Name: isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation
Uploaded: 2015-04-29T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation at JoVE.com

这项工作是由加拿大自然科学和工程研究理事会资助项目（NSERC，不授予298515-2011）。 AL是一个博士的接收者从自然科学和加拿大（NSERC）工程研究理事会，和MS奖学金从健康研究的加拿大学院资助项目（CIHR，不授予。MOP123246）。

所有的程序都按照加拿大议会动物护理指南批准德中心杜RECHERCHE中心医院DE L&#39;蒙特利尔大学的体制动物护理委员会（CRCHUM）之后进行。 1.分离鉴定，培养小鼠腹腔巨噬细胞，并<ol><li>准备3.8％的啤酒中​​巯基乙酸盐。要做到这一点，暂停38克硫乙醇酸盐培养基1000毫升蒸馏水。带来溶液煮沸以完全溶解培养基。通过在121℃高压灭菌15分钟进行消毒。存储长达3个月在黑暗中?...

<ol>
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巨噬细胞是生存的关键，并提供了一​​个诱人的目标来操纵宿主免疫学的目标。 Toll样受体和其他识别分子的发现进行了巨噬细胞免疫学争论的中心。巨噬细胞对多种刺激，包括细胞因子，损伤相关分子模式分子（DAMPS）20和与病原体（的PAMP）21组相关联的分子响应。这些不同的刺激响应代表的巨噬细胞的活化过程中，与通常与突然改变有关的基因表达22。 <p class="jove_...

网状内皮细胞吞噬系统是由细胞的各种组织和器官如骨髓，血液，肝脏和脾脏。巨噬细胞广泛分布在身体周围，在那里他们尤其参与先天免疫和适应性免疫应答，以控制和明确的感染。除了 ​​其在宿主防御作用，巨噬细胞也发挥在伤口愈合和在维持组织稳态1,2-重要作用。此外，巨噬细胞不仅是重要的免疫功能，而且还积极参与铁稳态3。在人体中，铁的约80％存在于血红蛋白的红细胞内，其中，当衰老被巨噬细胞吞噬4。每日，这些巨噬细胞回收的红细胞来源的铁25毫克，并提供其传输到等离子体5。此外，感染和炎症期间，促炎症的巨噬细胞隔离血清铁，减少铁availabiliTY病原体，同时在全身和地方各级6-8。同时，研究表明，巨噬细胞和肝细胞为主名为生产铁调素抗菌肽被认为是铁代谢9，10主调节器。铁调素主要是增加了炎症刺激，是在慢性炎症11-13部分负责铁封存在巨噬细胞。在巨噬细胞铁调素的表达不是很好理解，我们研究Toll样受体在本条中的作用（Toll样受体）。该TLR是巨噬细胞主要发现，在它们的活化中发挥中心作用。另外，在肝脏中的LPS诱导的铁调素表达依赖于TLR4 13。因此，要执行我们的研究中，我们使用了基于小鼠腹腔巨噬细胞的分离方法。 巨噬细胞系中的巨噬细胞螺柱广泛使用IES;尽管如此延长培养可以挑起基因损失和在这些细胞系中降低免疫功能。因此，从腹腔巨噬细胞的分离是至关重要的。 小鼠腹腔提供了一个理想的地点收获巨噬细胞13-15。孤立的小鼠腹腔巨噬细胞是便于对他们的免疫功能的几项研究。然而，在腹膜的巨噬细胞的数量不足以进行了广泛的研究，并估计每鼠约1×10 6巨噬细胞。因此，为了提高巨噬细胞的输出，无菌引发剂，例如巯基乙酸注射入细胞收获前的腹腔。巯基乙酸注射后，每只小鼠巨噬细胞的产率提高了10倍。尽管提高巨噬细胞产率，Brewer的巯基醋酸盐培养基充当诱导炎性反应，从而导致在招募的巨噬细胞，这马刺激性年，但没有必要影响基因的表达。因此，对照组包括未处理的巨噬细胞必须包括在每个实验。在我们的手中，并没有在未经处理的巯基乙酸检测铁调素表达其强烈的炎症刺激引起腹腔巨噬细胞。此外，研究表明，Brewer的巯基乙募集大量的巨噬细胞，但不激活它们16。另一方面，啤酒巯基乙引起的巨噬细胞显示增加溶酶体酶，但在杀死摄取微生物17的降低。然而，当与非引起的巨噬细胞16相比，吞噬能力不受影响。 一旦在菜培养的腹膜巨噬细胞成为粘附的，因此允许从其他类型的细胞从腹腔中分离的它们的分离。接着，分离的巨噬细胞进行攻击用不同的TLR激动剂。最后，表达从培养的细胞中提取，并使用定量逆转录酶 - 聚合酶链式反应（QRT-PCR）的基因表达进行了分析。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Charles River Laboratories, Inc. (Wilmington, MA, USA)</td>
			<td>475</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioglycollate</td>
			<td>Sigma-Aldrich, (St. Louis, MO)</td>
			<td>19032-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% sodium pentobarbital (Used for mice anesthesia (80 mg/kg, i.p.))</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate-buffered saline (DPBS), placed on ice and will serve to harvest macrophages</td>
			<td>WISENT INC Canada (QC)</td>
			<td>311-425-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640 (Supplement with penicillin, streptomycin, L-glutamine, and 10 % fetal calf serum).</td>
			<td>WISENT INC Canada (QC)</td>
			<td>350-000-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>1 and 5 ml syringes</td>
			<td>BD USA (NJ)</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Six-well plates</td>
			<td>Corning Incorporated (NY, USA)</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-pm25</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyionosine&#8211;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-PIC</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FLIC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipoprotein synthetic FSL1 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FSL</td>
			<td></td>
		</tr>
		<tr>
			<td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &#956;g/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-LRNA-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Type B CpG oligonucleotide ODN1826 (1 &#956;M)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>11B16-MM</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIZOL</td>
			<td>Invitrogen, (Burlington, ON, Canada)</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr>
			<td>20g and 23g needles</td>
			<td>BD USA (NJ)</td>
			<td>305175</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes placed on ice</td>
			<td>Sarstedt (Newton, MA, USA)</td>
			<td>62.547.205</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cells Lysis Buffer</td>
			<td>Sigma-Aldrich, (St. Louis, MO)</td>
			<td>R7757-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80 antibody</td>
			<td>BIO-RAD ( CA, USA)</td>
			<td>MCA497APC</td>
			<td></td>
		</tr>
		<tr>
			<td>CD16/CD32 antibodies</td>
			<td>Pharmingen, {Mississauga, ON, CA)</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>Coulter Epics Elite counter, Coulter, (Hialeah, FL,USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Six-well plates</td>
			<td>Corning Incorporated (NY, USA)</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-pm25</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyionosine&#8211;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLRL-PIC</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FLIC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipoprotein synthetic FSL1 (100 ng/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-FSL</td>
			<td></td>
		</tr>
		<tr>
			<td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &#956;g/ml)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>TLR-LRNA-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Type B CpG oligonucleotide ODN1826 (1 &#956;M)</td>
			<td>InvivoGen (San Diego, USA)</td>
			<td>11B16-MM</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIZOL</td>
			<td>Invitrogen, (Burlington, ON, Canada)</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorf tubes</td>
			<td>Axygen Scietific (CA,USA)</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific (ON, Canada)</td>
			<td>UN1888</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>JT Baker (PA, USA)</td>
			<td>70566</td>
			<td></td>
		</tr>
		<tr>
			<td>75% ethanol (in DEPC treated water)</td>
			<td>Commercial Alchohols (QC, Canada)</td>
			<td>17394</td>
			<td></td>
		</tr>
		<tr>
			<td>0.01% diethyl pyrocarbonate (DEPC) treated water (let stand overnight and autoclave)</td>
			<td>Sigma-Aldrich, (St. Louis, MO)</td>
			<td>216.542.8</td>
			<td></td>
		</tr>
		<tr>
			<td>Omniscript RT-PCR system</td>
			<td>Qiagen, (Mississauga, ON, Canada)</td>
			<td>205113</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotor Gene 3000</td>
			<td>Montreal Biotech, (Kirkland, QC, Canada)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect SYBR Green I PCR kits</td>
			<td>Qiagen, (Mississauga, ON, Canada)</td>
			<td>204141</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation

