Los autores agradecen a Dr. Minghan Tong y S. Kato para ratones y reactivos que amablemente. Agradecemos a los miembros del laboratorio de Zou para discusiones útiles. Este trabajo fue apoyado en parte por subvenciones de 973 programa del Ministerio Chino de ciencia y tecnología (MOST) [2014CB964704 y 2015CB964503], programa de investigación clínica del Hospital de 9 personas, Shanghai Jiao Tong University School of Medicine. Gracias por la ayuda de instalaciones centrales para la biología de la célula e instalaciones centrales de Biología química, CAS centro de excelencia en Ciencia Molecular de la célula, Instituto Shanghai de Bioquímica y biología celular, Academia China de Ciencias.

Todos los procedimientos relativos a los animales se realizaron según el protocolo aprobado por el Panel administrativo de Stanford en el laboratorio Animal Care (APLAC) y fueron aprobados por el cuidado de animales y uso del Instituto de Shanghai de Bioquímica y celular Biología.
1. preparación
<ol><li>Generar ratones de eliminación específicas de Raptor en osteoclastos (Raptorfl/fl; Ctsk-cre, en adelante RapCtsk) apareamiento Raptorfl/fl ratones ratones de Ctsk-cre . Usar los ratones Raptorfl/fl como el control de peso en este estudio.</li>
	<li>Tener un recipiente con hielo para mantener el hueso aislado.</li>
	<li>
Preparar medio de cultivo.
	<ol>
<li>Preparar medio de cultivo MEM α complementando alfa medio esencial mínimo (α-MEM) con 1 x glutamina, penicilina-estreptomicina y 10% de suero de ternero fetal.</li>
		<li>Preparar el medio de inducción de macrófagos de médula ósea consisten en 50 mL de medio MEM α y M-CSF 20 ng/ml.</li>
		<li>Preparar el medio de inducción de osteoclastos que consta de M-CSF y RANKL a 20 ng/mL, respectivamente, en 50 mL de medio de α-MEM.</li>
	</ol>
</li>
	<li>
Preparar los siguientes buffers antes de la tinción de la trampa.
	<ol>
<li>Preparar la solución de fix de 6,5 mL de acetona, 2,5 mL de solución de citrato y 0,8 mL de solución de formaldehído (vol/vol) de 37%.</li>
		<li>Preparar solución de tinción de la trampa.<ol>
<li>Precaliente agua desionizada a 37 ° C.</li>
			<li>Añadir 100 μl de solución de base de GBC rápido granate y 100 μl de solución de nitrito de sodio en un microtubo de 1,5 mL y mezclar por inversión suave para 30 s. deje la mezcla reposar 2 minutos.</li>
			<li>Precalentamiento 9 mL de agua desionizada a 37 ° C. Añadir 200 μL de la solución base de GBC diazotado rápido granate de paso 1.4.2.2, 100 μl de solución de fosfato de naftol AS-BI, 400 μL de solución de acetato y 200 μL de solución de tartrato.</li>
			<li>Mantener la trampa de coloración de la solución de la mezcla en un baño de agua a 37 ° C.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Preparar el siguiente buffer antes de cuantificación de actividad de trampa del medio de cultivo.
	<ol>
<li>Preparar el tampón tartrato (50 mL): 2 mL de ácido L-tartárico de 0,33 M, 48 mL de tartrato de sodio de 0,33 M dibásico deshidratar. Ajustar el pH a 4.9.</li>
		<li>Preparar (200 mL) de tampón 4-nitrofenil fosfato disódico (pNFF): 1,502 g de glicina, 41 mg de MgCl2, 27,2 mg de vivencias2y 180 mL de agua desionizada. Ajustar el pH a 10.4 con 3 M NaOH y el volumen a 200 mL con agua desionizada.</li>
		<li>Preparar pNFF buffer con un sustrato de la fosfatasa (para una placa de 96 pocillos): 49,37 mg de sustrato de la fosfatasa y 175 μl de tampón de pNFF de paso 1.5.2.</li>
		<li>Preparación de buffer de sustrato (9 mL/96-well plate): 6,24 mL de agua desionizada, 360 μL de solución de acetato y 2,4 mL de buffer de tartrato. Mezclar por vortex y precaliente a 37 ° C antes de usar.</li>
		<li>Agregar 90 μL de tampón de pNFF con fosfatasa substrate(1.5.3) a 9ml de sustrato precalentado buffer(1.5.4) para hacer una mezcla de sustrato y de vortex por 10 s.</li>
