This work was supported by the DFG (Deutsche Forschungsgemeinschaft, German Research Foundation) SFB 854 (Sonderforschungsbereich, collaborative research center). 
Thanks to Hans-Holger G&#228;rtner, Audiovisuelles Medienzentrum, Otto-von-Guericke University Magdeburg, Magdeburg, Germany, for technical support.

与萨克森州和萨克森 - 安哈尔特州，Regierungspraesidium德累斯顿/哈勒的国家批准进行这项研究，根据德国法律的保护动物（24D-9168.11-1 / 2008-24）第8。 1细胞分离 1.1准备股骨和胫骨的<ol><li>用5％异氟烷浓度通过在一个封闭的仓汽化异氟烷麻醉小鼠。后的小鼠停止移动，持续3秒，执行颈椎脱位。 </li><li>消毒后肢用乙醇（96％）。去除皮肤和肌肉分开的骨头用无菌手术刀。 <ol><li>开始与腹股沟皮肤切口，并延伸朝向内踝切口并执行围绕下部小腿皮肤的圆形解剖。由近端脱皮彻底去除腿部的皮肤。 </li><li>从分离用锋利的手术刀股骨股四头肌，同时删除前腓骨和腓肠肌肌肉群并通过定位和解剖韧带disarticulate腿在髋关节。 </li><li>启动向前，继续关节周围小心地旋转腿和横断韧带，从而脱节的联合。 </li></ol></li><li>洗股骨和胫骨用96％的乙醇最少90秒，以保证无菌细胞制剂。继续洗涤过程中使用无菌PBS冲洗掉剩余的乙醇。 注意：所有的后续步骤必须是无菌的，以避免污染。 </li></ol> 1.2收获骨髓<ol><li>切割每个骨的近端和远端具有一对细剪刀来访问的股骨和胫骨的轴。小心这一步，因为这些骨头断裂非常容易。 </li><li>冲洗骨头用温介质（M199 + 10％胎牛血清（FCS），+ 1％青霉素/链霉素）。使用无菌28-G针，1ml注射器，和10-15毫升培养基每间骨冲洗。 </li><li>握住骨用细镊子和，直到它变成半透明一个冲洗之一。小心施加压力的注射器柱塞的端部，以减少细胞应激。 </li><li>通过70微米尼龙网过滤骨髓并收集滤液。以提取骨髓的最大数量，从所述远端和近端反复冲洗。 </li><li>冲洗用PBS筛，并收集滤液。小心撞出的碎片和细胞集团轻轻搅拌和移液。 </li></ol> 1.3栽培<ol><li>离心细胞悬浮液在250×g离心在室温10分钟。弃上清，悬浮细胞与大约25毫升介质。重复一次。 </li><li>第二清洗步骤之后重悬细胞在6ml培养基。混合50微升的细胞溶液与50μl的台盼蓝。计数细胞在计数室在光学显微镜下。计算C的数使用此公式中的溶液ELLS： <img alt="式（1）" fo:content-width="1.3in" src="/files/ftp_upload/52347/52347eq1.jpg" /></li><li>种子上6孔超低附着板表面的细胞，以防止永久附着在板的底部。使用每毫升10 6个细胞的浓度以每孔最多至6ml。 </li><li>补充悬架20毫微克/毫升的M-CSF，促进细胞分化。 </li><li>培养在37℃下将细胞5天，5％二氧化碳和每日观察。 </li></ol>细胞收获1.4 注意：这些步骤应在冰上进行。继续无论1.4.1或1.4.2相应。 <ol><li>在这一点上，收获整个培养，包括分化的巨噬细胞，将粘附到培养板，即使它们是超低附着表面板。 <ol><li>通过温和移液收获整个培养和4℃的PB中的溶液分离S，0.5％牛血清白蛋白（BSA）和2mM EDTA中。重复，直到这些板块都清楚剩余的贴壁细胞 - 确认在显微镜下，如果不能确定。 </li></ol></li><li>可替代地，通过收获与细胞在悬浮液中，其中主要含有单核细胞的上清液丢弃粘附的巨噬细胞。 <ol><li>收获非贴壁细胞用的​​无EDTA的PBS中的溶液和0.5％牛血清白蛋白。不适用为了避免撞出轻度粘连的巨噬细胞用力过猛。 </li></ol></li></ol> 1.5 FACS分析（可选） 注意：可以通过使用MACS的耗尽CD117 +茎和祖细胞的细胞悬浮液。使用制造商协议，在此过程中。 <ol><li>根据要么步骤1.4.1或1.4.2收获细胞。 </li><li>离心细胞，在250×g离心10分钟，在4℃，并再次重复该步骤。 </li><li>悬浮至少250,000个细胞在300微升FACS的Buffer每个探头。 </li><li>转移的细胞溶液到96孔板（30每孔微升）。 </li><li>离心细胞悬浮液在250×g离心5分钟，在4℃。 </li><li>除去上清液，并添加25微升抗体，每孔（使用制造商稀释度）。 </li><li>染色1.5.1-1.5.6将细胞与抗体（按照生产商协议后步骤的细胞表型。常用标记的单核细胞/巨噬细胞识别包括的CD11b，F4 / 80（ 图3），CD115（图2），和GR- 1。 注意：对于小区的解决方案的进一步的描述中，使用象CD4，CD8A，的CD11c，CD25，CD45，CD45R，CD62L，CD80，CD86，CD145，CD117，MHCⅡ，的Ly6C，Ly6G和CD192标记的建议。蜂窝特性进行先前描述的12。 </li><li>孵育探针20分钟，在4℃。 </li><li>离心机的探针，在250×g离心5分钟，在4℃下重悬用200μl的FACS缓冲液中。重复次是步两次。 </li><li>转移探讨FACS管并进行FACS分析12。 </li><li>执行在第0天，第3和5 FACS分析12来分析细胞的分化。 </li></ol> 2.尾静脉注射 2.1准备<ol><li>暖在加热板上的动物，在37℃进行约10分钟。观察动物，同时升温至识别过热的迹象。 </li><li>悬浮的单核细胞，分离的步骤1.1- 1.4，在150微升的NaCl，并加载细胞到1 ml胰岛素注射器用30G针。装载注射器，以确保所有的单核细胞被注射之前轻轻旋涡振荡溶液。总是处理冰上细胞，避免热介导的附着和激活单核细胞。 </li></ol> 2.2抑制<ol><li>避免在选择鼠标静脉注射干扰其他老鼠在笼子里。 </li><li>将鼠标放到限制器。避免过多公关essure和小心的后肢。 （ 图4） 注：另外，使用全身麻醉，以保证无压力的动物处理。 </li><li>确保鼠标有呼吸的空间关闭限制器之前。 </li></ol> 2.3注塑<ol><li>消毒注射部位以消毒剂。喷在小鼠的尾部，静置至少1分钟。 </li><li>通过应用温和的压力，侧尾侧注射部位以上，为更好的可视性静脉停止尾部静脉血流。 </li><li>打开尾90度，注射前。 （ 图5） </li><li>注入以45度角。慢慢的注入，也没有每克超过5微升，以免伤及动物。 </li><li>如果有一个泡罩，这是一个失败的注入的标志，立即停止和更近侧重复注射。 </li><li>通过应用柔和的P止血注射侧ressure约1分钟。 </li><li>在该过程结束时，打开该卷收器，并放置在鼠标在其笼中。 </li><li>观察小鼠20分钟，以确保动物没有被注射伤害和胸骨斜卧被保持。延长观察时间，直到鼠标重新获得足够的意识。返回鼠标给其他动物的公司只后它已完全恢复。 </li></ol>

