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18 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates
Eleonore Ostermann 1, Cécile Macquin 1, Seiamak Bahram 1, Philippe Georgel 1
1ImmunoRhumatologie Moléculaire, INSERM UMR_S 1109, Centre de Recherche d'Immunologie et d'Hématologie, Université de Strasbourg

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

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Bioengineering

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo
Nihal E. Vrana 1, Agnes Dupret-Bories 1,2, Christophe Chaubaroux 1, Elisabeth Rieger 1,2, Christian Debry 1,2, Dominique Vautier 1,3, Marie-Helene Metz-Boutigue 1,3, Philippe Lavalle 1,3
1Biomatériaux et Bioingénieriee, INSERM, 2Service Oto-Rhino-Laryngologie, Hôpitaux Universitaires de Strasbourg, 3Faculté de Chirurgie Dentaire, Université de Strasbourg

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

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Neuroscience

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities
Khadija Elhabazi 1, Safia Ayachi 1, Brigitte Ilien 1, Frédéric Simonin 1
1Biotechnology and Cellular Signalling, UMR 7242 CNRS, Université de Strasbourg

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of naïve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

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Medicine

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice
Ipek Yalcin 1, Salim Megat 1,2, Florent Barthas 1,2, Elisabeth Waltisperger 1, Mélanie Kremer 1,2, Eric Salvat 1,2,3, Michel Barrot 1
1Institut des Neurosciences Cellulaires et Intégratives UPR3212, Centre National de la Recherche Scientifique, 2Université de Strasbourg, 3Hôpitaux Universitaires de Strasbourg

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The “cuff model” of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

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Developmental Biology

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo
Renee Wei-Yan Chow *1,2,3,4, Paola Lamperti *1,2,3,4, Emily Steed 1,2,3,4, Francesco Boselli 1,2,3,4, Julien Vermot 1,2,3,4
1Institut de Génétique et de Biologie Moléculaire et Cellulaire, 2UMR7104, Centre National de la Recherche Scientifique, 3U964, Institut National de la Santé et de la Recherche Médicale, 4Université de Strasbourg

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

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Chemistry

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators
Tristan Hessberger *1, Lukas B. Braun *1, Christophe A. Serra 2, Rudolf Zentel 1
1Department of Organic Chemistry, Johannes Gutenberg University, 2CNRS, ICS UPR 22, Université de Strasbourg

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

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Genetics

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity
Tiago Baptista 1,2,3,4, Didier Devys 1,2,3,4
1Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France, 2Centre National de la Recherche Scientifique, Illkirch, France, 3Institut National de la Santé et de la Recherche Médicale, Illkirch, France, 4Université de Strasbourg

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

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Chemistry

Photogeneration of N-Heterocyclic Carbenes: Application in Photoinduced Ring-Opening Metathesis Polymerization
Julien Pinaud 1, Emeline Placet 1, Patrick Lacroix-Desmazes 1, Thi Kim Hoang Trinh 2,3, Jean Pierre Malval 2,3, Abraham Chemtob 2,3, Loïc Pichavant 4, Valérie Héroguez 4
1ICGM, Université de Montpellier, CNRS, ENSCM, 2Institut de Science des Matériaux de Mulhouse (IS2M UMR 7361 CNRS), Université de Haute-Alsace, 3Université de Strasbourg, 4Laboratoire de Chimie des Polymères Organiques (LCPO UMR 5629 ENSCBP), Université de Bordeaux

We describe a protocol to photogenerate N-heterocyclic carbenes (NHCs) by UV irradiation of a 2-isopropylthioxanthone/imidazolium tetraphenylborate salt system. Methods to characterize the photoreleased NHC and elucidate the photochemical mechanism are proposed. The protocols for ring-opening metathesis photopolymerization in solution and miniemulsion illustrate the potential of this 2-component NHC photogenerating system.

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Bioengineering

Intra-Omental Islet Transplantation Using h-Omental Matrix Islet filliNG (hOMING)
Anaïs Schaschkow 1, Carole Mura 1, Michel Pinget 1, Karim Bouzakri 1, Elisa Maillard 1
1Centre Européen d'Etude du Diabète, Université de Strasbourg

Here, we present a protocol for in vivo validation of hydrogel-based cell therapy, illustrated by the example of islet transplantation. h-Omental Matrix Islet filliNG (hOMING) implantation allows implantation of a cell-hydrogel mixture between the omental layers, near to blood vessels, to maximize engraftment in a proper metabolic environment.

