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.

All animal procedures described in this protocol were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUCs) at Memorial Sloan-Kettering Cancer Center.
1. Aspiration of Bone Marrow (BM) Cells from the Femur
<ol>
	<li>Anesthetize the mouse which will undergo femoral bone marrow aspiration with isoflurane (1-4%) administered with a precision vaporizer. Depth of anesthesia should be monitored every 5-10 min throughout the procedure by observing that there is no change in respiratory rate associated with surgical manipulation and/or ear, toe, and tail pinch. Anesthesia is induced with isoflurane and also maintained throughout the procedure with isoflurane. A pre-emptive pre-procedural dose of buprenorphine at a dose of 0.05-0.1 mg/kg subcutaneously every 6-12 hr can be used to prevent pain associated with the procedure as an adjunct to the Carprofen.</li>
	<li>Apply ophthalmic ointment to the eyes of the mouse following induction of anesthesia to prevent corneal drying.</li>
	<li>The fur is carefully clipped from an area of skin approximately 150% larger than the area of the intended aspiration site. Loose fur is removed with a moist gauze pad. Please keep the mouse on a circulating warm water pad or other safe thermostatically controlled surface to prevent hypothermia during the procedure.</li>
	<li>Disinfect the entire leg containing the femur which will undergo aspiration with three sets of alternating scrubs (alternating with either a povidone-iodine (Betadine) or a chlorhexidine (Nolvasan) scrub and 70% isopropyl alcohol or 70% ethanol soaked gauze sponges).</li>
	<li>Wet a 0.5 ml Tuberculin Syringe (volume: 0.5 cc; gauge: 27.5 G) with sterile phosphate-buffered saline (PBS) before aspirating the BM. Fill the syringe with 200-500 &#956;l of PBS and immediately expel the PBS. Repeat this procedure 2-3 times.</li>
	<li>Keep the tibia bent from the femur by pushing the tibia with either the ring finger or the fifth finger. Confirm that a suitable anesthetic plane has been attained. The syringe is held using the thumb and the index finger. This allows the condyles to be exposed and facilitates insertion of the needle. </li>
	<li>After wetting the syringe, insert the needle through the patellar tendon so that the needle is lodged securely between the two condyles of the femur. By holding the diaphysis close to the epiphysis of the femur with the thumb and the index finger, the needle is inserted into the shaft of the femur easily.</li>
	<li>Swivel the needle outward and upward so that it is parallel with the shaft of the femur. This action facilitates retrieval of bone marrow contents from the femur shaft.</li>
	<li>Turn the needle clockwise and counterclockwise while pushing it slowly into the femoral marrow cavity. Confirm the correct positioning of the needle by gently moving the syringe laterally.</li>
	<li>Gently pull the needle plunger back, creating negative pressure, while moving the needle back and forth within the BM cavity. Note: The volume of BM aspirated will be approximately 5 &#181;l which typically corresponds to 0.4-0.8 x 106 mononuclear cells. Successful aspiration will be confirmed visually by the appearance of blood in the top of the needle in the base of the syringe. If no blood is seen in the syringe it is likely that a small bone or tissue fragment is stuck in the needle. This can be removed from the needle by moving the plunger up and down in PBS (this is one reason why the syringe should be prefilled with PBS before aspirating the BM). If the tissue cannot be dislodged from the syringe, use a new needle and syringe (again wet the syringe with 200-500 &#181;l of PBS).</li>
	<li>Once BM is successfully aspirated from the femur, remove the needle and syringe from the femur and mouse.</li>
	<li>Move aspirated BM to a microfuge tube prefilled with 500 &#181;l of PBS. For most applications, the BM cells should then be kept on ice until further processing if possible.</li>
	<li>
	 Following completion of the procedure, administer analgesic with carprofen 5 mg/kg subcutaneously. Then remove mouse from the anesthesia and place on a heated pad until fully recovered. NOTE: There should be no complication or distress experienced following the aspiration procedure if done properly.
	</li>
	<li>
	 Before returning mice to the housing area, ensure they are able to ambulate and reach food and water. Observe the mice for signs of distress or infection post procedure in the next 24 hr. Signs include: constant bleeding, anemia, lethargy. If any of these signs are seen post procedure, the animal(s) should be euthanized. Note: BM aspiration/sampling can be repeated but the repeat procedure should be performed on the opposite femur to prevent repeated trauma to the same leg. There is little information available regarding the frequency that BM aspiration can be performed. Femoral bone marrow aspiration is generally repeated no more frequently than every 2 weeks.
	</li>
</ol>
2. Assessment of Cellular Content in Aspirated BM Cells
<ol>
	<li>Pellet cells retrieved from BM aspiration and place in a microfuge tube by centrifugation at 300 x g for 5 min at 4 oC or RT.</li>
	<li>Aspirate the supernatant and then resuspend the pellet in 500 &#181;l of ACK red blood cell lysis buffer (&#8220;ammonium-chloride-potassium&#8221; lysis buffer).</li>
	<li>Incubate the cells in red cell lysis buffer for 10 min and then add 1 ml of PBS and spin down the mixture again at 300 x g for 5 min at 4 oC or RT. Note: The red blood cell lysed pellet now consists of BM mononuclear cells. These can be resuspended for FACS staining, cell counting, transplantation, cytospin analysis and/or any other use (just as BM cells harvested from sacrificed mice would be used).</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>