<ol>
	<li>Herold, 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=Transplantation+of+monocytes:+a+novel+strategy+for+in+vivo+augmentation+of+collateral+vessel+growth.">Transplantation of monocytes: a novel strategy for in vivo augmentation of collateral vessel growth.</a> Hum Gene Ther. 15 (1), 1-12 (2004).</li><li>Herold, J., Tillmanns, H., Xing, Z., Strasser, R. H., Braun-Dullaeus, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+transduction+of+monocytes:+promising+vehicles+for+therapeutic+arteriogenesis.">Isolation and transduction of monocytes: promising vehicles for therapeutic arteriogenesis.</a> Langenbecks Arch Surg. 391 (2), 72-82 (2006).</li><li>Francke, A., Weinert, S., Strasser, R. H., Braun-Dullaeus, R. C., Herold, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+bone+marrow+derived+monocytes:+a+novel+approach+for+augmentation+of+arteriogenesis+in+a+murine+model+of+femoral+artery+ligation.">Transplantation of bone marrow derived monocytes: a novel approach for augmentation of arteriogenesis in a murine model of femoral artery ligation.</a> American Journal Of Translational Research. 5 (2), 155-169 (2013).</li><li>Leon, 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=Dendritic+cell+differentiation+potential+of+mouse+monocytes:+monocytes+represent+immediate+precursors+of+CD8-+and+CD8++splenic+dendritic+cells.">Dendritic cell differentiation potential of mouse monocytes: monocytes represent immediate precursors of CD8- and CD8+ splenic dendritic cells.</a> Blood. 103 (7), 2668-2676 (2004).</li><li>Timmerman, J. M., Levy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+vaccines+for+cancer+immunotherapy.">Dendritic cell vaccines for cancer immunotherapy.</a> Annu Rev Med. 50, 507-529 (1999).</li><li>Salem, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+dendritic+cells+for+Peptide-based+vaccination+in+cancer+immunotherapy.">The use of dendritic cells for Peptide-based vaccination in cancer immunotherapy.</a> Methods Mol Biol. 1139, 479-503 (2014).</li><li>Timmerman, J. M., 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=Idiotype-pulsed+dendritic+cell+vaccination+for+B-cell+lymphoma:+clinical+and+immune+responses+in+35+patients.">Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune responses in 35 patients.</a> Blood. 99 (5), 1517-1526 (2002).</li><li>Berthold, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+monocytes+by+Ficoll+density+gradient+centrifugation.">Isolation of human monocytes by Ficoll density gradient centrifugation.</a> Blut. 43 (6), 367-371 (1981).</li><li>Houthuys, E., Movahedi, K., De Baetselier, P., Van Ginderachter, J. A., Brouckaert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+and+purification+of+mouse+peripheral+blood+monocytes.">A method for the isolation and purification of mouse peripheral blood monocytes.</a> Journal Of Immunological Methods. 359 (1-2), 1-10 (2010).</li><li>Seeger, F. H., Tonn, T., Krzossok, N., Zeiher, A. M., Dimmeler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+isolation+procedures+matter:+a+comparison+of+different+isolation+protocols+of+bone+marrow+mononuclear+cells+used+for+cell+therapy+in+patients+with+acute+myocardial+infarction.">Cell isolation procedures matter: a comparison of different isolation protocols of bone marrow mononuclear cells used for cell therapy in patients with acute myocardial infarction.</a> European Heart Journal. 28 (6), 766-772 (2007).</li><li>Auffray, C., 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=CX3CR1++CD115++CD135++common+macrophage/DC+precursors+and+the+role+of+CX3CR1+in+their+response+to+inflammation.">CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation.</a> The Journal Of Experimental Medicine. 206 (3), 595-606 (2009).</li><li>Francke, A., Herold, J., Weinert, S., Strasser, R. H., Braun-Dullaeus, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mature+murine+monocytes+from+heterogeneous+bone+marrow+and+description+of+their+properties.">Generation of mature murine monocytes from heterogeneous bone marrow and description of their properties.</a> J. Histochem. Cytochem. 59 (9), 813-825 (2011).</li><li>Sneider, E. B., Nowicki, P. T., Messina, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+in+the+treatment+of+peripheral+arterial+disease.">Regenerative medicine in the treatment of peripheral arterial disease.</a> Journal Of Cellular Biochemistry. 108 (4), 753-761 (2009).</li><li>Van Royen, N., Piek, J. J., Schaper, W., Fulton, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+review+of+clinical+arteriogenesis+research.">A critical review of clinical arteriogenesis research.</a> J Am Coll Cardiol. 55 (1), 17-25 (2009).</li><li>Lawall, H., Bramlage, P., Amann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+peripheral+arterial+disease+using+stem+and+progenitor+cell+therapy.">Treatment of peripheral arterial disease using stem and progenitor cell therapy.</a> J Vasc Surg. 53 (2), 445-453 (2011).</li><li>Herold, 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=Tetanus+toxoid-pulsed+monocyte+vaccination+for+augmentation+of+collateral+vessel+growth.">Tetanus toxoid-pulsed monocyte vaccination for augmentation of collateral vessel growth.</a> Journal of the American Heart Association. 3 (2), e000611 (2014).</li></ol>

