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.

在本协议中所述的所有动物的程序均按照指引实验动物的护理和使用，并进行批准的机构动物护理和使用委员会（IACUCs）在纪念Sloan-Kettering癌症中心。 骨髓1。抽吸（BM）从股骨细胞<ol><li>麻醉鼠标将经股骨骨髓穿刺与异氟醚（1-4％）给予精确气化器。麻醉深度，应注意，有与手术操作和/或耳部，趾部和尾部夹送相关呼吸速率没有变化来监测每5-10分钟整个过程。麻醉诱导异氟醚，并维持与异氟醚的程序。甲先发制人的预程序剂量的丁丙诺啡为0.05-0.1毫克/公斤的剂量经皮下每6-12小时，可用于防止与程序作为形容词有关的疼痛联合国国家工作队的卡洛芬。 </li><li>适用眼药膏鼠标下面的麻醉诱导的眼睛，以防止角膜干燥。 </li><li>皮草小心地从皮肤比预期的送气上门的面积约150％的面积被裁剪。宽松的皮草与湿润的纱布垫去除。请保持在一个循环热水垫或其他安全的恒温控制表面的鼠标，以防止在手术过程中体温降低。 </li><li>消毒包含将经抽吸三套交替磨砂（带有一个聚维酮碘（聚维酮碘）或洗必太（Nolvasan）擦洗和70％的异丙醇或70％乙醇浸泡的纱布海绵交替）股骨整个腿。 </li><li>抽吸骨髓前用无菌磷酸盐缓冲盐水（PBS）:;:湿0.5ml的结核菌素注射器（27.5克体积计0.5毫升）。填充注射器200-500微升PBS，并立即驱逐的PBS。重复此步骤2-3吨IME配合使用。 </li><li>通过无论是与无名指或者小指按压胫骨保持在胫骨弯曲从股骨。确认一个合适的麻醉平面已经实现。注射器用拇指和食指保持。这允许髁被暴露并便于插入针头。 </li><li>润湿注射器后，通过髌腱插入针，使针牢固地提出股骨髁2之间。通过保持骨干接近用拇指和食指股骨的骨骺，针被插入股骨的轴容易。 </li><li>旋转针向外和向上，这样它是与股骨的轴平行。这个动作有利于从股骨轴的骨髓内容检索。 </li><li>转动针顺时针和逆时针同时推动它慢慢地进入股骨髓腔。轻轻移动学年确认针的正确定位RINGE横向。 </li><li>轻轻拉出针柱塞回，产生负压，而将针BM腔内来回。注:BM吸气的量将通常对应于0.4-0.8×10 6单核细胞约5微升。成功的抽吸将视觉上的血液在注射器的底部的针顶端的外观来确认。如果没有血液被认为是在注射器它很可能是一个小骨或组织碎片卡在针。这可以从针通过移动活塞向上和向下的PBS中（这就是为什么在注射器应该用PBS吸出的BM之前预填充）被除去。如果组织不能从注射器脱落，使用一个新的针头和注射器（再次弄湿与200-500微升PBS的注射器）。 </li><li>一旦BM成功从股骨吸气，去除股骨和鼠标的针头和注射器。 </li><li>移动吸气BM到离心管公关efilled用500微升PBS。对于大多数应用中，骨髓细胞然后应保持在冰上，直到进一步的处理，如果可能的。 </li><li>下面的程序完成后，管理与镇痛卡洛芬5毫克/公斤皮下注射。然后从麻醉中移除鼠标放置在加热垫，直到完全康复。注意:不应该有并发症或遇险经历了以下愿望的过程，如果处理得当。 </li><li>返回小鼠住房面积之前，确保他们能够走动，达到食物和水。在接下来的24小时观察老鼠困扰或感染后的程序的迹象。症状包括:不断出血，贫血，嗜睡。如果任何这些迹象都看到程序后，动物（s）应实施安乐死。注意:BM抽吸/取样，可重复，但重复步骤应在相对的股骨以防止重复创伤同一腿进行。有关于FR多少信息equency该BM抽吸可以被执行。股骨骨髓穿刺，一般重复不超过每2周更频繁。 </li></ol>在吸气骨髓细胞细胞含量2。评估<ol><li>从骨髓抽吸和地点取回在离心管中离心300×g离心5分钟，在4℃ 或室温沉淀细胞。 </li><li>吸出上清液，然后重悬沉淀中加入500μl的ACK红细胞裂解缓冲液（"氯化铵钾"裂解缓冲液）。 </li><li>孵育的细胞中红细胞裂解液10分钟，然后加入1毫升的PBS并再次自旋向下的混合物在300×g离心5分钟，在4℃ 或室温。注:红细胞溶解沉淀现在由骨髓单个核细胞。这些可以被重悬浮于FACS染色，计数细胞，移植，细胞离心涂片器的分析和/或任何其它用途（正如骨髓细胞从处死小鼠收获的将被使用）。 </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>

