Name: proplatelet formation dynamics of mouse fresh bone marrow explants
Uploaded: 2021-05-20T15:00:00
Description: Watch this Scientific Journal Video about Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants at JoVE.com

Les auteurs tiennent à remercier Jean-Yves Rinckel, Julie Boscher, Patricia Laeuffer, Monique Freund, Ketty Knez-Hippert pour leur assistance technique. Ce travail a été soutenu par la bourse ANR (Agence Nationale de la Recherche) ANR-17-CE14-0001-01 et ANR-18-CE14-0037.

Toutes les expérimentations animales ont été réalisées conformément aux normes européennes 2010/63/UE et au Comité CREMEAS d’Éthique de l’Expérimentation Animale de l’Université de Strasbourg (Comité Régional d’Ethique en Matière d’Expérimentation Animale Strasbourg).
1. Préparation des réactifs
<ol><li>Préparer les réactifs comme décrit dans le tableau 1.<ol>
<li>Pour le Stock I, dissoudre chaque poudre séparément. S’assurer que l’osmolar...

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	<li>Thiery, J. P., Bessis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+platelet+genesis;+in+vitro+study+by+cinemicrophotography.">Mechanism of platelet genesis; in vitro study by cinemicrophotography.</a> Reviews in Hematology. 11 (2), 162-174 (1956).</li><li>Becker, R. P., De Bruyn, P. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+platelet+formation+by+human+megakaryocytes+cultured+with+the+Mpl+ligand.">Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand.</a> Blood. 89 (7), 2336-2346 (1997).</li><li>Kaushansky, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+megakaryocyte+progenitor+expansion+and+differentiation+by+the+c-Mpl+ligand+thrombopoietin.">Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin.</a> Nature. 369 (6481), 568-571 (1994).</li><li>Strassel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hirudin+and+heparin+enable+efficient+megakaryocyte+differentiation+of+mouse+bone+marrow+progenitors.">Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors.</a> Experimental Cell Research. 318 (1), 25-32 (2012).</li><li>Aguilar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+environmental+stiffness+for+megakaryocyte+differentiation+and+proplatelet+formation.">Importance of environmental stiffness for megakaryocyte differentiation and proplatelet formation.</a> Blood. 128 (16), 2022-2032 (2016).</li><li>Scandola, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electron+microscopy+to+study+megakaryocytes.">Use of electron microscopy to study megakaryocytes.</a> Platelets. 31 (5), 589-598 (2020).</li><li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+megakaryocyte+development+in+the+native+bone+marrow+environment.">Characterization of megakaryocyte development in the native bone marrow environment.</a> Methods in Molecular Biology. 788, 175-192 (2012).</li><li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+megakaryocyte+morphology+and+proplatelet+formation+in+mice+with+megakaryocyte-restricted+MYH9+inactivation.">Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation.</a> Blood. 113 (14), 3182-3189 (2009).</li><li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proplatelet+formation+deficit+and+megakaryocyte+death+contribute+to+thrombocytopenia+in+Myh9+knockout+mice.">Proplatelet formation deficit and megakaryocyte death contribute to thrombocytopenia in Myh9 knockout mice.</a> Journal of Thrombosis and Haemostasis. 8 (10), 2243-2251 (2010).</li><li>Ortiz-Rivero, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C3G,+through+its+GEF+activity,+induces+megakaryocytic+differentiation+and+proplatelet+formation.">C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation.</a> Cell Communication and Signaling. 16 (1), 101 (2018).</li><li>Junt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317 (5845), 1767-1770 (2007).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+MYH9-related+disease:+mutations+in+nonmuscle+myosin+II-A.">Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A.</a> Blood. 119 (1), 238-250 (2012).</li><li>Malara, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+structure+and+nano-mechanics+determine+megakaryocyte+function.">Extracellular matrix structure and nano-mechanics determine megakaryocyte function.</a> Blood. 118 (16), 4449-4453 (2011).</li><li>Balduini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+receptors,+extracellular+proteins+and+myosin+IIA+orchestrate+proplatelet+formation+by+human+megakaryocytes.">Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes.</a> Journal of Thrombosis and Haemostasis. 6 (11), 1900-1907 (2008).</li></ol>

Nous décrivons ici une méthode in vitro simple et peu coûteuse pour évaluer l’efficacité des mégacaryocytes pour étendre les proplaquettaires qui se sont développées dans la moelle osseuse. Le modèle d’explantation de moelle osseuse pour souris présente quatre avantages principaux. Tout d’abord, aucune compétence technique avancée n’est requise. Deuxièmement, le temps nécessaire pour obtenir des proplaquettaires étendant les mégacaryocytes est assez court, seulement 6 heures pour la mét...