The authors declare that they have no competing financial interests.

我们描述了一个简单而具有成本效益的方法，以分离大量骨髓的小鼠单核细胞。相较于使用外周血其他协议，其中获得的单核细胞产率5 1.4×10 6，我们能够从一个单一的供体小鼠获得的11×10 6±3×10 6单核细胞更高的产量。 当考虑使用这种方法的挑战，它在非无菌条件下工作时，提供潜在的污染是重要的。如果冲洗和不遵守以下洗涤步骤正确，污染的细?...

The isolation of monocytes is important and critical for many in vitro and in vivo studies. These cells are targets for diseases such as peripheral arterial disease, coronary heart disease, or other ischemic diseases, since collateral vessel growth is strongly driven by local inflammation. Inflammatory responses include endothelial activation and local recruitment of leukocytes, mainly monocytes, which then mature to macrophages and create a highly arteriogenic environment by secreting multiple growth factors to induce the remodeling of an arteriole into a functional collateral artery1-3. Monocytes also mature to dendritic cells, which are frequently used for immunological studies4,5 and cancer research6,7.
Problematic in the approach for monocyte isolation from peripheral blood8 is the high number of donor animals needed to produce a sufficient amount of monocytes for most analyses. Former protocols describe methods such as density gradient centrifugation and cell depletion via MACS9 when isolating monocytes; however, these techniques can alter the characteristics and functionality of monocytes which can lead to difficulties in interpretation10,11. Moreover, these methods are difficult and can reduce experimental reproducibility.
Our aim with this protocol is to provide a simple and cost effective method to generate large amounts of bone marrow-derived monocytes. Due to the high cell yield of 11 x 106 &#177; 3 x 106 cells obtained by this protocol, we can substantially reduce the number of mice required during the isolation of bone marrow-derived monocytes. The procedure can be completed within a minimal amount of time, and without using expensive and complicated techniques as referenced above. Here, we extract monocytes from native bone marrow suspension of donor mice, cultivate the suspension on ultralow attachment plates, and supplement the solution with 20 ng/ml M-CFS. On day 5 of incubation, cells are harvested and characterized to confirm functional and phenotypic properties.
For experiments in the field of arteriogenesis, intravenous transplantation of these bone marrow-derived monocytes into mice is an effective method of systemic drug delivery, which can be combined with femoral artery ligation in common peripheral arterial disease models.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>6-well-ultra-low-attachment plate</td>
			<td>Corning Incorporated, NY, USA</td>
			<td></td>
			<td>6-well-ultra-low-attachment plate, with cap, sterile</td>
		</tr>
		<tr>
			<td>8- 12 week old, male, balb/c mice&#160;</td>
			<td>Charles River, Sulzfeld, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well-plate</td>
			<td>Greiner bio one GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blue dead cell stain</td>
			<td>Life technologies GmbH, Darmstadt, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumine</td>
			<td>GE Healthcare, Freiburg, Germany</td>
			<td></td>
			<td>Fraction V, pH 7,0</td>
		</tr>
		<tr>
			<td>Canules</td>
			<td>B. Braun, Melsungen AG, Melsungen, Germany</td>
			<td></td>
			<td>28G, 30G</td>
		</tr>
		<tr>
			<td>CD115</td>
			<td>eBioscience, San Diego, USA</td>
			<td>12-1152</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>eBioscience, San Diego, USA</td>
			<td>53-0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td>With cap, steril</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter GmbH, Krefeld, Germany</td>
			<td></td>
			<td>Allegra&#174; X-15R centrifuge</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet, Mannheim, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disinfection agent</td>
			<td>Sch&#252;lke&#38;Mayr GmbH, Norderstedt, Germany</td>
			<td></td>
			<td>Kodan Tinktur forte</td>
		</tr>
		<tr>
			<td>Disposable scalpel No.10&#160;</td>
			<td>Feather safety razor Co.Ltd, Osaka, Japan&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%&#160;</td>
			<td>Otto Fischar GmbH und Co KG, Saarbr&#252;cken, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Extraction unit Pipetus</td>
			<td>Hirschmann Laborger&#228;te GmbH &#38; Co.KG, Eberstadt, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>AbD Serotec, D&#252;sseldorf, Germany</td>
			<td>MCA497APC</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td>Manufactured by our group with single components</td>
			<td></td>
			<td>PBS, 0,5% BSA, 0,1% NaN3</td>
		</tr>
		<tr>
			<td>FACS device</td>
