O.A.-W. is supported by an NIH K08 Clinical Investigator Award (1K08CA160647-01), a U.S. Department of Defense Postdoctoral Fellow Award in Bone Marrow Failure Research (W81XWH-12-1-0041), the Josie Roberston Investigator Program, and a Damon Runyon Clinical Investigator Award with Support from the Evan&#8217;s Foundation.

Todos los animales procedimientos descritos en este protocolo se realizaron de conformidad con las Directrices para el Cuidado y Uso de Animales de Laboratorio y fueron aprobados por el Cuidado de Animales y el empleo Comisión Institucional (IACUC) en el Centro de Cáncer Memorial Sloan-Kettering. 1. Aspiración de médula ósea (MO) Las células del Fémur <ol><li> Se anestesia el ratón que se someterá a la aspiración de médula ósea femoral con isoflurano (1-4%), administrado con un vaporizador de precisión. Profundidad de la anestesia debe ser monitoreado cada 5-10 minutos durante todo el procedimiento por la observación de que no hay ningún cambio en la frecuencia respiratoria asociada con la manipulación quirúrgica y / o la oreja, dedo del pie, y de pinzamiento del rabo. La anestesia se indujo con isoflurano y también mantiene durante todo el procedimiento con isoflurano. Una dosis previa al procedimiento preventivo de la buprenorfina a una dosis de 0,05-0,1 mg / kg por vía subcutánea cada 6-12 h se puede utilizar para prevenir el dolor asociado con el procedimiento como un adjUNCT al Carprofeno. </li><li> Aplicar pomada oftálmica a los ojos del ratón después de la inducción de la anestesia para evitar el secado de la córnea. </li><li> La piel es cuidadosamente cortados por un área de piel de aproximadamente 150% mayor que el área de la zona de la aspiración previsto. Piel floja se elimina con una gasa húmeda. Por favor, mantenga el ratón sobre un relleno aislante de agua caliente que circula u otra superficie con control termostático de seguridad para prevenir la hipotermia durante el procedimiento. </li><li> Desinfectar la pierna entera que contiene el fémur, que se someterá a la aspiración con tres conjuntos de matorrales alterna (alternando con ya sea un povidona-yodo (Betadine) o una de clorhexidina (Nolvasan) de fregado y 70% de alcohol isopropílico o 70% de etanol empapado esponjas de gasa). </li><li> Moje un 0,5 ml de tuberculina jeringa (volumen: 0,5 cc; calibre: 27,5 G) con solución salina estéril tamponada con fosfato (PBS), antes de aspirar el BM. Llene la jeringa con 200-500 l de PBS y expulsar inmediatamente a la PBS. Repita este procedimiento 2-3 times. </li><li> Mantenga la inclinación de la tibia del fémur empujando la tibia, ya sea con el dedo anular o el dedo meñique. Confirme que un plano anestésico adecuado se ha alcanzado. La jeringa se lleva a cabo utilizando el pulgar y el dedo índice. Esto permite que los cóndilos a estar expuestos y facilita la inserción de la aguja. </li><li> Después de humedecer la jeringa, insertar la aguja a través del tendón rotuliano de manera que la aguja está alojada de forma segura entre los dos cóndilos del fémur. Por la celebración de la diáfisis cerca de la epífisis del fémur con el pulgar y el dedo índice, la aguja se inserta en el eje del fémur fácilmente. </li><li> Girar la aguja hacia fuera y hacia arriba de manera que es paralelo con el eje del fémur. Esta acción facilita la recuperación de los contenidos de la médula ósea del eje del fémur. </li><li> Gire la aguja hacia la derecha y hacia la izquierda mientras empuja lentamente en la cavidad medular femoral. Confirmar la colocación correcta de la aguja moviendo suavemente el SYRinge lateralmente. </li><li> Tire suavemente el émbolo de la aguja hacia atrás, creando una presión negativa, mientras se mueve la aguja de un lado a otro dentro de la cavidad del BM. Nota: El volumen de aspirado de BM será de aproximadamente 5 l que típicamente corresponde a 0,4-0,8 x 10 6 células mononucleares. El éxito de aspiración será confirmada visualmente por la aparición de sangre en la parte superior de la aguja en la base de la jeringa. Si no hay sangre se ve en la jeringa es probable que un fragmento de hueso o tejido pequeña se ha quedado atascado en la aguja. Esto puede ser retirado de la aguja moviendo el émbolo hacia arriba y hacia abajo en PBS (esta es una razón por la jeringa debe ser previamente llenada con PBS antes de aspirar el BM). Si el tejido no puede ser desalojado de la jeringa, use una nueva aguja y jeringa (de nuevo mojar la jeringa con 200-500 l de PBS). </li><li> Una vez que BM se aspira con éxito del fémur, retire la aguja y la jeringa del fémur y el ratón. </li><li> Mueva aspirado BM a un pr tubo de microcentrífugaefilled con 500 l de PBS. Para la mayoría de las aplicaciones, las células de la MO entonces deben mantenerse en hielo hasta su procesamiento posterior, si es posible. </li><li> Tras la finalización del procedimiento, administrar analgésico con carprofeno 5 mg / kg por vía subcutánea. A continuación, retire del ratón de la anestesia y colocar en un cojín calentado hasta que se recupere completamente. NOTA: No debe haber ninguna complicación o sufrimiento experimentado tras el procedimiento de aspiración si se hace correctamente. </li><li> Antes de devolver los ratones con el área de vivienda, aseguran que son capaces de deambular y llegar a los alimentos y el agua. Observar los ratones en busca de signos de malestar o procedimiento después de la infección en la siguiente 24 horas. Las señales incluyen: sangrado constante, anemia, letargo. Si alguno de estos síntomas son vistos después del procedimiento, el animal (s) debe ser sacrificado. Nota: la aspiración BM / muestreo se puede repetir, pero la repetición del procedimiento se debe realizar en el fémur opuesto para evitar traumatismos repetidos en la misma pierna. Hay poca información disponible sobre la frequency que la aspiración BM se puede realizar. Aspiración de médula ósea femoral generalmente se repite con más frecuencia de cada 2 semanas. </li></ol> 2. Evaluación del contenido celular en células de aspirado BM <ol><li> Precipitar las células recuperadas de BM aspiración y colocar en un tubo de microcentrífuga por centrifugación a 300 g durante 5 min a 4 ° C o RT. </li><li> Aspirar el sobrenadante y luego resuspender el sedimento en 500 l de ACK sangre roja tampón de lisis celular (&quot;amonio-cloruro de potasio&quot; tampón de lisis). </li><li> Se incuban las células en tampón de lisis de glóbulos rojos durante 10 min y a continuación, añadir 1 ml de PBS y centrifugar la mezcla de nuevo a 300 xg durante 5 min a 4 º C o temperatura ambiente. Nota: El pellet lisado de glóbulos rojos ahora consiste en células mononucleares de BM. Estos pueden ser resuspendidas para la tinción de FACS, el recuento de células, trasplante, análisis de citospina y / o cualquier otro uso (tal como se utilizarían las células BM recogidas de ratones sacrificados). </li></ol>