The authors have nothing to disclose.

Serial BM aspiration is a routine procedure critical to clinical investigation of hematologic disorders in humans. The ability to perform an analogous serial sampling of BM in mice for characterization of the cellular composition and constituents of BM throughout lengthy experiments is likewise very valuable. This procedure is useful for characterization of HSPC&#8217;s without sacrificing the mouse but also for detecting the presence of additional cell types in the BM in instances where the contents of the peripheral blood may not be reflective of the cells residing in the BM. Serial BM aspiration has been used very effectively for this later purpose in monitoring the presence of human cells xenografted into immunocompromised mice for instance3. Given that 0.4-0.8 x 106 cells are typically retrieved with each BM aspiration, these cells can be utilized for a number of purposes including cytological analysis (Figure 1A), diagnostic flow cytometry (Figure 1B), flow cytometric cell separation followed by further downstream use of sorted cells including culture of cells (Figure 1C), nucleic acid extraction, protein extraction for assays geared towards proteomic characterization of limited cell numbers, and even further transplantation of extracted cells. At the same time, it is important to note that the number of HSCs retrieved in each femoral aspiration is limited by the overall number of cells retrieved in this procedure. For instance, considering the definition of HSCs as lineage-negative Sca1+ cKIT+ CD150+ CD48- CD244- cells, the frequency of HSCs is approximately 10 HSCs/100,000 cells7. Based on this frequency, approximately 40-80 HSCs is expected to be retrieved with each aspiration procedure. Also, as noted in Figure 1C, the frequency of LSK and progenitor cells is lower, albeit not reaching statistical significance, in bone marrow cells obtained via femoral bone marrow aspiration compared with conventional bone marrow harvest. We believe this relative decrease in LSK and progenitor frequency seen with aspiration relative to surgical bone marrow harvest is related to contamination with peripheral blood that occurs with femoral bone marrow aspiration. These limitations of femoral bone marrow aspiration in evaluating rare cell populations in the marrow should be noted. One additional note is that we have typically performed this procedure in mice of 6 weeks of age or older. We believe that this procedure would be increasingly technically challenging with mice less than 6 weeks of age.
This technique is also well suited to assess the chimerism of the BM as opposed to the peripheral blood in the context of an ongoing competitive reconstitution experiment. In these assays, the functional potential of HSC&#8217;s from an experimental set of mice is tested against a set known number of HSC&#8217;s with the readout being peripheral blood chimerism as well as chimerism of HSC&#8217;s (see review by Purton et al.8). Chimerism of the peripheral blood may contrast from the chimerism in the BM if perturbations affecting the hematopoietic stem cell compartment result in impaired differentiation of HSPC&#8217;s into mature circulating blood cells. Given that definitive hematopoiesis is not established until at least 16 weeks following transplantation9 and that chimerism may change after even longer periods of time10, techniques which facilitate earlier access to the HSPC compartment such as illustrated here may be particularly useful. In one recent example, competitive transplantation of BM cells from mice with homozygous post-natal deletion of Dnmt3a by Challen et al. revealed reduced chimerism from Dnmt3a-null mice in the peripheral blood but a paradoxical increase in HSC chimerism of Dnmt3a-null HSC&#8217;s in the BM11. This result was indicative of a differentiation defect of Dnmt3a-/- HSC&#8217;s despite an increase in self-renewal. Moreover, this result was seen only at timed sacrifice of recipient transplanted mice in serial competitive transplantation experiments at intervals of every 16 weeks. Therefore assays which allow for sampling of BM cells in parallel with peripheral blood without requiring sacrifice of the mice and allowing ongoing observation of mice are quite useful.
As mentioned earlier, the technique demonstrated here is similar to the technique utilized for intrafemoral injection directly into the marrow cavity space of mice2,3. Direct intrafemoral injection has proven to be very useful in facilitating human xenograft studies in mice where it has been shown to provide improved engraftment in direct comparison with intravenous injection6,12,13. This technique has even allowed for identification of previously unknown class of rapidly populating human HSCs.
Currently it is not known whether repeated sampling of the BM in mice through the same femur influences the cellular composition of the BM or number of marrow cells retrieved with repeated aspirations. Moreover, the minimum amount of time advisable to perform repeat aspiration procedures from the same femur is not known. When performing repeated femoral bone marrow aspirations in the same mouse, alternate the femur used for BM aspiration and perform serial BM aspirates at intervals of &#62;2 weeks. Further efforts focused on understanding the potential effects, if any, of serial femoral BM aspiration procedures in mice at specific intervals on the types and number of cells retrieved from BM or architecture of the BM are needed.