	</ol>
</li>
</ol>2. disección (día -1)
<ol><li>Eutanasia por separado 3 one-month-old WT y RapCtsk ratones femeninos por CO2 . Realizar la inhalación de CO2 con el equipo adecuado por personal capacitado.</li>
	<li>Sumerja 6 ratones en un vaso de precipitados con 100 mL de etanol al 75% (vol/vol) durante 5 min evitar la contaminación bacteriana y luego colocar en una mesa de disección en una posición supina. Hacer una incisión en la parte distal de la tibia vertical y disecar la piel a lo largo de las extremidades con las tijeras oftálmicas.</li>
	<li>Cortar el ligamento intraarticular de la articulación de la cadera con unas tijeras y dislocar los miembros posteriores del tronco.</li>
	<li>Cortar el ligamento articular de la articulación de la rodilla y disociar cuidadosamente la tibia y el fémur de articulación de la rodilla. Retire con cuidado el tejido suave con las tijeras.</li>
	<li>Colocar todos los huesos de un genotipo en de un ratón en cada pocillo de una placa de 6 pozos con 2 mL de α-MEM en el hielo. Nota: Para preservar la viabilidad celular, el hueso debe ser mantenido en estas condiciones por menos de 1 h.</li>
</ol>3. aislamiento (día -1)
<ol><li>Llenar un pocillo de una placa de seis pozos con etanol al 75% (3 mL) y los otros 5 pozos con medio de α-MEM (3 mL).</li>
	<li>Poner todos los huesos de un genotipo en el lavado de etanol para 15 huesos s y lavar 5 veces con medio de α-MEM de 10 s cada vez.</li>
	<li>Cortar las epífisis con la tijera e Inserte una aguja de jeringa 0.45 mm en la cavidad y lavado médula hacia fuera con medio MEM de α en un tubo de centrífuga de 50 mL.</li>
	<li>Limpie la cavidad del hueso utilizando el mismo método desde el otro extremo del hueso. Repetir al menos dos veces para lavar la cavidad ósea completamente hasta que el hueso es pálido.</li>
	<li>La médula ósea para obtener un pellet de células a 800 x g y 4 ° C por 5 min aspirar el sobrenadante de la centrífuga.</li>
	<li>Añadir 3 mL de tampón de lisis de glóbulos rojos en el tubo de centrífuga y la pipeta suavemente para Resuspender la médula ósea. Mantenga el tubo de centrífuga en el hielo durante 8 min a lyse células de sangre rojas.</li>
	<li>Añadir 6 mL de medio MEM de α en el tubo de la centrífuga para detener la lisis celular. Centrifugar a 500 x g y 4 ° C por 10 min y aspirar el sobrenadante.</li>
	<li>Resuspender en 3 mL de medio de cultivo MEM α y colocar las células en una placa seis-bien (generalmente un ratón por pozo).</li>
	<li>Incubar a 37 ° C en un incubador de 5% CO2 durante la noche.</li>
</ol>4. galjanoplastia (día 0)
<ol><li>Transferir el sobrenadante a un tubo de centrífuga de 15 mL para recoger células sueltas a la mañana siguiente.</li>
	<li>Centrifugue a 800 x g y 4 ° C por 5 min y aspirar el sobrenadante.</li>
	<li>Resuspender en 4 mL de medio de cultivo MEM α.</li>
	<li>Tomar 20 μl de la solución de células y mezclar con 20 μl de azul tripán. Añadir 10 μl de la mezcla a la cámara de conteo y obtener el conteo de células por mL de solución.</li>
	<li>Añadir un volumen adecuado de medio de cultivo MEM α para obtener una solución de celdas de 500.000 células/mL.</li>
	<li>Añadir 500 μl y 50 μl de medio de inducción de macrófagos de médula ósea en cada pocillo de una placa de 24 pozos y placa de 96 pocillos, respectivamente.</li>
	<li>Añadir 500 μl y 50 μl de la solución celular en cada pocillo de una placa de 24 pozos y placa de 96 pocillos, respectivamente.</li>
	<li>Incubar a 37 ° C en un incubador de 5% CO2 durante 3 días.</li>
</ol>5. diferenciación (días 3-7)
<ol><li>Tres días después de la galjanoplastia, recoger 50 μl de medio de cada pocillo de la placa de 96 pozos y congelar a-20 ° C y luego aspirar el medio residual.</li>
	<li>Añadir 1 mL y 100 μl de los osteoclastos inducción de medio en cada pocillo de una placa de 24 pocillos y una placa de 96 pozos, respectivamente.</li>