<ol>
	<li>Sundberg, R., Hodgson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspiration+of+bone+marrow+in+laboratory+animals.">Aspiration of bone marrow in laboratory animals.</a> Blood. 4, 557-561 (1949).</li><li>Schmitz, M., Bourquin, J. -. P., Bornhauser, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+technique+for+intrafemoral+injection+and+bone+marrow+sampling+in+mouse+transplant+models.">Alternative technique for intrafemoral injection and bone marrow sampling in mouse transplant models.</a> Leukemia &amp; lymphoma. 52, 1806-1808 (2011).</li><li>Warner, J. 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=Direct+evidence+for+cooperating+genetic+events+in+the+leukemic+transformation+of+normal+human+hematopoietic+cells.">Direct evidence for cooperating genetic events in the leukemic transformation of normal human hematopoietic cells.</a> Leukemia. 19, 1794-1805 (2005).</li><li>Chiu, P. P., Jiang, H., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukemia-initiating+cells+in+human+T-lymphoblastic+leukemia+exhibit+glucocorticoid+resistance.">Leukemia-initiating cells in human T-lymphoblastic leukemia exhibit glucocorticoid resistance.</a> Blood. 116, 5268-5279 (2010).</li><li>Guan, Y., Gerhard, B., Hogge, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+isolation,+and+stimulation+of+quiescent+primitive+leukemic+progenitor+cells+from+patients+with+acute+myeloid+leukemia+(AML).">Detection, isolation, and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML).</a> Blood. 101, 3142-3149 (2003).</li><li>Nolta, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gold+standard+improves:+a+better+assay+for+HSCs.">The gold standard improves: a better assay for HSCs.</a> Blood. 106, 1141-1142 (2005).</li><li>Kiel, M. J., 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=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121, 1109-1121 (2005).</li><li>Purton, L., Scadden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+factors+in+murine+hematopoietic+stem+cell+assays.">Limiting factors in murine hematopoietic stem cell assays.</a> Cell stem cell. 1, 263-270 (2007).</li><li>Dykstra, B., 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=Long-term+propagation+of+distinct+hematopoietic+differentiation+programs+in+vivo.">Long-term propagation of distinct hematopoietic differentiation programs in vivo.</a> Cell stem cell. 1, 218-229 (2007).</li><li>Jordan, C., Lemischka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+and+systemic+analysis+of+long-term+hematopoiesis+in+the+mouse.">Clonal and systemic analysis of long-term hematopoiesis in the mouse.</a> Genes &amp; development. 4, 220-232 (1990).</li><li>Challen, G., 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=Dnmt3a+is+essential+for+hematopoietic+stem+cell+differentiation.">Dnmt3a is essential for hematopoietic stem cell differentiation.</a> Nature genetics. 44, 23-31 (2012).</li><li>Hess, D. 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=Selection+based+on+CD133+and+high+aldehyde+dehydrogenase+activity+isolates+long-term+reconstituting+human+hematopoietic+stem+cells.">Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells.</a> Blood. 107, 2162-2169 (2006).</li><li>Mazurier, F., Doedens, M., Gan, O. I., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+myeloerythroid+repopulation+after+intrafemoral+transplantation+of+NOD-SCID+mice+reveals+a+new+class+of+human+stem+cells.">Rapid myeloerythroid repopulation after intrafemoral transplantation of NOD-SCID mice reveals a new class of human stem cells.</a> Nat Med. 9, 959-963 (2003).</li></ol>

