This work was supported by Inserm. Marion Bouvet is a fellow of the French Ministry for Research and Technology. Annie Turkieh is a fellow granted by European Union FP7 HOMAGE (305507).

该协议符合我们的机构人类研究伦理委员会的指导方针。从地区输血中心（里尔，法国）获得从健康人供体棕黄色大衣。从淡黄色大衣得到的样品被声明为一个INSERM集合（N°DC2010-1209）。 1.材料和文化传媒准备<ol><li>稀释10倍的磷酸盐缓冲盐水（PBS），在无菌蒸馏水中，得到的1×PBS。 </li><li>作的RPMI 1640培养基补充有庆大霉素（40微克/毫升）和L-谷氨酰胺（2毫摩尔），有和没有10％合并的人血清。 </li></ol>单核细胞 - 巨噬2.原代培养（MDM） <ol><li>稀buffycoat（25毫升）用1×PBS。在Ficoll / leucosep管和离心谨慎地装载在1600×g离心20分钟，在室温下进行。 </li><li>收集单核细胞的界面处进新管中。洗涤3次，用10ml。一次含0.1％乙二胺四PBS中乙酸（EDTA）的通过连续离心以1000×g离心，370×g离心和160×g离心，每次10分钟，然后再一次在1×PBS中单独以160×g离心10分钟。 </li><li>重新悬浮于5ml RPMI-1640培养基将细胞沉淀无血清和种子细胞35毫米培养皿中，每皿1×10 6个细胞的密度。 </li><li>沉降90分钟，在孵化后，弃去含有非贴壁细胞上清。洗涤贴壁细胞，包括单核细胞，3倍，用1ml的PBS;然后加入1毫升含有10％的新鲜培养基（体积/体积）人血清与先前的无血清细胞。 </li><li>培养6天后的，以产生替代性分化的巨噬细胞（M2）中，添加重组人IL-4（15毫微克/毫升），保持6天。然后，治疗分化的巨噬细胞用脂多糖（100毫微克/毫升）在12天4小时，得到M1巨噬细胞。 </li></ol> 3.提取M1和M2巨噬细胞蛋白的二维电泳<ol><li>洗macroph年龄三次用25mM的Tris，pH 7.4中和废料中含有30毫摩尔Tris pH为8,4％3缓冲器 - [（3-胆酰胺丙基）二甲氨基] -1-丙磺酸盐（CHAPS），2M硫脲和7M尿素。 </li><li>使用合适的1.5 ml微量离心管5分钟，在冰中并储存在-20℃的混合器裂解细胞。 </li><li>使用市售的Bradford试剂测定蛋白质浓度。存储该蛋白质的100μl的等分试样在-20℃下直到使用。 </li></ol> 4.等电聚焦注：请在黑暗中的所有标记的​​程序。 <ol><li>减少5微克每一个样品（M1和M2巨噬细胞），2毫摩尔Tris（2-羧乙基）膦（TCEP）处理1小时，调整为9微升用裂解缓冲液中，在37℃。 </li><li>添加菁3（Cy3标记）与M1提取物和M2的巯基反应性染料提取物为0.8纳米/微克蛋白的浓度。添加花青-5（Cy5标记），以M1和M2中提取在0.8纳米/微克蛋白的浓度和孵育为30分钟，在37℃。 </li><li>停止通过加入含7M尿素，2M硫脲，4％CHAPS，130毫摩尔二硫苏糖醇（DTT）和2％pharmalytes样品缓冲液等体积​​的反应。 </li><li>混合Cy3标记M1的样品用Cy5标记M2的样品中的单手和Cy3标记M2的样本与另一个手Cy5标记M1的样品。 </li><li>再水化的固相pH梯度（IPG）条（240毫米，pH值3-10的线性梯度）用450微升标记的混合样品在含7M尿素，在等电2M硫脲，4％CHAPS缓冲聚焦（IEF）电池系统为24小时，不施加任何电流。 </li><li>执行，在300伏（V）聚焦3小时，然后在梯度1,000V，6小时，在梯度的8000 V 3小时，最后在8000 V 3小时。 </li></ol> 5.第二维注：请在黑暗中所有的电泳程序。 <ol><li>孵育IPG胶条的平衡缓冲液含有荷兰国际集团为0.1mM的Tris-HCl（pH8）中，6M尿素，2％（重量/体积）十二烷基硫酸钠（SDS）和30％（体积/体积）甘油10分钟。 </li><li>传输平衡的IPG胶条用于第二维（SDS-聚丙烯酰胺凝胶电泳（PAGE））到12.5％PAGE凝胶上，并密封与低熔点琼脂糖。 </li><li>进行电泳，在20℃下使用的Ettan-Daltsix系统，在70伏的恒定电压过夜，然后300伏，直到溴酚蓝前沿到达凝胶的底部。 </li></ol> 6.图像采集和生物信息学分析<ol><li>扫描凝胶投两低荧光玻璃板之间具有DIGE成像扫描器在五百八分之五百三十二处为Cy3和六百七十○分之六百三十三激发波长/发射波长为Cy5标记，以具有100微米的像素尺寸得到的图像。 </li><li>用商业软件进行图像分析如先前详述12。 </li><li>计算和规范点卷中的每个图像。分配一个标准化的现货量为道具检测凝胶中的每个点的总价值的钮用来。 </li><li>通过比较这两个基团（M1和M2）之间的归一化点体积值分析对于每种类型的巨噬细胞中的蛋白斑点体积的差异。考虑光斑的卷之间的差异是显著如果变化为1.5倍（p &lt;0.05，单向ANOVA分析）。 </li></ol>

