Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.

1.维持巨噬细胞系<ol><li>成长的RAW264.7并在DMEM J774A.1细胞系在5％CO 2的存在下在补充有10％胎牛血清（FBS）中，在37ºC。为了帮助保持无菌，加1％青霉素/链霉素（青霉素/链霉素）的培养基（尽管这不是严格必需的）。 </li><li> 关键的步骤:确保巨噬细胞生长在一个健康的状态，实现高效转染和siRNA诱导的基因敲除。利用通道3和10之间的单元格后新鲜解冻，这些巨噬细胞系具有最佳的转染效率，并保持强劲的转染后的生存在这个阶段;不使用更高的通道数（&gt; 20），作为转染效率和基因敲低既降低很大。 </li><li>对于优化的siRNA转染，细胞板在20％汇合和生长一至两天直到细胞达到不超过80％汇合。杂草丛生的细胞不会转efficiently。 </li></ol> 2.编程96-穿梭系统的转染<ol><li>启动连接到所述梭中的计算机上的96孔穿梭软件。从左上角的文件菜单中新的参数文件 。 </li><li>突出显示将要在显示的96孔板概略转染孔中。绿色方框中会显示该井被选中。 </li><li>选择对应于巨噬细胞系的使用（DS-136对RAW264.7或DS-130对J774A.1），然后选择适用 ，位于diagramed 96孔板下面的程序。请注意，选择一个节目后，在原理图上板的孔将被着色深蓝色。空圆圈表示尚​​未分配的程序并不会感到震惊的井。 </li></ol> 3.准备试剂转染<ol><li>通过混合SF细胞系准备工作Nucleofector™解决方案的96孔Nucleofector™解决方案使用所附的补充管的全部内容。允许该混合试剂的平衡在使用前室温。储存于4ºC过量完整Nucleofector™溶液长达3个月，以备将来使用。 </li><li>预暖的DMEM，RPMI-1640，0.25％胰蛋白酶/ EDTA，和磷酸盐缓冲盐水（PBS），在37ºC之前制备的巨噬细胞用于转染。 </li><li>通过融化冰准备的siRNA转染。为了最小化siRNA的冷冻解冻的数量，以等分试样冷冻新的siRNA首次使用它们。 </li><li>在最初的实验中，使用的siRNA以相对高的剂量为2微米（从20μM的股稀释1:10）。随后滴定的siRNA诱导强有力的基因敲除下到较低的剂量，虽然需要通常较高剂量为有效的基因敲除中的巨噬细胞相比，其他类型的细胞。 注:siRNA是可从许多来源;相当可以获得健壮的基因敲除使用siGenome SMARTPOOLs，这是四个siRNA的靶向每个基因库。作为阴性对照，使用各种非靶向siRNA池或siRNA的。 </li><li>以监测转染效率，转任pmaxGFP对照质粒（在Amaxa公司的试剂盒提供），或荧光标记的siRNA在平行于该实验的siRNA。期望的GFP质粒的30-50％的转染（用显微镜测定或在转染后流式细胞术当天）和荧光标记的siRNA的&gt; 99％的转染（转染和恢复之后通过流式细胞仪立即测定）。 </li></ol> 4.准备的巨噬细胞的转染<ol><li>通过胰蛋白酶消化分离从细胞培养皿贴壁的巨噬细胞。吸出培养基，并用10ml PBS洗细胞两次。除去PBS后，孵育细胞预温的0.25％胰蛋白酶/ EDTA（1毫升/ 10cm平板）1-2分钟，直至细胞开始分离。轻轻晃动酶标板，移动SOLU在孵化期间重刑最大化胰蛋白酶。 </li><li>培养后，通过移液添加9毫升DMEM + FBS的培养基，轻轻混匀，上下，并且收集细胞到无菌管中。 </li><li>算使用血球分离的巨噬细胞的数量。预计每10cm平板大约一千万的巨噬细胞。计算出所需的巨噬细胞的总数;每个孔的96孔的穿梭需要200,000个细胞。记得添加了一些额外的井的价值单元，以便吸取损失。 </li><li>吸取细胞的数目计算为一个离心管中。离心细胞10分钟，于150×g离心室温。不要离心太快，因为这会降低巨噬细胞的健康和转染效率。 </li></ol> 5.用siRNA巨噬细胞的核转<ol><li>使用无菌的96孔圆底板（这同时减少试剂损失便于混合），加入2微升每种siRNA，每孔为互感器ected。 注意:根据siRNA的数目，这可以在巨噬细胞的离心步骤来完成的。 </li><li>从离心细胞吸出培养基。轻轻悬浮细胞Nucleofector™解决方案SF（含补充液），用每200,000细胞沉淀20微升。