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

Los autores desean agradecer a Jean-Yves Rinckel, Julie Boscher, Patricia Laeuffer, Monique Freund, Ketty Knez-Hippert por su asistencia técnica. Este trabajo ha sido apoyado por ANR (Agence National de la Recherche) Grant ANR-17-CE14-0001-01 y ANR-18-CE14-0037.

Todos los experimentos con animales se realizaron de acuerdo con las normas europeas 2010/63/EU y el Comité CREMEAS sobre la Ética de los Experimentos con Animales de la Universidad de Estrasburgo (Comité Régional d'Ethique en Matière d'Expérimentation Animale Strasbourg).
1. Preparación de reactivos
<ol><li>Preparar reactivos como se describe en la Tabla 1.<ol>
<li>Para el Stock I, disuelva cada polvo por separado. Asegúrese de que la osmolaridad de la preparación ...

<ol>
	<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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transmural+passage+of+blood+cells+into+myeloid+sinusoids+and+the+entry+of+platelets+into+the+sinusoidal+circulation:+A+scanning+electron+microscopic+investigation.">The transmural passage of blood cells into myeloid sinusoids and the entry of platelets into the sinusoidal circulation: A scanning electron microscopic investigation.</a> American Journal of Anatomy. 145 (2), 183-205 (1976).</li><li>Muto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scanning+and+transmission+electron+microscopic+study+on+rat+bone+marrow+sinuses+and+transmural+migration+of+blood+cells.">A scanning and transmission electron microscopic study on rat bone marrow sinuses and transmural migration of blood cells.</a> Archivum Histologicum Japonicum. 39 (1), 51-66 (1976).</li><li>Cramer, E. 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>

Aquí describimos un método in vitro simple y de bajo costo para evaluar la eficiencia de los megacariocitos para extender los proplatlets que han crecido en la médula ósea. El modelo de explante de médula ósea para ratón tiene cuatro ventajas principales. En primer lugar, no se requieren habilidades técnicas avanzadas. En segundo lugar, el tiempo necesario para obtener proplatlets extensibles de megacariocitos es bastante corto, solo 6 horas para el método de explante, en comparación con un mínimo de ...

La técnica de explante de médula ósea fue desarrollada por primera vez por Thiéry y Bessis en 1956 para describir la formación de extensiones citoplasmáticas de megacariocitos de rata como un evento inicial en la formación de plaquetas1. Utilizando contraste de fase y técnicas cinematográficas, estos autores caracterizaron la transformación de megacariocitos redondos maduros en células trombocitogénicas "parecidas a calamares" con extensiones citoplasmáticas que muestran movimientos dinámicos de elongación y contracción. Estos brazos se vuelven progresivamente más delgados hasta que se vuelven filiformes con pequeñas hinchazones a lo largo de los brazos y en las puntas. Estas elongaciones típicas de megacariocitos, obtenidas in vitro y en medios líquidos, tienen ciertas similitudes con las plaquetas observadas en la médula ósea fija, donde los megacariocitos sobresalen largas extensiones a través de las paredes sinusoides en la circulación sanguínea2,3. El descubrimiento y clonación de TPO en 1994, permitió diferenciar megacariocitos en cultivo capaces de formar extensiones proplatletas similares a las descritas en los explantes de médula ósea4,5,6. Sin embargo, la maduración de los megacariocitos es mucho menos eficiente en condiciones de cultivo, en particular la extensa red de membrana interna de los megacariocitos maduros de la médula ósea está subdesarrollada en los megacariocitos cultivados, lo que dificulta los estudios sobre los mecanismos de la biogénesis plaquetaria7,8.
Detallamos aquí el modelo de explante de médula ósea, basado en Thiéry y Bessis, para seguir en tiempo real la formación de proplatlet de megacariocitos de ratón, que han madurado completamente en su entorno nativo, eludiendo así posibles artefactos in vitro y malas interpretaciones. Los resultados obtenidos en ratones adultos de tipo salvaje se presentan para ilustrar la capacidad de los megacariocitos para extender los proplatlets, su morfología y la complejidad de los proplatlets. También introducimos una estrategia de cuantificación rápida para la validación de la calidad para garantizar la precisión y robustez de los datos durante el proceso de registro de megacariocitos. El protocolo aquí presentado es la versión más reciente del método publicado como libro capítulo anteriormente9.

