Name: femoral bone marrow aspiration in live mice
Uploaded: 2014-07-05T15:00:00.000+00:00
Duration: 7 min 10 s
Description: Watch this Scientific Journal Video about Femoral Bone Marrow Aspiration in Live Mice at JoVE.com

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><tbody><tr><td>PBS</td><td>PAA</td><td>H15-002</td><td></td></tr><tr><td>Bovine serum albumin</td><td>PAA</td><td>K41-001</td><td></td></tr><tr><td>ACK lysis buffer</td><td>Homemade</td><td></td><td>in 1 L. Adjust pH 7.2 ~ 7.4 and filter sterile with 0.22 &amp;mu;m vacuum filter.</td></tr><tr><td>8.3 g Ammonium chloride</td><td>Fisher Scientific</td><td>A661-500</td><td></td></tr><tr><td>1 g Potassium bicarbonate</td><td>Fisher Scientific</td><td>P184-500</td><td></td></tr><tr><td>200 &amp;mu;l 0.5 M EDTA pH 8</td><td>Gibco</td><td>15575-038</td><td></td></tr><tr><td>RPMI 1640</td><td>PAA</td><td>E15-842</td><td></td></tr><tr><td>0.5 ml Tuberculin syringe 27.5 G</td><td>Becton Dickinson</td><td>305620</td><td></td></tr><tr><td>Sterile cell strainer 70 &amp;mu;m</td><td>Fisher Scientific</td><td>22363548</td><td></td></tr><tr><td>Isoflurane, USP</td><td>Attane</td><td>NDC:66794-014-25</td><td></td></tr><tr><td>Blunt-end needle</td><td>Stemcell Technologies</td><td>28110</td><td></td></tr><tr><td>PrecisionGlide needle 23 G</td><td>Becton Dickinson</td><td>305193</td><td></td></tr><tr><td>3 ml Syringe Luer-Lok tip</td><td>Becton Dickinson</td><td>309657</td><td></td></tr><tr><td>Non-tissue culture treated plate, 6 Well</td><td>Becton Dickinson</td><td>351146</td><td></td></tr><tr><td>12 x 75 mm 5 ml tubes&amp;nbsp;</td><td>Becton Dickinson</td><td>352054</td><td>FACS staining</td></tr><tr><td>12 x 75 mm 5 ml tubes with cell-strainer cap</td><td>Becton Dickinson</td><td>352235</td><td>FACS staining</td></tr><tr><td>NK1.1 APC-Cy7</td><td>Biolegend</td><td>108723</td><td></td></tr><tr><td>CD11b APC-Cy7</td><td>Biolegend</td><td>101225</td><td></td></tr><tr><td>CD45R (B220) APC-Cy7</td><td>Biolegend</td><td>103223</td><td></td></tr><tr><td>CD3 APC-Cy7</td><td>Biolegend</td><td>100222</td><td></td></tr><tr><td>Ly-6G and Ly-6C (Gr-1) APC-Cy7</td><td>Biolegend</td><td>108423</td><td></td></tr><tr><td>Ter119 APC-Cy7</td><td>Biolegend</td><td>116223</td><td></td></tr><tr><td>CD19 APC-Cy7</td><td>Biolegend</td><td>302217</td><td></td></tr><tr><td>CD4 APC-Cy7</td><td>BioLegend</td><td>317417</td><td></td></tr><tr><td>CD117 (c-KIT) PE</td><td>BioLegend</td><td>105808</td><td></td></tr><tr><td>Ly-6A/E (Sca-1) PE-Cy7</td><td>Biolegend</td><td>122513</td><td></td></tr><tr><td>CD34 APC</td><td>Biolegend</td><td>128612</td><td></td></tr><tr><td>CD16/32 e450</td><td>eBioscience</td><td>48-0161-82</td><td></td></tr><tr><td>DAPI (4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride)</td><td>Sigma-Aldrich</td><td>32670</td><td></td></tr><tr><td>MethoCult GF M3434</td><td>STEMCELLTECHNOLOGIES</td><td>3434</td><td>For methocellulose culture</td></tr><tr><td>Carprofen</td><td>Crescent Chemical Company</td><td>C11045850</td><td>1 dose (5mg/kg)&amp;nbsp;</td></tr><tr><td>Flow cytometer, LSRFortessa</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Puralube vet ointment (sterile petrolatum ophthalmic ointment)</td><td>Dechra-US</td><td>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...

Watch this Scientific Journal Video about Femoral Bone Marrow Aspiration in Live Mice at JoVE.com

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 ...

femoral-bone-marrow-aspiration-in-live-mice

Research

JoVE Journal

Medicine

37.8K 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

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Combina la microscopia Intravital fluorescente (IVFM) con modelos genéticos para estudiar dinámica del Engraftment de células hematopoyéticas a nichos de la médula ósea

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Investigación

Biología del Desarrollo

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Aislamiento y la inyección intravenosa de murino médula ósea derivados monocitos

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Inmunología e Infección

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Seguimiento de médula ósea de ratón monocitos<em&gt; En Vivo</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Fémur Window Cámara Modelo de<em&gt; En Vivo</em&gt; Seguimiento de la célula de la médula ósea murina

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Preparación de la médula ósea entera para el análisis de citometría de masa de células de linaje neutrófilo

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Procedimientos de trasplante de médula ósea en ratones para estudiar la hematopoyesis clonal

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-&#947;, IL-12 &#38; IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host&#39;s defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers.
This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases.
In this paper, we polarized the na&#239;ve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. ...

<meta charset="utf8"></head><body>Inyección intrafemoral para estudio de médula ósea en ratones vivos

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

<meta charset="utf8"></head><body>Las inyecciones intrafemorales simplificadas con ratones vivos permiten un análisis continuo de la médula ósea

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Las inyecciones intrafemorales simplificadas con ratones vivos permiten un análisis continuo de la médula ósea

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

Femoral Aspiración de médula ósea en ratones vivos

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Las inyecciones intrafemorales simplificadas con ratones vivos permiten un análisis continuo de la médula ósea

Las inyecciones intrafemorales simplificadas con ratones vivos permiten un análisis continuo de la médula ósea

Las inyecciones intrafemorales simplificadas con ratones vivos permiten un análisis continuo de la médula ósea