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.

Alle in diesem Protokoll beschriebenen Tier Verfahren wurden in Übereinstimmung mit den Richtlinien für die Pflege und Verwendung von Labortieren durchgeführt und wurden von der Institutional Animal Care und Verwenden Committee (IACUCs) am Memorial Sloan-Kettering Cancer Center genehmigt. 1. Aspiration von Knochenmark (BM) Zellen aus dem Femur <ol><li> Anesthetize die Maus, die Oberschenkelknochenmarkpunktion mit Isofluran (1-4%) mit einer Genauigkeit Verdampfer verabreicht unterziehen wird. Narkosetiefe sollte alle 5-10 min durch die Beobachtung, dass es keine Veränderung der Atemfrequenz mit chirurgischen Manipulation und / oder Ohr, Zehen und Schwanz Prise verbunden überwacht werden während des gesamten Verfahrens. Anästhesie mit Isofluran induziert und auch während des gesamten Verfahrens mit Isofluran aufrechterhalten. Ein Bezugs vorprozessualer Dosis von Buprenorphin in einer Dosis von 0,05-0,1 mg / kg subkutan alle 6-12 Stunden können verwendet werden, um Schmerzen, die mit dem Verfahren als adj zugeordnet verhindernUNCT zu der Carprofen. </li><li> Anwenden Augensalbe für die Augen der Maus nach der Induktion der Anästhesie von Hornhaut Trocknen zu verhindern. </li><li> Das Fell wird vorsichtig von einem Hautbereich etwa 150% größer als die Fläche des beabsichtigten Aspiration Seite abgeschnitten. Lose Fell mit einem feuchten Gaze entfernt. Bitte halten Sie die Maus auf einem umlauf warmem Wasser Pad oder andere sichere thermostatisch gesteuerte Oberfläche zu Unterkühlung während des Verfahrens zu verhindern. </li><li> Desinfizieren Sie die gesamte Bein mit der Oberschenkelknochen, die Streben mit drei Sätzen von Wechsel Peelings (abwechselnd entweder mit einem PVP-Jod (Betadine) oder Chlorhexidin (Nolvasan) Peeling und 70% igem Alkohol oder 70% Ethanol getränkten Gaze Schwämme) unterzogen werden. </li><li> Befeuchten Sie ein 0,5 ml Tuberkulin-Spritze (Volumen: 0,5 cm ³; Weite: 27,5 G) mit steriler phosphatgepufferter Kochsalzlösung (PBS), bevor Absaugen des BM. Füllen Sie die Spritze mit 200-500 ul PBS und sofort vertreiben die PBS. Wiederholen Sie diesen Vorgang 2-3 times. </li><li> Halten Sie das Schienbein gebogen vom Oberschenkelknochen durch Drücken der Tibia entweder mit dem Ringfinger oder dem fünften Finger. Zu bestätigen, dass eine geeignete Anästhesieebene erreicht ist. Die Spritze wird mit dem Daumen und dem Zeigefinger gehalten wird. Dies ermöglicht die Kondylen ausgesetzt sein und erleichtert das Einführen der Nadel. </li><li> Nach Benetzung der Spritze und stechen Sie die Nadel durch die Patellasehne, sodass die Nadel sicher zwischen den beiden Kondylen des Oberschenkelknochens eingelegt. Durch Halten der Diaphyse der Nähe der Epiphyse des Femurs mit dem Daumen und dem Zeigefinger wird die Nadel in den Schaft des Oberschenkelknochens leicht eingeführt. </li><li> Schwenken der Nadel nach außen und oben, so daß sie parallel mit der Achse des Femurs ist. Diese Maßnahme erleichtert Abruf von Knochenmark Inhalte aus dem Femurschaft. </li><li> Drehen Sie die Nadel im Uhrzeigersinn und gegen den Uhrzeigersinn und schieben Sie sie langsam in die Oberschenkelmarkhöhle. Überprüfen Sie die korrekte Positionierung der Nadel durch leichtes Bewegen des syRinge seitlich. </li><li> Ziehen Sie die Nadel Kolben zurück, wodurch Unterdruck, während Sie die Nadel hin und her in der BM Höhle. Hinweis: Das Volumen des angesaugten BM ungefähr 5 &amp;mgr; l entspricht, die typischerweise 0,4-0,8 x 10 &amp;sup6; mononukleare Zellen sein. Erfolgreiche Aspiration wird visuell durch das Auftreten von Blut in der Spitze der Nadel in der Basis der Spritze bestätigt werden. Wenn kein Blut in der Spritze gesehen ist es wahrscheinlich, daß ein kleiner Knochen oder Gewebefragment in der Nadel stecken. Dies kann von der Nadel durch Bewegen des Kolbens nach oben und unten in PBS (dies ist ein Grund, warum der Spritze sollte mit PBS vor dem Absaugen des BM vorgefüllt werden) entfernt werden. Wenn das Gewebe nicht aus der Spritze verdrängt werden, verwenden Sie eine neue Nadel und Spritze (wieder nass die Spritze mit 200-500 ul PBS). </li><li> Sobald BM erfolgreich von der Oberschenkelknochen angesaugt, entfernen Sie die Nadel und Spritze aus dem Oberschenkelknochen und Maus. </li><li> Bewegen saugten BM zu einem Mikrozentrifugenröhrchen prmit 500 ul PBS efilled. Für die meisten Anwendungen, die BM-Zellen sollten dann auf Eis bis zur weiteren Verarbeitung, wenn möglich gehalten werden. </li><li> Nach Abschluss des Verfahrens, verwalten Analgetikum mit Carprofen 5 mg / kg subkutan. Entfernen Sie dann mit der Maus aus der Narkose und legen Sie auf einem Heizkissen, bis vollständig erholt. HINWEIS: Es sollte keine Komplikationen oder Stress erlebt nach der Aspiration Verfahren, wenn es richtig gemacht ist. </li><li> Vor der Rückkehr Mäuse mit dem Gehäuse-Bereich, sicherzustellen, dass sie in der Lage, ambulate und Nahrung und Wasser zu erreichen sind. Beachten Sie die Mäuse auf Anzeichen von Stress oder Infektionen nach dem Eingriff in der nächsten 24 Stunden. Symptome sind: konstant Blutungen, Anämie, Lethargie. Wenn eines dieser Zeichen sind nach dem Eingriff zu sehen ist, sollte das Tier (e) eingeschläfert werden. Hinweis: BM Aspiration / Probenahme kann wiederholt werden, aber der Vorgang wiederholen sollte auf der gegenüberliegenden Oberschenkelknochen wiederholt Trauma der gleichen Bein verhindern, durchgeführt werden. Es gibt wenig Informationen über die frequency dass BM Aspiration durchgeführt werden. Oberschenkelknochenmarkpunktion ist in der Regel nicht häufiger als alle 2 Wochen wiederholt. </li></ol> 2. Beurteilung der Zellgehalt in Aspirated BM Cells <ol><li> Pellet-Zellen aus BM Aspiration und in ein Mikrozentrifugenröhrchen durch Zentrifugation bei 300 g für 5 min abgerufen bei 4 ° C oder RT. </li><li> Saugt den Überstand und das Pellet in 500 &amp;mgr; l von roten Blutkörperchen ACK-Lysepuffer (&quot;Ammoniumchlorid-Kalium&quot; Lysepuffer). </li><li> Die Zellen in rot Zelllysepuffer Inkubation für 10 min und dann 1 ml PBS und Spin-Down der Mischung wieder bei 300 g für 5 min bei 4 ° C oder RT. Hinweis: Die roten Blutkörperchen lysiert Pellet besteht nun aus BM mononukleären Zellen. Diese können für FACS-Färbung, Zellzählung, Transplantation, Zytospin Analyse und / oder einer anderen Nutzung (wie BM-Zellen von Mäusen geerntet geopfert würde verwendet werden) resuspendiert werden. </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>