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 aspiration of a live C57/B6 mouse was utilized to obtain BM mononuclear cells followed by conventional BM harvest of the same mouse after sacrifice. BM mononuclear cells obtained by the two methods were then analyzed by (1) cytological analysis of BM cells, (2) determining the relative frequency of hematopoietic stem/progenitor cells (HSPCs), and (3) ex vivo culture of sorted HSPCs. In the latter experiment, lineage-negative Sca1+ c-KIT+ (LSK) cells were sorted from mononuclear cells obtained by BM aspiration as well as from conventional BM harvest. 150 LSK cells obtained by each method were then plated in methylcellulose semi-solid media containing myeloid-erythroid cytokines for 7 days in technical duplicate. The data shown in Figure 1 A,B illustrates that similar types and proportions of bulk cells and HSPCs were seen by morphologic and flow cytometric analysis using either femoral BM aspiration or conventional BM harvest.
In order to quantitatively determine the percentage of LSK and myeloid progenitor cells seen with conventional bone marrow harvest versus femoral bone marrow aspiration, we compared the percentage of LSK and MP subpopulations as a frequency of total live cells in 3 independent aspirations and conventional marrow harvests. This data, displayed in Figure 1C, reveals a decrease in LSK and MP subpopulations from bone marrow collected by femoral aspiration compared with conventional marrow harvest (although this difference was not statistically significant for any population).
Sorting of LSK cells followed by ex vivo culture in methylcellulose resulted in similar colony numbers and cell types obtained by femoral bone marrow aspiration and conventional BM harvest (as shown in Figure 1D). The colony types observed and enumerated include CFU-GEMM (the granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming unit), CFU-GM (the granulocyte, monocyte colony forming unit), and BFU-E (erythroid burst-forming unit).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51660/51660fig1highres.jpg" width="500" /> 
Figure 1. Femoral bone marrow (BM) aspiration in live mice to facilitate examination of marrow contents without sacrifice of mice. (A) Wright Giemsa stained cytospins of mononuclear cells obtained through BM aspiration of live mice. Approximately 0.4-0.8 x 106 mononuclear cells may be obtained in each individual femoral aspiration. Shown here are typically marrow contents of wildtype 6-week old C57/B6 mice obtained by femoral BM aspiration (right) versus conventional BM cell isolation through sacrifice of mice (left) (scale bar represents 10 &#181;m). (B) Femoral BM aspiration provides an adequate number of cells for assessment of the hematopoietic stem/progenitor compartment as illustrated here (Right: cells from BM aspirate; Left: cells obtained by harvesting of marrow from sacrificed mice). Lineage-negative Sca-1+ c-KIT+ (LSK) cells containing hematopoietic stem cells as well as lineage-negative Sca-1- c-KIT+ myeloid progenitors and their subtypes as defined by CD34 and Fc&#947;R expression were analyzed (parent gate is living, lineage-negative cells). Percentages shown refer to percent of cells within gate. (C) Quantification of LSK (cells, myeloid progenitor (MP) cells, common myeloid progenitors (lineage-negative Sca-1- c-KIT+ CD34+ Fc&#947;R intermediate+), granulocyte-macrophage progenitor cells (lineage-negative Sca-1- c-KIT+ CD34+ Fc&#947;R+), and megakaryocyte-erythroid progenitor cells (lineage-negative Sca-1- c-KIT+ CD34- Fc&#947;R-), as a frequency of live cells from conventional marrow harvest (n=3) and femoral bone marrow aspiration (n=3). Error bars represent standard deviation. (D) Representative photographs of methylcellulose colony assay results one week after plating of 150 LSK cells in myeloid/erythroid containing methylcellulose (above) and numbers and types of colonies observed using LSK cells from each method. The colony types observed and enumerated include CFU-GEMM (the granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming unit), CFU-GM (the granulocyte, monocyte colony forming unit), and BFU-E (erythroid burst-forming unit). <a href="https://www.jove.com/files/ftp_upload/51660/51660fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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.