<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 última etapa de la megacaropoyesis conduce a extensiones citoplasmáticas de megacariocitos maduros, los llamados proplatlets. Mucho se ha aprendido sobre la formación de proplatlet utilizando megacariocitosdiferenciados in vitro; sin embargo, existe una creciente evidencia de que los sistemas de cultivo convencionales no recapitulan fielmente el proceso de diferenciación/maduración que tiene lugar dentro de la médula ósea. En este manuscrito, presentamos un método de explante descrito inicialmente en 1956 por Thiéry y Bessis para visualizar megacariocitos que han madurado en su entorno nativo, evitando así posibles artefactos y malas interpretaciones. Las médulas óseas frescas se recogen enjuagando los fémures de los ratones, se cortan en secciones transversales de 0,5 mm y se colocan en una cámara de incubación a 37 ° C que contiene un tampón fisiológico. Los megacariocitos se hacen gradualmente visibles en la periferia del explante y se observan hasta 6 horas bajo un microscopio invertido acoplado a una cámara de video. Con el tiempo, los megacariocitos cambian su forma, con algunas células que tienen una forma esférica y otras que desarrollan extensiones gruesas o extienden muchos proplatelets delgados con ramificaciones extensas. Se llevan a cabo investigaciones cualitativas y cuantitativas. Este método tiene la ventaja de ser simple, reproducible y rápido, ya que numerosos megacariocitos están presentes, y clásicamente la mitad de ellos forman proplalets en 6 horas en comparación con 4 días para los megacariocitos de ratón cultivados. Además del estudio de ratones mutantes, una aplicación interesante de este método es la evaluación directa de los agentes farmacológicos en el proceso de extensión del proplatelet, sin interferir con el proceso de diferenciación que puede ocurrir en los cultivos.

Resultados cualitativos. Al comienzo del experimento, todas las células se compactan en la sección de médula ósea. Las células tardan 30 minutos en hacerse claramente visibles en la periferia de los explantes. Los megacariocitos son entonces reconocibles por su gran tamaño y su evolución puede ser estudiada a lo largo del tiempo (tamaño, forma, dinámica, extensión proplatelet y liberación plaquetaria)(Figura 2A). Los megacariocitos pequeños tienen un diámetro en...

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

Aquí, detallamos el método de explante de médula ósea, desde la preparación de muestras hasta el análisis microscópico de diapositivas, para evaluar la capacidad de los megacariocitos que se han diferenciado en su entorno fisiológico para formar proplatlets.