在感染和炎症，循环单核细胞离开血流并迁移入组织，在那里它们分化成巨噬细胞。巨噬细胞表达表面Toll样受体（TLR），其识别通过进化保守在宽范围的微生物的分子模式。 TLR的发挥其通常与基因表达的改变相关巨噬细胞活化中发挥中心作用。巨噬细胞是在许多疾病的关键，并已成为用于治疗的有吸引力的目标。在下面的协议中，我们描述了一种方法来分离使用啤酒巯基醋酸盐培养基小鼠腹腔巨噬细胞。后者将提升单核细胞迁移进入腹膜，因此这将提高巨噬细胞的产量提高了10倍。几个研究已经进行了使用骨髓，脾脏或腹膜巨噬细胞。然而，巨噬细胞被证明是在分离更成熟，更稳定在它们的功能性和表型。因此，巨噬细胞小鼠腹腔分离提出了一个重要的细胞群，可以在不同的免疫和代谢的研究服务。一旦分离，巨噬细胞刺激不同TLR配体，因此基因的表达进行评价。

我们首先特点分离小鼠腹腔巨噬细胞用流式细胞仪。要做到这一点，我们使用（F4 / 80）的抗体特异性识别只由巨噬细胞表达的标志物。这种特征是必需的，以确定分离的巨噬细胞的百分比和区分它们在分离过程得到的细胞之间。如图（ 图1），细胞的百分比表达抗原的F4 / 80被一致地为95％以上。接着，为了研究巨噬细胞中的基因表达，所述分离细胞用几种TLR配体：Pam3CSK4（TLR1 / 2），L...

Watch this Scientific Journal Video about Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation at JoVE.com

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Research

JoVE Journal

Immunology and Infection

Isolation of Mouse Peritoneal Cavity Cells

小鼠腹膜腔细胞的分离

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

科研

免疫与感染

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

在调节Toll样受体激活Neu1唾液酸酶活动的检测

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

定量成像小样本人体血液中单核细胞和树突状细胞系特定的Toll样受体介导的信号

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation, Processing and Analysis of Murine Gingival Cells

小鼠牙龈细胞的分离，处理和分析

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

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 ...

Isolation and Activation of Murine Lymphocytes

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

嗜肺军团菌外膜囊泡：隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

应用系统进化分析研究真核基因的起源

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...