Die Autoren haben nichts zu offenbaren.

Serien BM Anspruch ist ein Routineverfahren entscheidend für die klinische Untersuchung von hämatologischen Erkrankungen beim Menschen. Die Fähigkeit, einen analogen seriellen Abtastung BM in Mäusen für die Charakterisierung der zellulären Zusammensetzung und Bestandteile BM gesamten langwierigen Experimente ebenfalls sehr wertvoll. Dieses Verfahren ist nützlich für die Charakterisierung von HSPC, ohne Einbußen bei der Maus, sondern auch zum Nachweis der Anwesenheit von zusätzlichen Zelltypen im Knochenmark in...

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.

Oberschenkel BM Aspiration von einer Live-C57/B6 Maus wurde genutzt, um BM mononuklearen Zellen, gefolgt von herkömmlichen BM Ernte der gleichen Maus nach der Opfer zu erhalten. BM mononukleären Zellen durch die beiden Verfahren erhalten wurden dann mit (1) zytologische Analyse der BM-Zellen untersucht, (2) Bestimmen der relativen Häufigkeit von hämatopoetischen Stammzellen / Vorläuferzellen (HSPCs) und (3) ex-vivo-Kultur von sortierten HSPCs. Im letzteren Experiment linien negativen Sca1 + c-Kit wurden + ...

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.

Oberschenkelknochenmark-Aspiration im Live-Mäuse

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 Aufrufe.  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.

Serie: 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.

Kombination von intravitalen Fluoreszenz-Mikroskopie (IVFM) mit genetischen Modellen Engraftment Dynamik der blutbildenden Zellen des Knochenmarks Nischen zu studieren

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

Forschung

Entwicklungsbiologie

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).

Isolierung und intravenöse Injektion von Maus-Knochenmark stammenden Monozyten

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

Immunologie und Infektion

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.

Tracking-Maus-Knochenmark-Monozyten<em&gt; In-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.

Femur Fensterkammer Modell für<em&gt; In Vivo</em&gt; Zelltracking im Knochenmark der Maus

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.

Vorbereitung des ganzen Knochenmarks für die Mass Cytometry Analyse von Neutrophilen-Linienzellen

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.

Knochenmarktransplantationsverfahren bei Mäusen zur Untersuchung der klonalen Hämatopoese

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>Intrafemorale Injektion für Knochenmarkstudie an lebenden Mäusen

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>Vereinfachte intrafemorale Injektionen mit lebenden Mäusen ermöglichen eine kontinuierliche Knochenmarkanalyse

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.

Vereinfachte intrafemorale Injektionen mit lebenden Mäusen ermöglichen eine kontinuierliche Knochenmarkanalyse

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

Oberschenkelknochenmark-Aspiration im Live-Mäuse

Zusammenfassung

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

Vereinfachte intrafemorale Injektionen mit lebenden Mäusen ermöglichen eine kontinuierliche Knochenmarkanalyse

Vereinfachte intrafemorale Injektionen mit lebenden Mäusen ermöglichen eine kontinuierliche Knochenmarkanalyse

Vereinfachte intrafemorale Injektionen mit lebenden Mäusen ermöglichen eine kontinuierliche Knochenmarkanalyse