Name: isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation
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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><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&amp;rsquo;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>6-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&amp;ndash;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 &amp;mu;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 &amp;mu;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>20 G and 23 G 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>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），LPS...

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

36.3K 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

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

癌症研究

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.

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 ...

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 ...

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 ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

筛选生物活性纳米颗粒在噬菌体免疫细胞中的Toll样受体信号传导抑制剂

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

医学

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

巨噬细胞吞噬胸腺细胞吞噬的实验分析

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

从小鼠中分离和培养骨髓来源的巨噬细胞

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

Source: Zhen, Y. et al., <a href="https://app.jove.com/v/59731">Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages.</a> J. Vis. Exp. (2019)
This video demonstrates a procedure for analyzing peritoneal macrophage-mediated efferocytosis of apoptotic mouse thymocytes by fluorescence microscopy.

腹膜巨噬细胞对凋亡胸腺细胞的胞吐作用分析

Source: Zhen, Y. et al., Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages. J. Vis. Exp. (2019)
This video demonstrates a procedure ...