Dinámica de formación de proplatlets de explantes de médula ósea fresca de ratón

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

El objetivo general de este procedimiento es seguir en tiempo real la formación de proplatelet a partir de megacariocitos de ratón, que maduraron en su entorno nativo. Este método tiene la ventaja de ser simple, reproducible y rápido. Se pueden analizar muchos megacariocitos y visualizar la formación de proplatlets en solo seis horas, en lugar de esperar el primer día de cultivo como para los megacariocitos de ratón in vitro.Una aplicación interesante de este método es la posibilidad de estudiar el efecto del tratamiento farmacéutico o la mutación genética, específicamente en el paso de extensión proplatelet. Aquí no hay interferencias con el proceso de diferenciación, como en el caso del cultivo in vitro. Este video ayuda a garantizar estos pasos clave para enjuagar y cortar la médula ósea.También se explica cómo clasificar la morfología y los megacariocitos, lo que es realmente útil para la caracterización de defectos en la enfermedad trombofílica. Para comenzar, caliente el amortiguador de Tyrode a 37 grados centígrados y encienda la cámara de calentamiento del microscopio para llevar la temperatura a 37 grados centígrados. Prepare todas las herramientas necesarias, como un temporizador, cámaras de incubación, jeringas calibre 21 de cinco mililitros, fórceps, una cuchilla de afeitar, pipetas Pasteur, portaobjetos de vidrio y tubo de centrífuga de 15 mililitros.Enjuague la médula ósea con dos mililitros del tampón de Tyrode introduciendo una aguja de calibre 21 en la abertura del fémur en el lado de la rodilla, y presione lentamente el émbolo para recuperar un cilindro de médula intacto. Use una pipeta de plástico de tres minutos para transferir cuidadosa y suavemente la médula ósea intacta a un portaobjetos de vidrio. Cortar los extremos de la médula que pueden haber sido comprimidos en el momento del lavado.Luego corte secciones transversales delgadas de 0,5 milímetros de espesor con una cuchilla de afeitar afilada para evitar dañar los megacariocitos. Usando una pipeta de plástico, recoja 10 secciones en un tubo de un mililitro que contenga el tampón de Tyrode. Transfiera cuidadosamente las secciones a una cámara de incubación con un diámetro de 13 milímetros.Aspire el tampón y ajuste el volumen a 30 microlitros del tampón de Tyrode complementado con suero de 5% de ratón. Coloque las secciones a distancia. Selle la cámara autoadhesiva con un deslizamiento de cubierta de 22 por 55 milímetros, inclinando el deslizamiento de la cubierta para evitar la formación de burbujas de aire.Coloque la cámara en la cámara de calentamiento a 37 grados centígrados. Inicie el cronómetro y ejecute el experimento durante seis horas a 37 grados centígrados, haga videos para grabar la transformación de los megacariocitos. Después de 30 minutos, las células de la médula migran gradualmente a la periferia del explante formando una capa mono.Después de una hora de incubación, los megacariocitos se pueden identificar por su gran tamaño y núcleos polilobulados. El número de megacariocitos aumenta después de tres horas de incubación, y algunos tienen largas extensiones. Dibuja un mapa para localizar cada sección en la cámara de incubación.Después de una hora, identifique los megacariocitos visibles, que son células polilobuladas gigantes en la periferia de cada sección, y trace sus posiciones en el dibujo. Repita este procedimiento después de tres y seis horas. Los megacariocitos se cuentan manualmente y se clasifican según su morfología a las tres y seis horas después del sellado de la cámara de incubación.Se clasifican como pequeños, grandes con intención gruesa y proplatelet que se extienden. Con la ayuda del mapeo, su evolución se puede seguir a lo largo del tiempo. Los resultados se expresan como un porcentaje de cada clase en cada momento de observación.Clásicamente, la mitad de los megacariocitos visibles en la periferia extienden los proplatlets a las seis horas para la médula ósea de ratón de tipo salvaje. El destino de los megacariocitos redondos fue seguido por la captura de imágenes secuenciales a lo largo del tiempo para obtener imágenes de cómo forman los proplatlets. Una cosa importante a recordar al intentar este procedimiento es mantener los explantes a 37 grados Centígrados durante la duración del experimento.Una temperatura diferente puede cambiar los resultados. Curiosamente, el protocolo de explante se puede utilizar después de experimentos de microscopía introvertidos. Al final, los megacariocitos en los explantes de médula ósea serán naturalmente fluorescentes, y su comportamiento se puede investigar fácilmente.Esto permite combinar diferentes tratamientos en los mismos animales y así reducir el número de ratones. En conclusión, el método de explante es rápido, simple y proporciona mucha información sobre la capacidad de los megacariocitos naturales a los proplatlets externos. El hallazgo cualitativo y cuantitativo resultante es clave para nuestra comprensión de la enfermedad trombofílica.en un contexto fisiológico o patológico.

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.

Cultura de células dendríticas mieloides a partir de precursores de médula ósea

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

Investigación

Biología

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.

Generación de médula ósea procedentes de células murinas dendríticas para su uso en dos fotones de imágenes

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 preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

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.

Mensajeras de las células hematopoyéticas de la médula ósea

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.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

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.

Osteoclastos Derivación de médula ósea de ratón

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-interno celular masiva inyección de células madre embrionarias conduce a mayores tasas de quimerismo

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.

Expresión de proteínas fluorescentes de fusión en murinas médula ósea células dendríticas y macrófagos

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.

Aislamiento, purificación y diferenciación de los precursores de osteoclastos de la médula ósea de rata

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 Imágenes de dos fotones de megacariocitos y proplatatos en la médula ósea del cráneo del ratón

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