Name: proteomic profiling of macrophages by 2d electrophoresis
Uploaded: 2014-11-04T15:00:00
Description: Watch this Scientific Journal Video about Proteomic Profiling of Macrophages by 2D Electrophoresis at JoVE.com

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?...

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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 ...

Research

JoVE Journal

Immunology and Infection

Depletion and Reconstitution of Macrophages in Mice

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

2D and 3D Chromosome Painting in Malaria Mosquitoes

疟疾蚊子中的二维和三维染色体绘画

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Study of Phagolysosome Biogenesis in Live Macrophages

在现场研究巨噬细胞吞噬溶酶体生源

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

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

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

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

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

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 ...