche Scientifique (CNRS), Institut des Neurosciences Cognitives et Intégratives d’Aquitaine, Avenue des Facultés, Talence Cedex, France
Bio-protocol author page: a1987
 and Daniel Cattaert*
Daniel CattaertAffiliation 1: Université de Bordeaux, Bordeaux, France
Affiliation 2: Centre National de la Recherche Scientifique (CNRS), Institut des Neurosciences Cognitives et Intégratives d’Aquitaine, Avenue des Facultés, Talence Cedex, France
Bio-protocol author page: a1988
 (*contributed equally to this work) date: 2/5/2015, 3366 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1396.

[Abstract] Crayfish are omnivorous freshwater arthropods that naturally explore their environment during day and night, but also frequently hide under a shelter or in a hole in case of danger. They may be submitted to various stressors, including predation, social interactions or changes in environmental parameters ...

Preparation and Immunofluorescence Staining of the Trachea in Drosophila Larvae and Pupae

Author: Feng Chen
Feng ChenAffiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, USA
For correspondence: fchen66@alumni.stanford.edu
Bio-protocol author page: a3101
date: 5/5/2016, 3012 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1797.

[Abstract] The Drosophila melanogaster trachea is a branched network of rigid chitin-lined tubes that ramify throughout the body and functions as the fly’s respiratory organ. Small openings at the ends of the tracheal tubes allow gas exchange to occur by diffusion between internal tissues and the exterior environment. ...

Electroretinogram (ERG) Recordings from Drosophila

Authors: Elisheva Rhodes-Mordov*
Elisheva Rhodes-MordovAffiliation: Departments of Medical Neurobiology, the Institute of Medical Research Israel-Canada (IMRIC) and the Edmond and Lily Safra Center for Brain Sciences (ELSC), Faculty of Medicine of the Hebrew University, Jerusalem, Israel
Bio-protocol author page: a2672
Hadar Samra*
Hadar SamraAffiliation: Departments of Medical Neurobiology, the Institute of Medical Research Israel-Canada (IMRIC) and the Edmond and Lily Safra Center for Brain Sciences (ELSC), Faculty of Medicine of the Hebrew University, Jerusalem, Israel
Bio-protocol author page: a2673
 and Baruch Minke
Baruch MinkeAffiliation: Departments of Medical Neurobiology, the Institute of Medical Research Israel-Canada (IMRIC) and the Edmond and Lily Safra Center for Brain Sciences (ELSC), Faculty of Medicine of the Hebrew University, Jerusalem, Israel
For correspondence: baruchm@ekmd.huji.ac.il
Bio-protocol author page: a2674
 (*contributed equally to this work) date: 11/5/2015, 2994 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1636.

[Abstract] Phototransduction is a process in which light is converted into electrical signals used by the central nervous system. Invertebrate phototransduction is a process mediated by the phosphoinositide signaling cascade, characterized by Phospholipase C (PLC) as the effector enzyme and the Transient Receptor ...
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