我们要感谢他们的意见和有益的意见的Tsirka实验室成员。这项工作是支持由R01NS42168到SET，12PRE12060489 RB，的NSF-3MT IGERT和特纳论文的奖学金LT，NSF-3MT IGERT JCN。

1。解剖（0天） <ol><li> P0-2鼠标幼仔低温麻醉。与一个Kimwipe浸泡，70％乙醇消毒纸巾擦拭头幼崽。 </li><li>卸下磁头，用一把剪刀，使用4幼仔每10cm的培养板中。在冰冷的Hank氏缓冲液的培养皿中，将磁头。 </li><li>锚头使用弯钳眼窝，并小心地去除皮肤覆盖直microforceps颅骨。 </li><li>取出颅骨与直microforceps，小心使用不穿刺或损坏皮质。最有效的方法是小脑附近，位于头的基础，这个区域将被丢弃起开始拆除。 </li><li>用小铲取出大脑，在冰冷的Hank氏缓冲液新鲜的陪替氏培养皿中，放置在（4）的大脑。 </li><li>从这点上的所有步骤利用microforceps下狄氏CTION显微镜。从大脑的腹侧，锚持有小脑组织，使脑两侧的两个小切口。要小心，不要砍下所有的方式，通过组织，覆盖在这个区域的脑皮质。 </li><li>轻轻逗在一块从两个皮质的脑和小脑。这两个皮层应形成的凹状。 </li><li>分开的两个皮质和定向单一皮质，内侧为进一步解剖。海马是很难观察到，但它位于相反的嗅球尖端的皮质出现小结节。 </li><li>取出海马，其中有一个月牙形状。皮质翻转过来查看背侧。 </li><li>使用嗅球作为一个起点，删除所有脑膜和嗅球本身从皮质。 </li><li>解剖皮质应置于15 ml锥形管14毫升的山坳D汉克的缓冲液冰。 </li></ol> 2。细胞培养（0天） <ol><li>预涂10厘米的组织培养皿5微克/毫升无菌水稀释聚-D-赖氨酸（PDL）3小时，在37°C </li><li>吸PDL，一次用无菌水冲洗板。干板的紫外线下，在组织培养罩20分钟。这一步之前应进行解剖过程。 </li><li>汉克的缓冲吸液管从4大脑皮质，适用于1个胰蛋白酶/ EDTA溶液4毫升，磨碎组织P1000尖，地方管在37℃培养箱中培养15分钟。 </li><li>完整的的小胶质媒体每管加4毫升，停止酶消化，混合，和自旋向下的内容1.5K转速为5分钟。 </li><li>吸出上清液，并重复洗涤4毫升完全培养基。将细胞重新悬浮在10毫升的完整的​​小胶质细胞介质（含10％FBS的DMEM，1％丙酮酸钠，0.08％庆大霉素）和过滤器通过40微Ñ​​网状细胞过滤。 </li><li>板8皮层，每10毫升的密度为10厘米的组织培养板和在细胞培养箱中于37℃和5％CO 2的（成人小胶质协议）。 </li></ol> （3天） <ol><li>更改媒体在所有细胞培养皿完整的小胶质媒体。 </li></ol> （10天） <ol><li>加入利多卡因HBSS（分离的小胶质细胞）到培养基中，10厘米的组织培养板在室温（RT）和孵育10-15分钟的400微升60毫米。 </li><li>收集媒体/细胞悬液板，板洗一次，汉克的缓冲。收集洗涤缓冲液，收回剩余的小胶质细胞。 </li><li>添加5mM的EDTA（pH8.0）中的细胞悬浮液，以使最终浓度为50μM，或以1/100稀释。 </li><li>自旋向下的细胞悬浮液以1,000×g离心（1500转）5分钟，重新悬浮在含1％FBS的1毫升的DMEM。 </li><li>计数活细胞数（每成人糜croglia 3.1/3.2）和分裂的细胞组织培养板所需的实验密度。约1×10 6小胶质细胞从10厘米板包含从4大脑皮质的收获。免疫荧光法，是推荐的密度为2.5×10 4个细胞每18毫米的盖玻片。 </li><li>允许至少24个小时的小胶质细胞完全返回到前分枝，静息状态下使用。 </li></ol>协议文本:（成人小胶质细胞） 1。组织收集<ol><li>大衣组织培养板与聚-D-赖氨酸（PDL）3小时37°C或一夜之间在4°C。一次洗板立即使用前用无菌水。 </li><li> CO 2窒息，并确定完成，安乐死是安乐死鼠标。用70％乙醇清洁脊髓区域。 </li><li>用一把剪刀剪的脊髓顶部的皮肤上，然后进行切断脊髓区域T1到T12。切剩余的肌肉的脊髓的两侧。 </li><li>慢慢地切割的椎骨microscissors你轻轻地握住您的指尖脊髓。要小心，不要穿刺脊髓组织是非常柔软。慢慢地提取脊髓microforceps。 </li><li>脊髓淹没在培养皿中含有冰冷的HBSS。使用光学显微镜取出所有可见的使用microforceps脑膜</li><li>切脊髓成横向分部小到足以产生几个片段。碎片的厚度不应该超过2毫米，以促进高效率的消化。脊髓片转移到15毫升锥形管含1毫升冰冷的HBSS。保持管在冰上，直到消化步骤。 </li></ol> 2。组织消化<ol><li>吸HBSS从15毫升锥形管含有脊髓组织。要小心，不要吸任何一张纸巾。 </li><li>添加1毫升的胰蛋白酶（2。5 g / L的照射猪胰蛋白酶和0.2 g / L的EDTA的HBSS），并在37℃孵育30分钟。 </li><li>要停止消化，消除胰蛋白酶和初级小胶质细胞培养基中加入3毫升含有血清制止酶消化。 </li><li>吸取和，撞出组织。使用40微米的细胞过滤筛选的细胞悬液。 </li></ol> 3。细胞计数及电镀<ol><li>混合等体积的细胞悬浮液和DAPI溶液。 </li><li>使用血球计数细胞。 </li><li>板5 7×10 5个细胞/ ml在PDL涂层35×10毫米的菜肴之间的密度。在一个较低的密度电镀阻止细胞的生长，即使当细胞培养在12孔板中，有一个较小的区域相比，35×10毫米的菜肴。 </li></ol>