	<li>Incubar a 37 ° C durante 2 días.</li>
	<li>Recoger 50 μl del medio de cada pozo y aspirar el medio residual.</li>
	<li>Añadir el medio de inducción de osteoclastos en cada pozo.</li>
	<li>Incubar a 37 ° C por 1 día. Nota: En este momento, grandes, multinucleados osteoclastos puede considerarse bajo un microscopio invertido (figura 2A).</li>
	<li>Si no se observan osteoclastos, cambiar el medio de inducción de los osteoclastos y observar todos los días hasta que aparecen los osteoclastos.</li>
	<li>Recoger 50 μl del medio de cada pocillo de la placa de 96 pocillos después de osteoclastos han formado.</li>
</ol>6. tartrato fosfatasa ácida resistente (TRAP) tinción
Nota: Los osteoclastos maduros pueden presentar después de 6-7 días de cultivo en vitro (paso 4).
<ol><li>Medio de aspirar y lavar suavemente 3 veces con 1 x PBS.</li>
	<li>Fijar las células en cada pocillo de la placa de 96 pocillos con 100 μl de solución de solución de 30 s a temperatura ambiente.</li>
	<li>Después de la fijación, Aspire la solución de fix y lavar suavemente 3 veces con agua desionizada precalentada a 37 ° C. Aspire agua desionizada.</li>
	<li>Añada 100 μl de mancha de trampa en cada pocillo de la placa de 96 pocillos y colocar la placa en una incubadora a 37 ° C por 30 min y protector de la luz.</li>
	<li>Después de la tinción, Aspire la mancha trampa y suavemente Lave 3 veces con agua desionizada.</li>
	<li>Osteoclastos de imagen usando un microscopio invertido con 40 aumentos.</li>
</ol>7. la trampa cuantificación de actividad de medio de cultivo
<ol><li>Recoger el sobrenadante de la cultura de 30 μl de cada pocillo de la placa de 96 pocillos en el paso 5.1, 5.4 y 5.8; Utilizar 30 μl de medio de inducción no cultivadas de los osteoclastos como control negativo. Coloque las muestras en una nueva placa de 96 pocillos.</li>
	<li>Agregar 90 μL de substrato mixture(1.5.5) en cada pocillo de la placa de 96 pocillos.</li>
	<li>Colocar la placa en una incubadora a 37 ° C por 1 h y escudo de la luz.</li>
	<li>Detener la reacción agregando 50 μl de NaOH M 3 en cada pocillo de la placa de 96 pocillos.</li>
	<li>Medida theabsorbance en la longitud de onda de 405 nm.</li>
</ol>8. Western Blotting
Nota: Después de 6-7 días de cultivo en vitro en la placa de 24 pocillos, osteoclastos maduros deben ser visibles.
<ol><li>Aspire el medio.</li>
	<li>Lave suavemente con pre-enfriado de 1 x PBS 3 veces.</li>
	<li>Aspire el PBS.</li>
	<li>Añada 100 μl de 1 x SDS lysis buffer que contiene inhibidor de la proteasa cóctel.</li>
	<li>Lisados de calor a 105 ° C por 10 min y centrifugar a 12.000 x g y 4 ° C durante 10 minutos recogen el sobrenadante que contiene las proteínas.</li>
	<li>Separe los lisados que contiene 30 μg de proteína 10% SDS-PAGE11 y luego se transfieren a una membrana de polivinilideno (PVDF) de fluoruro.</li>
	<li>Bloquear las membranas con leche descremada en polvo 5% durante 30 minutos.</li>
	<li>Incubar las membranas con el Raptor, anti-P-S6, S6 anti, anti-β-actina en el 1:1,000 de dilución de leche descremada 5% a 4 ° C durante la noche.</li>
	<li>Lavar las membranas con Tris amortiguada salina con Tween-20 (TBST) por 10 min a temperatura ambiente.</li>
	<li>Repita el paso 8.9 dos veces.</li>
	<li>Incubar las membranas con peroxidasa de rábano (HRP)-conjugado de anticuerpos IgG anti-ratón o conejo en 1:5,000 dilución en la leche descremada en polvo 5% durante 1 h a temperatura ambiente.</li>
	<li>Lavar las membranas 3 veces con TBST durante 10 min a temperatura ambiente.</li>
	<li>Para detección quimioluminiscente, añadir sustrato quimioluminiscente occidental de HRP en las membranas e incubar 5 min a temperatura ambiente. Drenar el sustrato sobrante y exponer la bienvenida a la película de rayos x.</li>
</ol>