作者什么都没有透露。

串行BM愿望是例行程序，血液系统疾病在人类临床研究的关键。以在整个实验中冗长的细胞组成和BM的成分表征执行BM的类似序列的采样在小鼠中的能力同样是非常有价值的。这个过程是对HSPC的表征有用而不牺牲小鼠而且，用于检测额外的细胞类型中的BM中存在的实例，其中的外周血的内容可能不反光居住在骨髓中的细胞。串行BM愿望已被用于非常有效地为这一目的后对监测人类细胞的存在异种移植到免疫功能低下小鼠为例3。鉴于0.4-0.8×10 6细胞通常检索与每个BM吸出，这些细胞可用于多种用途，包括细胞学分析（ 图1A），直径诺斯替流式细胞术（ 图1B），流式细胞术细胞分离接着进一步下游使用排序后的细胞，包括细胞（ 图1C）的培养，核酸提取，蛋白质提取测定面向有限的细胞数目的蛋白质组鉴定，甚至进一步移植提取细胞。同时，需要注意的是在每个所述股骨抽吸检索造血干细胞的数量是通过在此过程中检索到的单元的总数的限制是重要的。例如，考虑到造血干细胞的谱系阴性SCA1 + CKIT + CD150 + CD48-CD244-细胞的定义中，造血干细胞的频率为约10 HSCs/100，000细胞7。基于这个频率，约40-80造血干细胞有望被检索与每个抽吸过程。此外，如在图1C中所指出的，LSK和祖细胞的频率较低，尽管没有达到统计学显着性，在骨MARRO通过股骨骨髓穿刺获得瓦特细胞与传统的骨髓收获相比。我们相信，在这LSK相对减少和祖频率见过相对于手术的骨髓收获的愿望是与污染发生股骨骨髓穿刺外周血。在骨髓中的评估罕见的细胞群股骨骨髓穿刺这些限制应该注意。还有需要注意一点的是，我们已经典型地执行这个程序在6周龄以上小鼠。我们相信，这一程序将与小鼠小于6周龄越来越技术上具有挑战性。 这种技术也非常适合于评估在BM的嵌合体，而不是外周血中持续竞争的重组实验的情况下。在这些测定中，HSC的从一个实验组小鼠的官能电位对HSC与一组已知的数字测试＃8217; s的读出是外周血嵌合体以及HSC公司的嵌合体（见审查潘顿等[8]）。外周血嵌合体可以从嵌合在BM对比，如果影响到造血干细胞区室中的结果在HSPC的受损分化成成熟的循环血细胞的干扰。鉴于明确的造血不成立，直到移植后9和嵌合体的至少16周可能的时间10甚至更长的时间，技术，这有利于较早获得的HSPC隔室如这里所示的可能特别有用之后发生变化。最近的一个例子，骨髓细胞从小鼠由化酶Dnmt3a缺失小鼠外周血Challen 等。发现嵌合体减少，但在化酶Dnmt3a的HSC嵌合一种矛盾的纯合子增加产后删除化酶Dnmt3a竞争移植空HSC公司在BM 11。这个结果反映化酶Dnmt3a的分化缺陷- / -恒生综合的虽然增加了自我更新。此外，这一结果是在每16周的时间间隔只见在收件人移植小鼠的定时牺牲串行竞争力的移植实验。因此，分析其中允许骨髓细胞的采样中外周血并行而不需要鼠标的牺牲，让持续观察小鼠是非常有用的。 如前面提到的，这里展示了技术类似于用于intrafemoral直接注射到小鼠2,3的骨髓腔空间的技术。直接intrafemoral注射已被证明是在促进小鼠在那里它已被证明是提供改进的移植与静脉注射6,12,13直接比较人异种移植研究中非常有用。这种技术甚至允许香港海关识别以前未知的类的快速填充人类造血干细胞。 目前，它是不知道的BM小鼠通过相同的股骨重复抽样是否会影响骨髓或检索与反复的愿望骨髓细胞数量的细胞组成。此外，不知道时间的最小量最好从同一股骨执行重复抽吸过程。当在相同的鼠标进行重复股骨骨髓的愿望，交替使用BM愿望股骨和&gt; 2周的间隔进行串行骨髓抽吸。进一步的努力集中在理解的潜在影响，如果有的话，串行股骨骨髓吸出小鼠的类型和从骨髓或骨髓检索架构细胞数量的具体区间的程序是必要的。

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.