La technique de l’explantation de moelle osseuse a été développée pour la première fois par Thiéry et Bessis en 1956 pour décrire la formation d’extensions cytoplasmiques mégacaryocytaires de rat comme un événement initial dans la formation de plaquettes1. En utilisant le contraste de phase et des techniques cinématographiques, ces auteurs ont caractérisé la transformation de mégacaryocytes ronds matures en cellules thrombocytogènes « semblables à des calmars » avec des extensions cytoplasmiques montrant des mouvements dynamiques d’allongement et de contraction. Ces bras deviennent progressivement plus minces jusqu’à ce qu’ils deviennent filiformes avec de petits gonflements le long des bras et aux extrémités. Ces allongements typiques des mégacaryocytes, obtenus in vitro et en milieu liquide, présentent certaines similitudes avec les plaquettes observées dans la moelle osseuse fixe, où les mégacaryocytes dépassent de longues extensions à travers les parois sinusoïdales dans la circulation sanguine2,3. La découverte et le clonage du TPO en 1994, ont permis de différencier les mégacaryocytes en culture capables de former des extensions proplaquettaires ressemblant à celles décrites dans les explantes de moelle osseuse4,5,6. Cependant, la maturation des mégacaryocytes est beaucoup moins efficace dans les conditions de culture, notamment le vaste réseau membranaire interne des mégacaryocytes matures de la moelle osseuse est sous-développé dans les mégacaryocytes en culture, entravant les études sur les mécanismes de la biogenèse plaquettaire7,8.
Nous détaillons ici le modèle d’explantation de moelle osseuse, basé sur Thiéry et Bessis, à suivre en temps réel la formation de proplaquettaires de mégacaryocytes de souris, qui ont complètement mûri dans leur environnement natif, contournant ainsi d’éventuels artefacts in vitro et des interprétations erronées. Les résultats obtenus chez des souris adultes de type sauvage sont présentés pour illustrer la capacité des mégacaryocytes à étendre les proplaquettaires, leur morphologie et la complexité des proplaquettaires. Nous introduisons également une stratégie de quantification rapide pour la validation de la qualité afin d’assurer l’exactitude et la robustesse des données pendant le processus d’enregistrement des mégacaryocytes. Le protocole présenté ici est la version la plus récente de la méthode publiée sous la forme d’un chapitre de livre précédemment9.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mL syringes</td>
			<td>Terumo</td>
			<td>SS+05S1</td>
			<td></td>
		</tr>
		<tr>
			<td>21-gauge needles</td>
			<td>BD Microlance</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2.6H2O</td>
			<td>Sigma</td>
			<td>21108</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverwall Incubation Chambers</td>
			<td>Electron Microscopy Sciences</td>
			<td>70324-02</td>
			<td>Depth : 0,2 mm</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H-3375</td>
			<td>pH adjusted to 7.5</td>
		</tr>
		<tr>
			<td>Human serum albumin</td>
			<td>VIALEBEX</td>
			<td>authorized medication : n&#176; 3400956446995</td>
			<td>20% (200mg/mL -100mL)</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>Sigma</td>
			<td>BVBW8448</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover Glass</td>
			<td>Electron Microscopy Sciences</td>
			<td>72200-40</td>
			<td>22 mm x 55 mm</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica Microsystems SA, Westlar, Germany</td>
			<td>DMI8 - 514341</td>
			<td>air lens</td>
		</tr>
		<tr>
			<td>microscope camera</td>
			<td>Leica Microsystems SA, Westlar, Germany</td>
			<td>K5 CMS GmbH -14401137</td>
			<td>image resolution : 4.2 megapixel</td>
		</tr>
		<tr>
			<td>Mouse serum</td>
			<td>BioWest</td>
			<td>S2160-010</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4.H2O</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>PSG 100x</td>
			<td>Gibco, Life Technologies</td>
			<td>1037-016</td>
			<td>10,000 units/mL penicillin, 10,000 &#956;g/mL streptomycin and 29.2 mg/mL glutamine</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose D (+)</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
	</tbody>
</table>