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R., 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=TRANCE+is+a+novel+ligand+of+the+tumor+necrosis+factor+receptor+family+that+activates+c-Jun+N-terminal+kinase+in+T+cells.">TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells.</a> J Biol Chem. 272 (40), 25190-25194 (1997).</li><li>Zoncu, R., Efeyan, A., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTOR:+from+growth+signal+integration+to+cancer,+diabetes+and+ageing.">mTOR: from growth signal integration to cancer, diabetes and ageing.</a> Nat Rev Mol Cell Biol. 12 (1), 21-35 (2011).</li><li>Bhaskar, P. T., Hay, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+TORCs+and+Akt.">The two TORCs and Akt.</a> Dev Cell. 12 (4), 487-502 (2007).</li><li>Dai, Q., 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=Inactivation+of+Regulatory-associated+Protein+of+mTOR+(Raptor)/Mammalian+Target+of+Rapamycin+Complex+1+(mTORC1)+Signaling+in+Osteoclasts+Increases+Bone+Mass+by+Inhibiting+Osteoclast+Differentiation+in+Mice.">Inactivation of Regulatory-associated Protein of mTOR (Raptor)/Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Osteoclasts Increases Bone Mass by Inhibiting Osteoclast Differentiation in Mice.</a> J Biol Chem. 292 (1), 196-204 (2017).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (1989).</li><li>Weischenfeldt, J., Porse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+Marrow-Derived+Macrophages+(BMM):+Isolation+and+Applications.">Bone Marrow-Derived Macrophages (BMM): Isolation and Applications.</a> CSH Protoc. 2008, (2008).</li><li>Bradley, E. W., Oursler, M. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANKL-based+osteoclastogenic+assays+from+murine+bone+marrow+cells.">RANKL-based osteoclastogenic assays from murine bone marrow cells.</a> Methods Mol Biol. 1130, 307-313 (2014).</li><li>Hsu, H., 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=Tumor+necrosis+factor+receptor+family+member+RANK+mediates+osteoclast+differentiation+and+activation+induced+by+osteoprotegerin+ligand.">Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.</a> Proc Natl Acad Sci U S A. 96 (7), 3540-3545 (1999).</li><li>Underwood, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+where+comes+the+osteoclast?.">From where comes the osteoclast?.</a> J Pathol. 144 (4), 225-226 (1984).</li><li>Wein, M. N., 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=Control+of+bone+resorption+in+mice+by+Schnurri-3.">Control of bone resorption in mice by Schnurri-3.</a> Proc Natl Acad Sci U S A. 109 (21), 8173-8178 (2012).</li></ol>