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There are no declared conflict of interest.

本文所描述的协议详细说明了分析巨噬细胞中，M1（促炎）和M2（抗炎）的两个亚型的各种刺激的影响的方法。的M1和M2巨噬细胞的原代培养物是从单核细胞分化得到的先前公布的7。 的2D DIGE凝胶电泳的方法需要特殊的材料和设备，如等电聚焦，以扫描在对Cy3和Cy5，一个扫描器，用于荧光激发/发射波长下的凝胶两次细胞对于等电聚焦，低荧光板用于SDS-PAGE上和2D软件。这...

巨噬细胞是异质的，塑料的细胞，能够获得不同的功能表型。 在体内，这些细胞对大量的各种微环境signalssuch作为微生物产物，细胞因子， 等等 。1的响应。 在体外 ，所述促炎性表型（M1）的巨噬细胞可通过脂多糖（LPS）和抗炎表型（M2），通过某些细胞因子，如白介素-4（IL-4）诱导。此外，巨噬细胞可以从一个激活的M1至M2的表型转换，而相反地，在特定的信号2。 根据不同的表型，巨噬细胞将具有不同的功能。 M1巨噬细胞产生促炎细胞因子，如肿瘤坏死因子α（TNF-α），杀灭微生物或肿瘤细胞的3个细胞。与此相反，M2巨噬细胞阻止这些炎性应答状在伤口愈合和纤维化由产生抗inflammatorÝ因子如TGF-β3,4。 从健康供体的人外周血单核细胞通过Ficoll密度梯度离心法如使用适于从Boyum 6的技术先前描述5中分离得到。在培养的巨噬细胞后6天的原代培养7可以分化成M1或M2的表型。 蛋白质表达或根据各种受控刺激，如宿主的致病性或微生物毒素的巨噬细胞的两种亚型之间的蛋白质变化的分析，将是有益的破译亲和抗炎的巨噬细胞的功能。 蛋白质组学是用于直接监控的蛋白质，是具体地上调或下调的下各种刺激培养的人体巨噬细胞的独特工具。荧光染料已解决了某些二维凝胶电泳的局限性，如低灵敏度和图像分析8 &lt;<html> / SUP&gt;。染料与半胱氨酸残基反应而增加的检测灵敏度相比，那些与赖氨酸残基9的反应。在先前的研究中，我们证明了DIGE饱和标记的有用稀少样品10的分析结果相比较，以经典的银染的二维电泳11。这种技术是在快速分析巨噬细胞的两种亚型之间或从同一亚型未处理和处理的巨噬细胞之间的蛋白质修饰很有帮助。 此蛋白质组技术的优点是具有由2D凝胶12分析获得的蛋白质的大小和翻译后修饰的信息。应当考虑到，这不是一个高通量技术，限制了可被分析的样品的数目。基于质谱的高通量测定法评论了发展最近13可以改善这一点。 _content“&gt;这里，我们提出了如何通过电泳，等电聚焦的过程中执行从培养的巨噬细胞的蛋白提取2D DIGE分析和SDS-PAGE，以及对适当的2D软件的有用信息。

