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，在室温18。 注：丢弃如果混浊发展其指示细菌污染。该溶液可以保存长达一年在黑暗中，如果保持无菌。 </li><li>使用连接到23政针1毫升注射器，注射1毫升3.8％巯基乙酸布鲁尔介质进入腹腔每个亩腔本身并等待3天。使用新的注射器和针头每个鼠标。 </li><li>通过腹膜内注射戊巴比妥钠（100毫克/千克）麻醉小鼠，并用颈脱位安乐死的小鼠。通过检查呼吸频率确认正确的麻醉。通常快速呼吸表明，鼠标不深度麻醉。 </li><li>洗每只小鼠用70％乙醇的腹部。使用剪刀，沿着腹膜的底部中线进行横向切割。 </li><li>使用镊子，拉回来的腹部皮肤暴露腹膜透明皮肤。使用连接到20G针5毫升注射器，注入5毫升冷的DPBS到腹膜各小鼠的空腔。 </li><li>对腹腔进行轻柔的按摩，然后小心吸液不进行打孔的任何器官。取出针和分配的腹膜液到50ml锥形离心管中。 </li><li>离心，在400×g离心在冷冻离心机10分钟。细胞必须在整个过程中留下来的冷。弃上清，悬浮细胞沉淀在RPMI培养基1640 </li><li>使用血细胞计数器，计数细胞并调整至细胞密度为1×10 6个细胞/ ml。 </li><li>通过流式细胞表征分离的细胞的表型流式细胞仪使用每鼠和antibodie 1×10 6细胞对F4 / 80（对巨噬细胞的表面抗原表达的）。 </li></ol> 2.细胞治疗<ol><li>直接，分离后，加入1×10 6细胞到每个孔中。离开的小鼠腹腔巨噬细胞，在37℃进行1至2小时，他们中培养粘附到6孔板中。轻轻洗涤3次，用温PBS除去非粘附细胞。 </li><li>随后，培养的细胞在900微升无血清DMEM 24小时在下面TLR​​配体的存在下：（Pam3CSK4-TLR1 / 2（0.5毫克/毫升）;聚（I：C）-TLR3（10毫克/毫升） ; LPS-TLR4（100纳克/毫升）;鞭毛-TLR5（100纳克/毫升）; FSL1-TLR6 / 2（100纳克/毫升）; ssRNA40-TLR7（1微克/毫升）; ODN1826-TLR9（1μM）。 注：准备为每个​​配体的10倍原液。中加入100μl后者向每个孔，由此进行10倍稀释。 </li></ol> 3. R​​NA分离<ol><li>通过移液管加入1ml TRIzol试剂至各孔，并通过细胞裂解物多次在6孔板直接删除所有介质和裂解细胞。孵育均化样品进行5至10分钟，在RT下，以允许核蛋白复合物的完全分离。 </li><li>转移到裂解1.5毫升无RNA酶的DNA酶和无离心管。加入每1ml的TRIzol0.2毫升氯仿。摇管用手剧烈持续15秒，并培育它们在RT 5至10分钟。离心机在12000×g离心15分钟，在4℃。观察分离成较低红相（苯酚 - 氯仿）的混合物中，相间和无色上层水相。的RNA仍然只在水相中。 </li><li>陈德良sfer仔细上层水相转移到新的管中，而不会干扰相间。通过用0.5ml异丙醇混合沉淀从它的RNA。孵育的样品在室温下10分钟，并离心以12,000 xg离心在4℃下10分钟。将RNA沉淀物形成白色沉淀在管的侧面和底部。 </li><li>去除上清液。用1ml 75％的乙醇洗涤一次RNA沉淀。混合通过涡旋和离心样品在7500×g离心5分钟，在4℃。简要地干燥的RNA（空气干燥10分钟）。 RNA溶解于0.1​​ml DEPC（无RNA酶的水），并孵育10分钟，在55℃。 </li><li>使用分光光度计定量RNA。为此，稀释样品于DEPC水100倍，并读取在260纳米的波长。那么RNA浓度将是：外径260×40毫微克/ UL×稀释因素。 </li><li>均衡的RNA浓度和合成cDNA按使用RT-PCR系统用于第一链cDNA合成试剂盒制造商的指示。如每制造商的说明，测量基因mRNA水平实时PCR（45次）的实时DNA检测系统。正常化基因表达水平有两个看家基因，例如β-actin和GAPDH。 注：规范化RNA浓度为500微克/毫升，并使用0.5微克cDNA合成。 </li></ol>

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The authors have no competing financial interests.

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. ...

isolation-murine-peritoneal-macrophages-to-carry-out-gene-expression

Research

JoVE Journal

Immunology and Infection

36.1K 次观看.  Institut du cancer de Montréal. 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.

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

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

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

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

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

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

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, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

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

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 ...

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

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

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

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

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

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

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

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

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 ...

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...