Todos los autores afirman que tienen no hay conflictos de intereses.

El ensayo de osteoclastogenic es el método más utilizado para aislar y cultura osteoclastos en vitro12,13. Mientras que varias inducciones basadas en RANKL osteoclasto se han descrito13,14,15, el presente estudio describe un protocolo con algunas modificaciones basadas en los métodos anteriores.
En el estudio anterior, eran placas ...

El hueso es un órgano siempre-que cambia y es remodelado por osteoblastos y osteoclastos durante toda la vida. Osteoclastos se encargan de la resorción de la matriz mineralizada y osteoblastos sintetizan y secretan nuevo hueso matrices1. El equilibrio entre la reabsorción ósea y formación ósea es crucial para la salud de los huesos incluyendo el mantenimiento del hueso masa y respuesta a la estimulación y lesiones. Si este equilibrio se interrumpe, puede ocurrir una serie de enfermedades metabólicas óseas, incluyendo osteoporosis y enfermedades periodontales. En estas enfermedades, pérdida de masa ósea resultante de la resorción ósea osteoclástica supera el hueso formando capacidad de osteoblastos2,3. Así, con el fin de desarrollar objetivos farmacéuticos para tratar trastornos esqueléticos como la osteoporosis, es fundamental para entender la generación y la biología de osteoclastos4.
Osteoclastos son las células multinucleated gigante únicas localizadas en o cerca de la superficie del hueso y pertenecen a la familia de monocito/macrófago1. Ibbotson K. J., et al. informó un método para generar células similares a osteoclastos en vitro con medio que contiene 1,25-dihidroxi-vitamina D35. La identificación del factor estimulante de colonias de macrófagos (M-CSF) y el receptor activador del ligando de factor nuclear-κ B (RANKL) como factores esenciales de la formación de osteoclastos ha aumentado dramáticamente la eficiencia de la osteoclastogénesis in vitro 1 , 6 , 7. la capacidad de cultura osteoclastos en vitro ha mejorado nuestra comprensión de la generación y regulación de los osteoclastos.
El mecanicista/mamíferos objetivo de rapamicina (mTOR) funciones en dos complejos estructuralmente y funcionalmente distintos, a saber mTORC1 y mTORC28,9. Los dos complejos son distintos unos de otros debido a sus diferentes componentes y sustratos aguas abajo. mTORC1 contiene la única proteína reguladoras asociadas de mTOR (Raptor), mientras mTORC2 contiene al rapamicina insensible compañero de mTOR (Powder)9. mTORC1 pueden integrar y transmitir señales importantes para regular la diferenciación, proliferación y crecimiento celular. Recientemente, hemos demostrado que eso mTORC1 desempeña un papel clave en la red de la resorción ósea catabólica por la eliminación de Raptor para inactivar mTORC1 en osteoclastos10. Sin embargo, los mecanismos subyacentes requieren estudio adicional. En el presente estudio, se utilizó un método basado en RANKL osteoclastogenic generar osteoclastos macrófagos derivados de médula ósea (BMMs) de tipo salvaje (WT) y ratones RapCtsk y estudiar la influencia de mTORC1 inactivación en osteoclastos formación.

<table>
	<tbody>
		<tr>
			<td>Raptorfl/fl mice</td>
			<td>The Jackson Laboratory</td>
			<td>013188</td>
			<td></td>
		</tr>
		<tr>
			<td>Ctsk-cre mice</td>
			<td></td>
			<td></td>
			<td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Corning</td>
			<td>10-022-CVR</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibico</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin streptomycin</td>
			<td>Gibico</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>BioInd</td>
			<td>04-001-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse M-CSF protein</td>
			<td>R&#38;D</td>
			<td>Q3U4F9</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse RANKL protein</td>
			<td>R&#38;D</td>
			<td>Q3TWY5</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Beyotime</td>
			<td>C3702</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma-Aldrich</td>
			<td>302643</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate solution</td>
			<td>Sigma-Aldrich</td>
			<td>915</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Garnet GBC Base solution</td>
			<td>Sigma-Aldrich</td>
			<td>3872</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Nitrite Solution</td>
			<td>Sigma-Aldrich</td>
			<td>914</td>
			<td></td>
		</tr>
		<tr>
			<td>Naphthol AS-BI Phosphate Solution</td>
			<td>Sigma-Aldrich</td>
			<td>3871</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetate solution</td>
			<td>Sigma-Aldrich</td>
			<td>3863</td>
			<td></td>
		</tr>
		<tr>
			<td>Tartrate solution</td>
			<td>Sigma-Aldrich</td>
			<td>3873</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
			<td>Corning</td>
			<td>21-031-CVR</td>
			<td></td>
		</tr>
		<tr>
			<td>L-tartaric acid</td>
			<td>Sigma-Aldrich</td>
			<td>251380</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tartrate dibasic dehydrate</td>
			<td>Sigma-Aldrich</td>
			<td>s4797</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Shanghai Chemical Co. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase substrate</td>
			<td>Sigma-Aldrich</td>
			<td>P4744</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Raptor</td>
			<td>Cell Signaling Technology</td>
			<td>2280</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-P-ribosomal protein S6 (S235/236)</td>
			<td>Cell Signaling Technology</td>
			<td>2317</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ribosomal protein S6</td>
			<td>Cell Signaling Technology</td>
			<td>2211</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-&#946;-actin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-130300</td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde</td>
			<td>Xilong scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polyvinylidene fluoride (PVDF) membrane</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Western Chemiluminescent HRP Substrate (ECL)</td>
			<td>Millipore</td>
			<td>00000367MSDS</td>
			<td></td>
		</tr>
		<tr>
			<td>IX71</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Envision</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.45-mm Syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito forcep</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rankl-based osteoclast culture assay of mouse bone marrow to investigate the role of mtorc1 in osteoclast formation