<table border="0" cellpadding="0" cellspacing="0" width="472">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:208px;"><a name="RANGE!A1:C43">Name</a></td>
			<td style="width:155px;">Company</td>
			<td style="width:111px;">Catalog number</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RPMI 1640</td>
			<td>Invitrogen</td>
			<td>31870-074</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PBX 10 X</td>
			<td>Invitrogen</td>
			<td>14200-083</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-glutamine-200mM-100X</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">gentamycin 10 mg/mL</td>
			<td>Invitrogen</td>
			<td>15710-049</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">human serum</td>
			<td>Invitrogen</td>
			<td>34005100</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ficoll d=1,077</td>
			<td>ATGC</td>
			<td>L6115</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Leucosep</td>
			<td>Dutscher</td>
			<td>16760</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#160;6-wells plate PRIMARIA&#160;</td>
			<td>Becton Dickinson</td>
			<td>353846</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IL-4</td>
			<td>Promocell</td>
			<td>B-61410</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">lipopolysaccharide</td>
			<td>Sigma-Aldrich</td>
			<td>L-2654</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Giemsa</td>
			<td>Fluka</td>
			<td>48900</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EDTA MM372,2</td>
			<td>Research Organics&#160;</td>
			<td>3.00E+01</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter 0,22&#181;m</td>
			<td>Millipore</td>
			<td>SCGPTORE</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">100 mL cylinder</td>
			<td>Corning</td>
			<td>430182</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TCEP</td>
			<td>Interchim</td>
			<td>UP242214</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bradford reagent</td>
			<td>Bio-Rad</td>
			<td>5000006</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cy3+Cy5-reactive dye</td>
			<td>GE Healthcare</td>
			<td>25-8009-83</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IPG strip 3-10 24cm</td>
			<td>GE Healthcare</td>
			<td>17-6002-44</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protean IEF cell</td>
			<td>Bio-Rad</td>
			<td>165-4000</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Low-melting agarose</td>
			<td>Invitrogen</td>
			<td>15517-014</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ettan-Daltsix system</td>
			<td>GE Healthcare</td>
			<td>80-6485-08</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ettan DIGE Imager scanner</td>
			<td>GE Healthcare</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Progenesis Samespot</td>
			<td>Non linear dynamics</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">50 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">15 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CHAPS</td>
			<td>Sigma-Aldrich</td>
			<td>C5070</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ur&#233;e</td>
			<td>Merk</td>
			<td>108484-500</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Thiour&#233;e</td>
			<td>Sigma-Aldrich</td>
			<td>T7875</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DTT</td>
			<td>Bio-Rad</td>
			<td>1610611</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">APS</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tris Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tris HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T3253</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pharmalytes 3-10</td>
			<td>GE Healthcare</td>
			<td>17-0456-01</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">SDS</td>
			<td>Sigma-Aldrich</td>
			<td>L3773</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>114391</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G6279</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acrylamide 40%</td>
			<td>Bio-Rad</td>
			<td>161-0148</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2D clean Up</td>
			<td>GE Healthcare</td>
			<td>80-6454-51</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dim&#233;thylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>22705-6</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">electrode wicks</td>
			<td>Bio-Rad</td>
			<td>165-4071</td>
		</tr>
	</tbody>
</table>

proteomic profiling of macrophages by 2d electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

执行适当的差动蛋白质组学分析，待分析样品的处理应加以核实。 在给出的例子中，需要的巨噬细胞的细胞培养物的质量为形态学和分子方面的先前公布的5。单核细胞分化成巨噬细胞和培养物的均一性，随后通过相显微镜。 图1显示的M1和M2巨噬细胞的原代培养物的一个例子。如图所示，我们证实M1（ 图1A，B）和M 2（ 图1C，D） ​​的</s...

Watch this Scientific Journal Video about Proteomic Profiling of Macrophages by 2D Electrophoresis at JoVE.com

Proteomic Profiling of Macrophages by 2D Electrophoresis

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

巨噬细胞通过二维电泳蛋白组学分析

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

proteomic-profiling-of-macrophages-by-2d-electrophoresis

Research

JoVE Journal

Immunology and Infection

12.5K 次观看.  University Lille Nord de France. Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

视频: Proteomic Profiling of Macrophages by 2D Electrophoresis

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

在小鼠体内的巨噬细胞的枯竭和重建

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

科研

免疫与感染

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

胞浆的定量与液泡<em&gt;沙门氏菌</em&gt;在原发性巨噬细胞差透

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

从HIV-1感染细胞的外来体基于SILAC蛋白质组学表征

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...