Los autores no tienen nada que revelar.

De serie aspiración BM es un procedimiento de rutina crítica para investigación clínica de los trastornos hematológicos en seres humanos. La capacidad de realizar un muestreo en serie análoga de BM en ratones para la caracterización de la composición celular y componentes de la BM a lo largo de largos experimentos es también muy valiosa. Este procedimiento es útil para la caracterización de HSPC de sin sacrificar el ratón, sino también para detectar la presencia de tipos de células adicionales en el BM en ...

All blood cells are derived from hematopoietic stem cells (HSCs). Procedures which allow access to the bone marrow (BM), the site of the vast majority of HSC&#8217;s in mice and men, without requiring sacrifice of animals, provide a resource to monitor the source of hematopoiesis serially. The overall goal of the procedure described here is to provide a detailed protocol for the sampling of BM cells from the femur of live mice which provides material representative of the cells which would be retrieved by conventional harvest of BM cells through sacrifice of the mice. The advantage of this procedure over conventional harvest of BM cells is that this procedure does not require the sacrifice of mice and therefore allows for longitudinal study of the bone marrow compartment of mice over time. 
Although this murine bone marrow aspiration procedure has been used in a number of studies and described previously (see Sundberg et al. for a historical review1), formal step-by-step procedures illustrating this technique have not been previously published. This protocol enables routine serial sampling of the BM for purposes such as assessing engraftment in the BM of cells exogenously introduced into the mouse (as would be done in xenograft studies for instance2,3), analysis of chimerism in the BM for comparison with that of the peripheral blood (where results are not necessarily congruent), monitoring the abundance of specific cell types normally enriched in the marrow such as hematopoietic stem and progenitor cells, and retrieval of BM cells for ex vivo culture and/or transplantation. In addition, evaluation of marrow contents may be very useful in murine models of hematological disease as aspiration is helpful in assessing hematological disease burden at time points when disease may not be evident in the peripheral blood3-5. Moreover, this technique may be used to evaluate response of hematologic disease in the marrow to drugs administered in therapeutic studies. Thus, the procedure described here is ideal for investigators wishing to obtain access to bone marrow hematopoietic cells from mice for cytological analysis, flow cytometric analysis, in vitro culturing, and/or in vivo transplantation studies without requiring sacrifice or harm to the mice. 
The protocol for sampling of BM described here is quite similar to the technique used for intra-femoral injection of material directly into the marrow cavity3,6. The key difference being that this protocol details the procedure for removal of cells from the marrow as opposed to instilling material directly into the marrow cavity space as is done with intrafemoral injection.