Los osteoclastos son único hueso-resorbing células que diferencian del linaje monocito/macrófago de la médula ósea. Disfunción de los osteoclastos puede resultar en una serie de enfermedades metabólicas del hueso, incluyendo la osteoporosis. Para desarrollar objetivos de productos farmacéuticos para la prevención de pérdida de masa ósea patológica, se deben entender los mecanismos por el cual los osteoclastos diferencian de precursores. La capacidad de aislar y un gran número de osteoclastos en vitro la cultura es fundamental para determinar el papel de genes específicos en la diferenciación de los osteoclastos. Inactivación del mecanicista/mamíferos objetivo de rapamicina complejo 1 (TORC1) en osteoclastos puede disminuir el número de osteoclastos y aumenta la masa ósea; sin embargo, los mecanismos subyacentes requieren estudio adicional. En el presente estudio, se describe un protocolo basado en RANKL para aislar y cultura osteoclastos de médula ósea de ratón y estudiar la influencia de mTORC1 inactivación en la formación de osteoclastos. Este protocolo con éxito dio lugar a un gran número de osteoclastos gigantes, normalmente dentro de una semana. Supresión de Raptor deterioró la formación de osteoclastos y disminución de la actividad de la fosfatasa ácida tartrato resistente secretor, indicando que eso mTORC1 es fundamental para la formación de osteoclastos.

Mediante el presente Protocolo, un gran número de osteoclastos gigantes fueron visto el día 6; Si no se observan osteoclastos gigantes, un día más de diferenciación de osteoclastos puede ser necesario (figura 1). Formación de osteoclastos exitosa fue confirmada por trampa de tinción (figura 2A). Osteoclastos son células de color rojo/morado vino gigantes con más de 3 núcleos. Más de 250 osteoclastos se obtuvieron en ca...

Watch this Scientific Journal Video about A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation at JoVE.com

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Este manuscrito describe un protocolo para aislar y la cultura osteoclastos en vitro de médula ósea de ratón y para estudiar el papel de lo mamíferos/mecanicista Diana de la rapamicina 1 complejo en la formación de osteoclastos.

Una base de RANKL osteoclasto cultura ensayo de médula ósea de ratón para investigar el papel de mTORC1 en la formación de osteoclastos

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

a-rankl-based-osteoclast-culture-assay-mouse-bone-marrow-to

Research

JoVE Journal

Developmental Biology

12.7K vistas.  National Clinical Research Center of Stomatology. Este manuscrito describe un protocolo para aislar y la cultura osteoclastos en vitro de médula ósea de ratón y para estudiar el papel de lo mamíferos/mecanicista Diana de la rapamicina 1 complejo en la formación de osteoclastos.

Video: A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Un enfoque optogenética para Evaluar formación de conexiones neuronales en un sistema de co-cultura

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Investigación

Biología del Desarrollo

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Un método enzimático para rescatar a las células madre mesenquimales de médula ósea Las muestras Clotted

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

Uso del análisis confocal de<em&gt; Xenopus laevis</em&gt; Encargado de investigar moduladores de Wnt y Shh morfógeno Degradados

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

Células Progenitoras de aislamiento Mesangiogenic (PSM) de Humana de Médula Ósea

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

Cultura de murinos embrionarias Metatarsianos: Un modelo fisiológico de osificación endocondral

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

El tratamiento de drogas y<em&gt; En Vivo</em&gt; Imagen de osteoblasto-osteoclastos Interacciones en un modelo Medaka pescado osteoporosis

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

Ordenamiento temporal de los datos de expresión dinámica de la expresión espacial detallada Mapas

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

La estimulación de la señalización de Notch en el ratón osteoclastos precursores

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

Un ensayo de transferencia de puntos de 5 mC cuantificando el nivel de metilación de ADN de la distredenciación de condrocitos<em&gt; In Vitro</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Bioingeniería de los microambientes humanizados de la médula ósea en el ratón y su visualización por imágenes en directo

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Una base de RANKL osteoclasto cultura ensayo de médula ósea de ratón para investigar el papel de mTORC1 en la formación de osteoclastos

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