proplatelet formation dynamics of mouse fresh bone marrow explants

La dernière étape de la mégacarylopoïèse conduit à des extensions cytoplasmiques à partir de mégacaryocytes matures, appelés proplaquettaires. On a beaucoup appris sur la formation de proplaquettaires à l’aide de mégacaryocytes différenciés in vitro; cependant, il existe de plus en plus de preuves que les systèmes de culture conventionnels ne récapitulent pas fidèlement le processus de différenciation/maturation qui a lieu à l’intérieur de la moelle osseuse. Dans ce manuscrit, nous présentons une méthode d’explantation initialement décrite en 1956 par Thiéry et Bessis pour visualiser les mégacaryocytes qui ont mûri dans leur environnement natif, contournant ainsi les artefacts potentiels et les mauvaises interprétations. Les moelles osseuses fraîches sont collectées en rinçant les fémurs des souris, tranchées en sections transversales de 0,5 mm et placées dans une chambre d’incubation à 37 °C contenant un tampon physiologique. Les mégacaryocytes deviennent progressivement visibles à la périphérie de l’explantation et sont observés jusqu’à 6 heures sous un microscope inversé couplé à une caméra vidéo. Au fil du temps, les mégacaryocytes changent de forme, certaines cellules ayant une forme sphérique et d’autres développant des extensions épaisses ou étendant de nombreuses proplaquettaires minces avec une ramification étendue. Des enquêtes qualitatives et quantitatives sont menées. Cette méthode a l’avantage d’être simple, reproductible et rapide car de nombreux mégacaryocytes sont présents, et classiquement la moitié d’entre eux forment des proplaquettaires en 6 heures contre 4 jours pour les mégacaryocytes de souris en culture. En plus de l’étude de souris mutantes, une application intéressante de cette méthode est l’évaluation simple des agents pharmacologiques sur le processus d’extension proplaquettaire, sans interférer avec le processus de différenciation qui peut se produire dans les cultures.

Résultats qualitatifs. Au début de l’expérience, toutes les cellules sont compactées dans la section de la moelle osseuse. Il faut 30 min pour que les cellules deviennent clairement visibles à la périphérie des explants. Les mégacaryocytes sont alors reconnaissables à leur grande taille et leur évolution peut ensuite être étudiée dans le temps (taille, forme, dynamique, extension proplaquettaire et libération plaquettaire)(Figure 2A). Les petits mégacaryocyt...

Watch this Scientific Journal Video about Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants at JoVE.com

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

Ici, nous détaillons la méthode d’explantation de moelle osseuse, de la préparation de l’échantillon à l’analyse microscopique des lames, afin d’évaluer la capacité des mégacaryocytes qui se sont différenciés dans leur environnement physiologique à former des proplaquettaires.

Dynamique de formation proplaquettaire des explants de moelle osseuse fraîche de souris

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...