现场C57/B6小鼠股骨骨髓的愿望被用来获取骨髓单个核细胞其次是传统的BM收获相同的鼠标牺牲后的。通过这两种方法获得的骨髓单核细胞，然后通过骨髓细胞（1）的细胞学分析进行分析，（2）确定的造血干/祖细胞的相对频率（HSPCs），和（3） 体外排序HSPCs的培养。在后者的实验中，谱系阴性SCA1 +的c-kit +（LSK）细胞由骨髓抽吸以及从传统的骨髓收获得到的单核细胞进行排序。然后通过各种方法获得的150 LSK细胞涂布在含有粒细胞 - 红细胞的细胞因子7天，在技术重复的甲基纤维素半固体培养基。在图1 A中所示的数据，B示出了大容量电池和HSPCs的相似类型和比例是由形态学看到和使用任一股骨骨髓抽吸或常规骨髓收获的流式细胞分析。为了定量地确定LSK和髓系祖细胞的看到与传统的骨髓收获与股骨骨髓穿刺的百分比，我们比较了LSK和MP亚群的比例为总活细胞在3个独立的愿望和传统的骨髓收获的频率。这个数据，在图1C显示，揭示了LSK和MP骨髓亚群与传统的骨髓收获相比，收集股骨吸下降（尽管这种差异没有统计学任何人口显著）。 LSK细胞随后在体外培养中的甲基纤维素分拣导致类似的集落数，并通过股骨骨髓穿刺和常规骨髓收获得到的细胞类型（如显示在图1D）。观察和列举的殖民地类型包括CFU-GEMM（粒细胞，红细胞，单核细胞，megakaryocyte菌落形成单位），CFU-GM（粒细胞，单核细胞集落形成单位）和BFU-E（红细胞系爆裂形成单位）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51660/51660fig1highres.jpg" width="500" /> 图1股骨骨髓（BM）中抽吸活体小鼠，以方便检查骨髓内容，而不小鼠的牺牲。（A）赖特姬姆萨染色，通过活体小鼠的骨髓抽吸获得单个核细胞的细胞离心涂片。约0.4-0.8×10 6单核细胞可以在每个单独的股骨抽吸得到。这里显示的是典型的骨髓通过小鼠的牺牲者股骨骨髓抽吸（右）与常规骨髓细胞分离获得的野生型6周龄C57/B6小鼠的内容（左）（标尺为10微米）。（B）股骨骨髓的愿望提供细胞足够数量的用于评估的造血干/祖隔间换货所示，这里（右:细胞从骨髓吸出物;左:通过收获从处死小鼠的骨髓获得的细胞）。谱系阴性的Sca-1 + C-KIT +（LSK）由CD34和Fcγ受体的表达进行了分析定义含有造血干细胞以及谱系阴性的Sca-1的c-kit +骨髓祖细胞和它们的亚型细胞（亲栅极活，谱系阴性细胞）。所示百分比是指细胞的百分比内门。LSK（C）的定量（细胞，髓样祖（MP）的细胞，常见髓系祖细胞（谱系阴性的Sca-1的c-kit + CD34 +的FcγR中间+），粒细胞-巨噬细胞祖细胞（谱系阴性的Sca-1的c-kit + CD34 +的FcγR+）和巨核细胞，红系祖细胞（谱系阴性的Sca-1的c-kit + CD34-的FcγR-），如从常规骨髓收获活细胞的频率（n = 3）和股骨骨髓穿刺（N = 3）。误差棒代表标准日的甲基纤维素集落检测viation（D）代表的照片150 LSK细胞在含有甲基纤维素（上图）和数字和菌落类型的使用LSK细胞从每个方法观察髓/红系的电镀后的一个星期的效果。观察并计数菌落类型包括CFU-GEMM（粒细胞，红细胞，单核细胞，巨核细胞集落形成单位），CFU-GM（粒细胞，单核细胞集落形成单位）和BFU-E（红细胞系爆裂形成单位）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51660/51660fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</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.

股骨骨髓的愿望在活体小鼠

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 次观看.  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.

视频: Femoral Bone Marrow Aspiration in Live Mice

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

造血干细胞的自动定量 - 在脱钙骨的组织学图像间质细胞的相互作用

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

科研

发育生物学

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.

结合活体荧光显微镜 (IVFM) 和遗传模型研究造血细胞对骨髓生态位的植入动力学

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

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

医学

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.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

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

免疫与感染

髓系肿瘤中骨髓微环境的分离和表征

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

作者聚焦:分析小鼠血液系统恶性肿瘤的骨髓微环境 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

识别骨髓增生异常综合征和急性髓系白血病中的骨髓微环境群体

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

癌症研究

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.

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

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.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

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.

小鼠骨髓移植程序研究克隆造血症

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

股骨骨髓的愿望在活体小鼠

摘要

探索更多视频

此视频中的章节

造血干细胞的自动定量 - 在脱钙骨的组织学图像间质细胞的相互作用

结合活体荧光显微镜 (IVFM) 和遗传模型研究造血细胞对骨髓生态位的植入动力学

骨转移模型

跟踪小鼠骨髓单核细胞

识别骨髓增生异常综合征和急性髓系白血病中的骨髓微环境群体

识别骨髓增生异常综合征和急性髓系白血病中的骨髓微环境群体

识别骨髓增生异常综合征和急性髓系白血病中的骨髓微环境群体

股骨窗口商会模型

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

小鼠骨髓移植程序研究克隆造血症