<table border="0" cellpadding="0" cellspacing="0" width="963">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PBS</td>
			<td style="width:171px;">PAA</td>
			<td style="width:117px;">H15-002</td>
			<td style="width:407px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin</td>
			<td style="width:171px;">PAA</td>
			<td style="width:117px;">K41-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">ACK lysis buffer</td>
			<td style="width:171px;">Homemade</td>
			<td style="width:117px;"></td>
			<td>in 1 L. Adjust pH 7.2 ~ 7.4 and filter sterile with 0.22 &#956;m vacuum filter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">8.3 g Ammonium Chloride</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:117px;">A661-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">1 g Potassium Bicarbonate</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:117px;">P184-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">200 &#956;l 0.5M EDTA pH 8</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:117px;">15575-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">RPMI 1640</td>
			<td style="width:171px;">PAA</td>
			<td style="width:117px;">E15-842</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">0.5ml Tuberculin cyringe 27G 1/2</td>
			<td style="width:171px;">Becton Dickinson</td>
			<td style="width:117px;">305620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sterile cell strainer 70 &#956;m</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:117px;">22363548</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Isoflurane, USP</td>
			<td style="width:171px;">Attane</td>
			<td style="width:117px;">NDC:66794-014-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Blunt-End Needle</td>
			<td style="width:171px;">Stemcell Technologies</td>
			<td>28110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PrecisionGlide needle 23G</td>
			<td style="width:171px;">Becton Dickinson</td>
			<td style="width:117px;">305193</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">3ml syringe Luer-Lok Tip</td>
			<td style="width:171px;">Becton Dickinson</td>
			<td style="width:117px;">309657</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Non-Tissue Culture Treated Plate, 6 Well</td>
			<td style="width:171px;">Becton Dickinson</td>
			<td style="width:117px;">351146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">12 x 75 mm 5 ml tubes&#160;</td>
			<td style="width:171px;">Becton Dickinson</td>
			<td style="width:117px;">352054</td>
			<td>FACS staining</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">12 x 75 mm 5 ml tubes with cell-strainer cap</td>
			<td style="width:171px;">Becton Dickinson</td>
			<td style="width:117px;">352235</td>
			<td>FACS staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">NK1.1 APC-Cy7</td>
			<td>Biolegend</td>
			<td>108723</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">CD11b APC-Cy7</td>
			<td>Biolegend</td>
			<td>101225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">CD45R (B220) APC-Cy7</td>
			<td>Biolegend</td>
			<td>103223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">CD3 APC-Cy7</td>
			<td>Biolegend</td>
			<td style="width:117px;">100222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Ly-6G and Ly-6C (Gr-1) APC-Cy7</td>
			<td>Biolegend</td>
			<td style="width:117px;">108423</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Ter119 APC-Cy7</td>
			<td>Biolegend</td>
			<td>116223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">CD19 APC-Cy7</td>
			<td>Biolegend</td>
			<td>302217</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">CD4 APC-Cy7</td>
			<td>BioLegend</td>
			<td>317417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">CD117 (c-KIT) PE</td>
			<td>BioLegend</td>
			<td>105808</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Ly-6A/E (Sca-1) PE-Cy7</td>
			<td>Biolegend</td>
			<td style="width:117px;">122513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">CD34 APC</td>
			<td>Biolegend</td>
			<td style="width:117px;">128612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">CD16/32 e450</td>
			<td style="width:171px;">eBioscience</td>
			<td style="width:117px;">48-0161-82</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">DAPI (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td>32670</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">MethoCult GF M3434</td>
			<td style="width:171px;">STEMCELLTECHNOLOGIES</td>
			<td style="width:117px;">3434</td>
			<td>For methocellulose culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Carprofen</td>
			<td style="width:171px;">Crescent Chemical Company</td>
			<td>C11045850</td>
			<td>1 dose (5mg/kg)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Flow Cytometer, LSRFortessa</td>
			<td style="width:171px;">Becton Dickinson</td>
			<td style="width:117px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Puralube Vet Ointment (Sterile Petrolatum Ophthalmic Ointment)</td>
			<td style="width:171px;">Dechra-US</td>
			<td style="width:117px;">17033-211-38</td>
			<td></td>
		</tr>
	</tbody>
</table>