一旦重悬，立即用细胞最佳转染。 </li><li>传输重悬的细胞进入无菌槽;使用多通道移液管，移20微升的重新悬浮的细胞混合物放入96孔板中含有siRNA的各孔中。移液器向上和向下轻轻混匀。 </li><li>转移20微升细胞/ siRNA的混合物放入96孔Nucleocuvette™板的关键步骤:避免在转移步骤期间产生气泡，因为这会核转期间引起的错误。 </li><li>将所有的转板实验样品后，覆盖96孔板用所提供的盖和版本Ÿ轻轻拍打在坚硬的表面，从样本中删除任何气泡。 </li><li>将96孔板盖子放入96孔Nucleofector班车并选择程序屏幕的右下方"上传并开始"。前核转，按照程序提示保存结果。 注意:在核转过程中，井成功转染将由一个绿色圆圈加号所描绘的96孔原理图在屏幕上。一个红色的圆圈，减号指的错误，很可能是由于气泡或错误吸取试剂的正确数量。 </li></ol> 6.恢复和巨噬细胞的电镀<ol><li>立即核转后，用多道移液器，加入80微升预温（37℃）的RMP1-1640媒体到每个孔中。媒体添加到每孔中的侧面，允许媒体慢慢与转染的细胞，这是非常微妙的，在这个阶段混合。不要添加媒体太快，因为这将大大降低生存能力。 </li><li>将96孔板用转染的样品和RPMI-1640成37ºC培养箱，用5％CO 2下2分钟;这是至关重要的，以使细胞进一步操作之前恢复。 </li><li>从培养箱中取出96孔板中并小心地从每孔中转移到混合物用多道移液器96孔组织培养板中。 </li><li>使用多通道移液管，加入100微升预温（37℃）的DMEM + FBS培养基中的每个孔中。 </li><li>孵育96孔组织培养板用在37ºC培养箱，用5％CO 2下24-36小时的样品（优化siRNA个体的精确定时，尽管该时间段通常可以很好）。 </li></ol> 7. LPS刺激巨噬细胞<ol><li>继24-36小时后核转孵化，准备LPS（或其他的PAMP）的新鲜培养基。准备足够的新鲜培养基对所有井（200微升米EDIA /孔的96孔板加一点点额外的移液）。稀释的LPS至20毫微克/毫升的DMEM + FBS的最终浓度。 </li><li>从96孔板小心吸出培养基，并添加含有内毒素的细胞的新鲜培养基。 </li><li>监控刺激后在不同时间对LPS反应或其它的PAMP。 6小时是足够的，以产生细胞因子如IL-6或TNFα的一个健壮的炎性细胞因子应答。 </li></ol> 8.监控诱导先天免疫反应，基因敲除和活力的巨噬细胞<ol><li>并监控LPS诱导的细胞因子的产生，吸管从细胞上清液中加入96孔储藏板。细胞因子产生可以然后通过ELISA进行测量。 </li><li>一旦除去上清液，用荧光素二乙酸酯监测细胞生存力（当被细胞酯酶活细胞裂解，这种化合物变成荧光）。确定采用t控制细胞活力的siRNA靶向基因他的做法。注:转染过程本身应该有生存力的影响不大，如果执行正确。 <ol><li>添加荧光素二乙酸酯（5毫克/毫升的库存在丙酮）至每个孔中（1:在PBS 100的最终稀释度）。 </li><li>在室温下孵育一至五分钟，然后监测荧光在使用板读数器（460nm处激发，515nm处发射）的细胞。 </li><li>可选:要确保在孔中的荧光均匀，裂解使用缓冲剂如RIPA细胞;这提供了在井均匀的荧光信号，因为有些板读取器被配置为监测井的一小部分;如果细胞不是均匀地镀敷，荧光素二乙酸酯可以得到不准确的读数。 </li></ol></li><li>作为替代，以监测细胞生存力，监视的siRNA诱导的基因使用从贴壁细胞的RNA通过qPCR拦截。 <ol><li>之后将上清液从细胞中除去（ 步骤8.1以上），广告ðRLT缓冲液（其用于裂解和保存的RNA）直接连接到贴壁细胞以裂解它们。 </li><li>使用RNA制备试剂盒，如的RNeasy迷你试剂盒提取RNA，并使用定量PCR用基因特异性引物并监控基因敲除相对于对照siRNA处理。使用引物对Βactin或GAPDH用于标准化。 </li></ol></li><li>作为另一种选择，监察先天免疫应答的其它方面，通过加入FITC标记的大肠杆菌监视巨噬细胞的吞噬能力菌颗粒上直接使用试剂盒的贴壁细胞。按照制造商的协议，它只需几个小时，监视使用读板器或显微镜巨噬细胞吞噬。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。这篇文章是由赞助Lonza公司