			<td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td>
			<td></td>
			<td>BD FACS Canto II</td>
		</tr>
		<tr>
			<td>FACS tubes&#160;&#160;&#160;&#160;</td>
			<td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon&#174; pipette</td>
			<td>Becton Dickenson Labware, NY, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Rubis, Stabio, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>R&#246;sner-Matby Meditrade GmbH, Kiefersfelden, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gr1</td>
			<td>eBioscience, San Diego, USA</td>
			<td>53-5931</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating plate&#160;</td>
			<td>Labotect GmbH, G&#246;ttingen, Germany&#160;</td>
			<td></td>
			<td>Hot Plate 062</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany</td>
			<td></td>
			<td>Incu safe</td>
		</tr>
		<tr>
			<td>Isofluran</td>
			<td>Baxter Deutschland GmbH, Unterschlei&#223;heim, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Carl Zeiss SMT GmbH, Oberkochen, Germany</td>
			<td></td>
			<td>Axiovert 40 &#176;C</td>
		</tr>
		<tr>
			<td>Macrophage-Colony Stimulating Factor</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/srp3110?lang=de&amp;region=DE">SRP3110&#160;</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical shaker</td>
			<td>IKA, Staufen, Germany</td>
			<td></td>
			<td>ms2 minishaker</td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>PAA Laboratories GmbH, Pasching, Austria</td>
			<td></td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Micro test tubes</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiological work bench</td>
			<td>Thermo Electron, LED GmbH, Langenselbold, Germany</td>
			<td></td>
			<td>Hera safe</td>
		</tr>
		<tr>
			<td>Monocyte wash buffer&#160;</td>
			<td>Manufactured by our group with single components</td>
			<td></td>
			<td>PBS, 0,5% BSA, 2mM EDTA</td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Berlin Chemie AG, Berlin, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3 (sodium acide)</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Paul Marienfeld GmbH und Co.KG, Lauda-K&#246;nigshofen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon cellsieve</td>
			<td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td>
			<td></td>
			<td>Cell strainer, 70 &#181;m mesh size</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life technologies GmbH, Darmstadt, Germany</td>
			<td></td>
			<td>pH 7.4, sterile</td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td></td>
			<td>10&#181;L/100&#181;L/200&#181;L/1000&#181;L</td>
		</tr>
		<tr>
			<td>Pipetting heads</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td>Cellstar 5 ml, 10 ml</td>
		</tr>
		<tr>
			<td>Suction unit</td>
			<td>Integra bioscience, Fernwald, Germany</td>
			<td></td>
			<td>Vacusafe comfort</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Word Precision Instruments, Inc., Sarasota, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>B. Braun, Melsungen AG, Melsungen, Germany</td>
			<td></td>
			<td>1mL Omnifix&#174; -F insuline syringe</td>
		</tr>
		<tr>
			<td>Tubes with cap</td>
			<td>Greiner bio one GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td>15mL/50mL Cellstar tubes</td>
		</tr>
		<tr>
			<td>Warm water bath</td>
			<td>Julabo Labortechnik GmbH, Seelbach, Germany</td>
			<td></td>
			<td>Julabo SW22</td>
		</tr>
	</tbody>
</table>

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.

来自鼠骨髓中提取的细胞溶液包含各种细胞类型。主要的细胞类型是淋巴细胞，粒细胞和单核细胞。细胞类型可以通过尺寸和粒度，其示于图1中为5天后分化的收获两个天然悬浮液和细胞来估计。注意培养过程中的换档细胞组成。然而，人口的准确分类必须依靠细胞标记鲜明的表达。 用于识别的MPS-系统的细胞中最重要的标记是CD115，其功能是作为对M-CSF受体和专?...

Watch this Scientific Journal Video about Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes at JoVE.com

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

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).

隔离和小鼠骨髓的静脉注射单核细胞衍生

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

isolation-intravenous-injection-murine-bone-marrow-derived

Research

JoVE Journal

Immunology and Infection

23.3K 次观看.  Otto von Guericke University Magdeburg. 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).

视频: Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

科研

免疫与感染

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

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 ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

鼠后肢长骨解剖和骨髓隔离

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

使用胶原酶E从毛里结肠分离拉米纳普里亚单核细胞

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

骨髓来源巨噬细胞的分离和 体外 培养用于NO-氧化还原生物学研究

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...