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

Nanyang Technological University

Research

JoVE Journal

Medicine

37.8K Views.  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.

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

Developmental Biology

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).

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

Immunology and Infection

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.

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.

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.

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.

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. ...

Intrafemoral Injection for Bone Marrow Study in Live Mice

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.

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.

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

Isolation and Culture of Bone Marrow Stromal Cells from a Mouse Tibia

Source: Maridas, D. E., et al. <a href="https://app.jove.com/v/56750">Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.</a> J. Vis. Exp.(2018)
In this video, we demonstrate the isolation and culture of bone marrow stromal cells from the long bones of mice. The isolated adherent BMSCs can be used for further analysis.

Izolacja i hodowla komórek zrębu szpiku kostnego z kości piszczelowej myszy

Source: Maridas, D. E., et al. Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice. J. Vis. ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Isolation and Culture

Bone Marrow Harvest from Mouse Hind Limb

Source: Zhu, Y. P., Padgett, et al. <a href="https://www.jove.com/t/59617">Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells.</a> J. Vis. Exp. (2019).
This video describes a detailed protocol to isolate fresh whole bone marrow from the mouse&#39;s hind limbs to investigate whole BM defects for patients with blood disorders such as leukemia.

Pobieranie szpiku kostnego z tylnej kończyny myszy

Source: Zhu, Y. P., Padgett, et al. Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells. J. Vis. Exp. (2019).
This ...

Femoral Bone Marrow Aspiration in Live Mice

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

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

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Tracking Mouse Bone Marrow Monocytes In Vivo

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

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

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

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

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

Izolacja i hodowla komórek zrębu szpiku kostnego z kości piszczelowej myszy

Pobieranie szpiku kostnego z tylnej kończyny myszy