众多研究已经发表，其中各个基因已在小鼠巨噬细胞被设为目标的siRNA。而脂质介导的转染已用于siRNA递送给个体基础上的巨噬细胞的细胞系，这些方法存在的生存能力，有限的基因敲除，以及变异性的潜在影响，从基因到基因。为了开发更强有力的测定法，可以用于靶向基因在中等或高通量的方式，我们的优化使用Amaxa公司nucleofector 96-穿梭系统，它显示出在基因敲除更多的一致性和生存能力有限...

在这个协议中，我们描述了有效的方法，以抑制使用的siRNA的基因在小鼠巨噬细胞系和监视这些处理上的先天免疫反应的影响。这些程序在96孔格式进行，允许RNAi筛选在中等或高通量的方式。 在对感染的反应，人类装入立即先天免疫反应和较慢的但更具体的适应性免疫反应。这种快速先天免疫反应涉及吞噬先天免疫细胞，包括巨噬细胞1的募集和活化。经典的活化巨噬细胞参与了急性炎症反应，产生抗微生物蛋白和化合物，细胞因子和趋化因子，并呈递抗原2,3。另外活化的巨噬细胞在调节机体免疫力，保持宽容，组织修复的作用，并且伤口愈合4-8。由于他们的职能范围广泛的巨噬细胞可起到多种疾病，包括动脉粥样硬化，关节炎，癌症和9的作用。因此，巨噬细胞的研究一直是研究的重要领域中的各种疾病领域的一段时间。 尽管其在先天免疫应答的重要性，巨噬细胞可以是具有挑战性的细胞来工作。特别是，它难以得到利用巨噬细胞中的脂质试剂有效转染无关联的毒性10,11。此外，即使当的siRNA被有效地传递到巨噬细胞中RNAi诱导的基因经常拦截的鲁棒性可以相当温和，可以从基因而异基因。 为了克服这些技术挑战，我们有两种小鼠巨噬细胞系RAW264.7 17和18 J774A.1优化转染和技术击倒12-16。此方法使用Amaxa公司Nucleofector 96孔班车系统转染;该系统使用专门的试剂和电穿孔的组合转染细胞在96孔格式19。转染后，我们描述的方法来最大化细胞恢复和生存能力，并最大限度地随后的siRNA诱导的基因敲除。为了说明这种方法的实用性，我们描述了一种协议，用于siRNA递送到这些巨噬细胞系，刺激这些细胞用脂多糖（LPS），并监视在生产几种促炎性细胞因子的水平的先天免疫应答。我们提供一种其中我们针对Toll样受体（TLR）家族，其成员感测LPS和其他相关的病原体分子模式（PAMP），以调节先天免疫的样本数据。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Amaxa nucleofector 96-well shuttle system</td>
			<td>Lonza</td>
			<td>AAM-1001S</td>
		<td></td></tr>
		<tr >
			<td >Amaxa SF cell line 96-well nucleofector kit</td>
			<td>Lonza</td>
			<td>V4SC-2096</td>
		<td></td></tr>
		<tr >
			<td >RAW264.7 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
		<td></td></tr>
		<tr >
			<td >J774A.1 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-67</td>
		<td></td></tr>
		<tr >
			<td >siGenome smartpool siRNA</td>
			<td>Dharmacon</td>
			<td>varies depending on gene</td>
		<td></td></tr>
		<tr >
			<td >Non-targeting control siRNA pool</td>
			<td>Dharmacon</td>
			<td>D-001206-13-20</td>
		<td></td></tr>
		<tr >
			<td >Block-iT fluorescent oligo for electrooration</td>
			<td>Invitrogen</td>
			<td>13750062</td>
		<td></td></tr>
		<tr >
			<td >Ultrapure E. coli O111:B4 LPS</td>
			<td>List Biological Laboratories</td>
			<td>421</td>
		<td></td></tr>
		<tr >
			<td >DMEM, high glucose</td>
			<td>Invitrogen</td>
			<td>11995-065</td>
		<td></td></tr>
		<tr >
			<td >RPM1-1640</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
		<td></td></tr>
		<tr >
			<td >Penicillin-Streptomycin Solution (Pen/Strep)</td>
			<td>Fisher</td>
			<td>SV30010</td>
		<td></td></tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200072</td>
		<td></td></tr>
		<tr >
			<td >96 well tissue culture plates</td>
			<td>Fisher</td>
			<td>07-200-89 </td>
		<td></td></tr>
		<tr >
			<td >96 well round bottom sterile plates (not coated)</td>