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Environment

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging
Claire Villette 1, Loïc Maurer 1,2, Dimitri Heintz 1
1Plant Imaging and Mass Spectrometry (PIMS), Institut de biologie moléculaire des plantes, CNRS, Université de Strasbourg, 2Département mécanique, ICube Laboratoire des sciences de l'ingénieur, de l'informatique et de l'imagerie, UNISTRA/CNRS/ENGEES/INSA

This method uses mass spectrometry imaging (MSI) to understand metabolic processes in S. alba leaves when exposed to xenobiotics. The method allows the spatial localization of compounds of interest and their predicted metabolites within specific, intact tissues.

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Biochemistry

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis
Kyle G. Kroeck 1,2, Weihua Qiu 1,2, Claudio Catalano 1,2, Thi Kim Hoang Trinh 1,2, Youzhong Guo 1,2
1Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, 2Institute for Structural Biology, Drug Discovery and Development, School of Pharmacy, Virginia Commonwealth University

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

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Biology

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy
Cyril Scandola 1, François Lanza 1, Christian Gachet 1, Anita Eckly 1
1Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS

Here, we present a protocol to analyze ultrastructure of the megakaryocytes in situ using transmission electron microscopy (TEM). Murine bone marrows are collected, fixed, embedded in epoxy resin and cut in ultrathin sections. After contrast staining, the bone marrow is observed under a TEM microscope at 120 kV.

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Biology

Isolation of Mouse Megakaryocyte Progenitors
Quentin Kimmerlin 1, Manuela Tavian 2, Christian Gachet 1, François Lanza 1, Nathalie Brouard 1
1Université de Strasbourg, INSERM UMR S1255, EFS Grand-EST, 2UMR-S1113 –IRFAC, Université de Strasbourg

This method describes the purification by flow cytometry of MEP and MKp from mice femurs, tibias, and pelvic bones.

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Biology

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation
Anaïs Pongerard 1, Lea Mallo 1, Christian Gachet 1, Henri de La Salle 1, François Lanza 1, Catherine Strassel 1
1BPPS UMR-S 1255, FMTS, Université de Strasbourg, INSERM, EFS Grand Est

This protocol describes in detail all the steps involved in obtaining leukofilter-derived CD34+ hematopoietic progenitors and their in vitro differentiation and maturation into proplatelet-bearing megakaryocytes that are able to release platelets in the culture medium. This procedure is useful for in-depth analysis of cellular and molecular mechanisms controlling megakaryopoiesis.

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Biology

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants
Inès Guinard 1, François Lanza 1, Christian Gachet 1, Catherine Léon 1, Anita Eckly 1
1Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS

Here, we detail the bone marrow explant method, from sample preparation to microscopic slide analysis, to evaluate the ability of megakaryocytes which have differentiated in their physiological environment to form proplatelets.

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Biology

Megakaryocyte Culture in 3D Methylcellulose-Based Hydrogel to Improve Cell Maturation and Study the Impact of Stiffness and Confinement
Julie Boscher 1, Christian Gachet 1, François Lanza 1, Catherine Léon 1
1Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS

It is now acknowledged that the three-dimensional environment of cells can play an important role in their behavior, maturation and/or differentiation. This protocol describes a three-dimensional cell culture model designed to study the impact of physical containment and mechanical constraints on megakaryocytes.

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Biology

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow
Alicia Bornert 1, Fabien Pertuy 1, François Lanza 1, Christian Gachet 1, Catherine Léon 1
1INSERM, EFS Grand Est, Université de Strasbourg

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

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Developmental Biology

An Efficient Protocol for CUT&RUN Analysis of FACS-Isolated Mouse Satellite Cells
Kamar Ghaibour *1, Joe Rizk *1, Claudine Ebel 1, Tao Ye 1, Muriel Philipps 1, Valérie Schreiber 1, Daniel Metzger 1, Delphine Duteil 1
1CNRS, Inserm, IGBMC UMR 7104- UMR-S 1258, Université de Strasbourg

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&RUN).

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