L’objectif global de cette procédure est de suivre en temps réel la formation de proplaquettaires à partir de mégacaryocytes de souris, qui ont mûri dans leur environnement natif. Cette méthode a l’avantage d’être simple, reproductible et rapide. On peut analyser de nombreux mégacaryocytes et visualiser la formation de proplaquettaires en seulement six heures, au lieu d’attendre le premier jour de culture comme pour les mégacaryocytes de souris in vitro.Une application intéressante de cette méthode est la possibilité d’étudier l’effet du traitement pharmaceutique ou de la mutation génétique, en particulier à l’étape de l’extension proplaquettaire. Ici, il n’y a pas d’interférence avec le processus de différenciation, comme dans le cas de la culture in vitro. Cette vidéo permet d’assurer ces étapes clés du rinçage et de la coupe de la moelle osseuse.Il est également expliqué comment classer la morphologie et les mégacaryocytes, ce qui est vraiment utile pour la caractérisation des défauts dans la maladie thrombophile. Pour commencer, réchauffez le tampon de Tyrode à 37 degrés Celsius et allumez la chambre de chauffage du microscope pour amener la température à 37 degrés Celsius. Préparez tous les outils nécessaires, tels qu’une minuterie, des chambres d’incubation, des seringues de calibre 21 millilitres, des pinces, une lame de rasoir, des pipettes Pasteur, des lames en verre et un tube de centrifugeuse de 15 millilitres.Rincez la moelle osseuse avec deux millilitres de tampon de Tyrode en introduisant une aiguille de calibre 21 dans l’ouverture du fémur du côté du genou et appuyez lentement sur le piston pour récupérer un cylindre de moelle intact. Utilisez une pipette en plastique de trois minutes pour transférer soigneusement et doucement la moelle osseuse intacte sur une lame en verre. Coupez les extrémités de la moelle qui peuvent avoir été comprimées au moment de la chasse d’eau.Coupez ensuite de fines sections transversales de 0,5 millimètre d’épaisseur à l’aide d’une lame de rasoir tranchante pour éviter d’endommager les mégacaryocytes. À l’aide d’une pipette en plastique, collectez 10 sections dans un tube d’un millilitre contenant le tampon de Tyrode. Transférez soigneusement les sections dans une chambre d’incubation d’un diamètre de 13 millimètres.Aspirez le tampon et réglez le volume à 30 microlitres du tampon de Tyrode complété par du sérum de souris à 5%. Positionnez les sections à distance. Scellez la chambre auto-adhésive avec un glissement de couvercle de 22 par 55 millimètres, en inclinant le glissement du couvercle pour éviter la formation de bulles d’air.Placez la chambre dans la chambre de chauffage à 37 degrés Celsius. Démarrez le chronomètre et exécutez l’expérience pendant six heures à 37 degrés Celsius, Faites des vidéos pour enregistrer la transformation des mégacaryocytes. Après 30 minutes, les cellules de la moelle migrent progressivement vers la périphérie de l’explantation formant une monocouche.Après une heure d’incubation, les mégacaryocytes peuvent être identifiés par leur grande taille et leurs noyaux polylobulés. Le nombre de mégacaryocytes augmente après trois heures d’incubation, et certains ont de longues extensions. Dessinez une carte pour localiser chaque section de la chambre d’incubation.Après une heure, identifiez les mégacaryocytes visibles, qui sont des cellules polylobulées géantes à la périphérie de chaque section, et tracez leurs positions sur le dessin. Répétez cette procédure après trois et six heures. Les mégacaryocytes sont comptés manuellement et classés selon leur morphologie trois et six heures après la fermeture de la chambre d’incubation.Ils sont classés comme petits, grands avec une intention épaisse et proplaquettaires. Avec l’aide de la cartographie, leur évolution peut être suivie au fil du temps. Les résultats sont exprimés en pourcentage de chaque classe à chaque moment d’observation.Classiquement, la moitié des mégacaryocytes visibles à la périphérie s’étendent les proplaquettaires à six heures pour la moelle osseuse de souris de type sauvage. Le sort des mégacaryocytes ronds a été suivi par la capture d’images séquentielles au fil du temps pour imager comment ils forment des proplaquettaires. Une chose importante à retenir lors de la tentative de cette procédure est de maintenir les explants à 37 degrés Celsius pendant toute la durée de l’expérience.Une température différente peut modifier les résultats. Fait intéressant, le protocole explant peut être utilisé après des expériences de microscopie introvertie. En fin de compte, les mégacaryocytes dans les explantes de moelle osseuse seront naturellement fluorescents et leur comportement peut être facilement étudié.Cela permet de combiner différents traitements sur les mêmes animaux et ainsi de réduire le nombre de souris. En conclusion, la méthode d’explantation est rapide, simple et fournit de nombreuses informations sur la capacité des mégacaryocytes naturels aux proplaquettaires externes. Les résultats qualitatifs et quantitatifs qui en résultent sont essentiels à notre compréhension de la maladie thrombophile.dans un contexte physiologique ou pathologique.

proplatelet-formation-dynamics-of-mouse-fresh-bone-marrow-explants

Research

JoVE Journal

Biology

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Culture de cellules dendritiques myéloïdes à partir de précurseurs de moelle osseuse

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Recherche

Biologie

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Génération de la moelle osseuse des cellules dendritiques dérivées murin pour l&#39;utilisation dans l&#39;imagerie 2-photons

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing des cellules hématopoïétiques de la moelle osseuse

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Ostéoclastes Dérivation de moelle osseuse de souris

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

TRANS-intérieur cellulaire massive Injection de cellules souches embryonnaires conduit à des taux plus élevés de chimérisme

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Expression des protéines de Fusion Fluorescent en Murine dérivés de la moelle osseuse des cellules dendritiques et Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Isolement, purification et différenciation des précurseurs d’Ostéoclast de la moelle osseuse de rat

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

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

In Vivo Imagerie à deux photons de mégacaryocytes et de proplaquettaires dans la moelle osseuse du crâne de souris

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...