			<td>Fisher</td>
			<td>07-200-745</td>
		<td></td></tr>
		<tr >
			<td >Mouse IL-6 Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY406</td>
		<td></td></tr>
		<tr >
			<td >Mouse TNFa Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY410</td>
		<td></td></tr>
		<tr >
			<td >Fluorescein diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
		<td></td></tr>
		<tr >
			<td >RLT Bufer</td>
			<td>Qiagen</td>
			<td>79216</td>
		<td></td></tr>
		<tr >
			<td >Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
		<td></td></tr>
		<tr >
			<td >Vybrant Phagocytosis Assay Kit</td>
			<td>Invitrogen</td>
			<td>V-6694</td>
		<td></td></tr>
	</tbody>
</table>

using rna-interference to investigate the innate immune response in mouse macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

为了证明使用这种方法转染的效率，我们使用流式细胞仪（ 图1）监视的FITC标记的siRNA的摄取。 为了说明我们的用于监视先天免疫应答的方法的实用性，我们转染的靶向已知的先天性免疫调节基因插入的RAW264.7巨噬细胞系的siRNA，刺激了细胞用LPS，然后监测生产的促炎性细胞因子的IL- 6和TNFα。不同的TLR识别不同的PAMP，与从由TLR4 20识别革兰氏阴性细菌的脂?...

Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

using-rna-interference-to-investigate-innate-immune-response-mouse

Research

JoVE Journal

Immunology and Infection

10.6K 次观看.  National Jewish Health and University of Colorado School of Medicine. In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

视频: Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

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

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

细胞因子产生的病毒逃避解剖先天免疫信号

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

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. ...

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

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

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. ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

巨噬细胞报告器细胞分析，以检查聚合物表面的吸附蛋白层上的收费类似受体介导NF-kB/AP-1信号

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

生物工程

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

具有体外转录mRNA的初级巨噬细胞的高效转染

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

基因敲击由CRISPR/Cas9和细胞排序在巨噬细胞和T细胞线

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

聚乙胺包覆氧化铁纳米粒子作为载体, 在体外和体内向巨噬细胞输送小干扰 rna

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

医学

<meta charset="utf8"></head><body>用于体内基因调节的慢病毒载体制备

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

<meta charset="utf8"></head><body>作者聚焦:通过慢病毒载体介导的基因操作探索巨噬细胞免疫代谢

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

小鼠 体内 腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...