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>

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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，污染的细菌和真菌的可能性较大。此外，骨髓由异质细胞悬浮液，包括粒细胞，淋巴细胞和红细胞。通常这些细胞2天和第3天培养的间消失，由于缺乏生长因子。后生长因子从动分化，巨噬细胞是污染的主要来源。为了使吨他数的巨噬细胞的低，并尽量减少单核细胞向巨噬细胞的分化，使用超低附着板和上述的洗涤步骤是关键的。 所以很难确定一个标记物只单核细胞特异性的。标记之间的干扰必须使用一个以上的标志物，并有可变性，在粒度和细胞的大小在分析细胞悬浮液。标记与图1-3中所示的描述是对于在培养中有代表性的细胞类型良好的替代品。它可以是难以区分的各种细胞类型，特别是在5天所以关键是要使用其他标记物像CD115和F4 / 80。 考虑骨髓细胞的粘附无的条件下膨胀，巨噬细胞分化，可以延长，未分化的单核细胞可以积累。巨噬细胞分化表明污染;然而，杜E要即使在超低附着培养表面的粘合剂的性质，它们可以容易地丢弃，而只有从培养收获非粘着细胞。在以前的实验1,3，污染被巨噬细胞并没有导致严重的临床副作用。器官和细胞移植后的小鼠的肌肉组织学后处理表明，注入的巨噬细胞吸收在诸如脾，肝或肺器官。在这些器官的巨噬细胞的同化证明在小鼠中没有观察到的临床效果。这些巨噬细胞引发的分子机制可用于进一步调查有趣的问题。 当全身应用的单核细胞以及巨噬细胞的影响动脉中的外周动脉疾病，特别是。临床副作用，如栓塞或块状系统性炎症无法观察到甚至注射超过250万的单核细胞后，但这些细胞可引发炎症和Potentially造成严重的系统性影响。在人体试验中，这些影响可能有临床意义。动脉的触发可影响肿瘤生长或糖尿病性视网膜病变，并注射细胞溶液应当在未来的研究探讨的这些可能的全身效应。 以前的出版物9描述的外围血-和骨髓来源的单核细胞，这解释了为什么从骨髓单核细胞的特性可以从那些从外周血细胞中分离的不同的特性之间的差异。 隔离的方法是临床感兴趣的为好。抽血，而不是提取人骨髓是临床医学的背景下更实用。照顾动物福利后，如果需要，以应用镇痛是很重要的。用于静脉内注射，它实行同时处理动物和注射器在同一时间是至关重要的。如果一个ANIMAL正在经历多次注射，静脉的质量和尾部的皮肤受罪。 尽管有这些缺点，这种方法是非常有用的，研究的行为和单核细胞的特性。在动脉粥样硬化，免疫或炎症的现场实验可以受益于这种方法。仅30分钟，需要从骨髓提取到细胞接种在6孔超低附着表面板。伦理问题被最小化，因为高收率此协议的最小化需要作为细胞供体动物的数量。这种新方法将成为许多研究涉及单核细胞有帮助的。 我们一直专注于侧支血管生长通过单核细胞移植治疗增强。这个过程的多因素的性质可以解释为什么单因素的方法动脉的增强都产生不同的结果13,14。这导致了在王爱平细胞为基础的治疗。自体骨髓衍生的干细胞和祖细胞已经被确定为潜在的移植细胞，但仅中度临床益处已报道15。除了对剂量，分离方法，和给药途径的研究，进一步的研究仍然需要确定最佳的细胞类型。自体细胞移植的缺点可能是其不能激活先天和/或适应性免疫应答者，都为动脉的关键。增加局部炎症可能代表动脉16的最好刺激。 静脉内注射的单核细胞是内小鼠模型细胞移植的常用方法，如果全身给药是理想的。由于系统性影响的，副作用可能会出现为好。确保静脉是针对注射。是很常见的动脉和静脉，尤其是当注入大量色素麦克风混淆即动脉注射可引起血栓，从而导致流通中的系统性问题。

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受体和专门对单核细胞祖细胞，单核细胞和巨噬细胞表达。因此，它可能不被用于识别独特的MPS-子集，但能可靠地估计细胞驱入单核细胞/巨噬细胞分化的绝对量。参见图2 CD115的表达在不同的时间安排entiation。 成熟标记F4 / 80是有用的区分少年单核细胞，成熟的单核细胞和巨噬细胞。巨噬细胞表现出强劲的F4 / 80的表达相比，少年单核细胞的祖细胞，它们是负的标记。成熟的单核细胞显示出的F4 / 80的中间表现。因此CD115和F4 / 80标记的组合表示用于定量和监测细胞分化（ 见图3）一个可行的方法。 CD115和F4 / 80的结合是足够的常规培养观察和质量控制应用之前的细胞。在培养的第5天的骨髓来源的单核细胞的更详细的检查证实了他们的结构和功能特性的一致性的那些的外周血单核细胞12。 它小心固定鼠标在阻挡器，避免张力尾巴上是重要的。限制freedo米运动尽可能不妨碍呼吸，以方便注射。 <img alt="图1" src="/files/ftp_upload/52347/52347fig1highres.jpg" /> 图1.左面板:细胞类型在培养第1天的组合物的细胞类型，包括淋巴细胞，单核细胞和粒细胞。右侧面板:注意培养过程中的换档细胞组成。细胞群包括单核细胞和巨噬细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52347/52347fig2highres.jpg" /> 图2.时间轴分化过程中CD115的表达。注意，&gt; 95％的细胞是CD115 5天后分化的阳性，这表明广大<html>大部分细胞要么单核细胞或巨噬细胞。分类可以根据细胞大小和粒度，但特异性细胞标志物更加可靠。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52347/52347fig3highres.jpg" /> 图3.增加单核细胞/巨噬细胞的成熟标记F4 / 80 5天的表达。注意与中间的F4 / 80的表达，这与单核细胞的表型相一致的人口的外观。第7天:注意右移F4 / 80的表达，与巨噬细胞成熟一致的<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> E 4"SRC ="/文件/ ftp_upload / 52347 / 52347fig4highres.jpg"/&gt; 图4.鼠标固定在阻挡器尾静脉注射。在认真抑制鼠标，旋转尾部90度，直至脉的背侧可见。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52347/52347fig5highres.jpg" /> 的小鼠尾部图5的截面示意图。该图说明中的鼠尾船只的位置。动脉（红色）位于腹侧和背侧，和静脉都在尾部的侧面。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

23.5K 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, ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

抗体药物对小鼠骨髓及腹腔巨噬细胞活化作用的评价

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

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 ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

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 ...