femoral bone marrow aspiration in live mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

Femoral BM aspiración de un ratón C57/B6 vivo se utilizó para obtener células mononucleares de BM seguido de la cosecha BM convencional del mismo ratón después del sacrificio. Células mononucleares BM obtenidos por los dos métodos fueron analizadas mediante (1) el análisis citológico de células de la MO, (2) determinar la frecuencia relativa de células madre / progenitoras hematopoyéticas (HSPCs), y (3) el cultivo ex vivo de HSPCs ordenados. En el experimento, Sca1 de linaje negativo últi...

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Femoral Bone Marrow Aspiration in Live Mice

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Femoral Aspiración de médula ósea en ratones vivos

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

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Research

JoVE Journal

Medicine

37.7K vistas.  Memorial Sloan-Kettering Cancer Center. This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Video: Femoral Bone Marrow Aspiration in Live Mice

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Usando cuantitativa en tiempo real PCR para determinar quimerismo de células donantes en un modelo competitivo Trasplante de médula ósea murina

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Investigación

Medicina

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Evaluación previa a la clínica de inhibidores de tirosina cinasa para el tratamiento de la leucemia aguda

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

A-crítico tamaño femoral Defecto sencillo modelo en ratones

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

El busulfán como agente mielosupresor para Generación estable de alto nivel de Médula Ósea quimerismo en ratones

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

En vivo de imágenes y Seguimiento de tecnecio-99m Etiquetada células madre mesenquimales de médula ósea en Equinos tendinopatía

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

Establecimiento de un defecto segmentario Modelo femoral de tamaño crítico en ratones estabilizado por osteosíntesis Plate

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

Guiada por la base de datos-citometría de flujo para la evaluación de la maduración de células mieloides de la médula

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

La generación de fracturas femorales cerradas en ratones: un modelo para el estudio del hueso curativo

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the lung. Barometric plethysmography is a classic technique to evaluate the lung volume with a long history of clinical use. Volumetric capnography utilizes the profile of exhaled carbon dioxide to determine the volume of the conducting airways, or dead space, and provides an index of airways homogeneity. These techniques may be used independently, or in combination to evaluate the dependence of airways volume and homogeneity on lung volume. This paper provides detailed technical instructions to replicate these techniques and our representative data demonstrates that the airways volume and homogeneity are highly correlated to lung volume. We also provide a macro for the analysis of capnographic data, which can be modified or adapted to fit different experimental designs. The advantage of these measures is that their advantages and limitations are supported by decades of experimental data, and they can be made repeatedly in the same subject without expensive imaging equipment or technically advanced analysis algorithms. These methods may be particularly useful for investigators interested in perturbations that change both the functional residual capacity of the lung and airways volume.

Here, we describe two measures of pulmonary function &#8211; barometric plethysmography, which allows the measurement of lung volume, and volumetric capnography, a tool to measure the anatomic dead space and airways uniformity. These techniques may be used independently or combined to assess airways function at different lung volumes.

Combina la capnografía volumétrica y la pletismografía barométrica para medir la relación estructura-función de pulmón

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Análisis de citometría de flujo de subconjuntos de células inmunitarias dentro del bazo murino, médula ósea, ganglios linfáticos y tejido sinovial en un modelo de osteoartritis

Methods Article

Femoral Aspiración de médula ósea en ratones vivos