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Neurosci. 26, 1880-1887 (2006).</li><li>Sasmono, R., Oceandy, D., Pollard, J., Tong, W., Pavli, P., Wainwright, B., Ostrowski, M., Himes, S., Hume, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+macrophage+colony-stimulating+factor+receptor-green+fluorescent+protein+transgene+is+expressed+throughout+the+mononuclear+phagocyte+system+of+the+mouse.">A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.</a> Blood. 101, 1155-1163 (2003).</li><li>Ekdahl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+-+tuning+and+pruning+adult+neurogenesis.">Microglial activation - tuning and pruning adult neurogenesis.</a> Front Pharmacol. 3, 14 (2012).</li><li>Sierra, A., Gottfried-Blackmore, A., McEwen, B., Bulloch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+derived+from+aging+mice+exhibit+an+altered+inflammatory+profile.">Microglia derived from aging mice exhibit an altered inflammatory profile.</a> Glia. 55, 412-424 (2007).</li></ol>

作者什么都没有透露。

小胶质细胞调节中枢神经系统正常运作，以及各种病症的炎症反应。小胶质细胞突触重塑功能已被牵连在维护正常脑动态平衡15。在神经源性级联，他们参与了海马4，16齿状回神经祖细胞的间隙。因此，有必要制定一个文化系统，在新生儿和成人学习的小胶质细胞，这将覆盖的广袤的发展和成年后，整个小胶质细胞有多个分立功能。我们已经提出了一个快速，高效的小胶质细胞培?...

小胶质细胞是中枢神经系统，最密切相似周巨噬细胞的结构和功能1的的居民免疫细胞的。它最近已表明，出生后的小胶质细胞来源于原始粒细胞前体细胞和胚胎发育的第8天之前产生，颠覆了以前的概念，即出生后的造血祖细胞是小胶质细胞的来源在成人大脑2。他们在一些神经系统疾病中发挥关键作用，并能快速响应感染或受伤的释放促炎或抗炎性细胞因子3。因此，小胶质细胞在中枢神经系统中可以被操纵，影响疾病进展包括一个独立单元。发展强大的和可重复性的方法分离和培养新生儿或成人的小胶质细胞是重要的未来的研究。 小胶质细胞是已知的，在许多的脑疾病是至关重要的玩家。最近，ROL上课新兴的细胞在正常脑小胶质细胞吞噬多余的从1，4海马齿状回神经祖细胞的发育和功能。小胶质细胞也可以调节几个影响脊髓的神经系统疾病，如MS，神经性疼痛，脊髓损伤5-7。脊髓小胶质细胞相比，脑小胶质细胞激活信号，8，9，在本地环境中的差异可能是由于不同的反应。因此，重要的是要建立一个适当的文化研究脊髓小胶质细胞在体外系统。出生的小胶质细胞产生显着的促炎性细胞因子一氧化氮相比，成体细胞在体外刺激后IFN-γ或TNF-α的10,11进一步突出，需要使用成体细胞研究小胶质细胞的某些疾病的情况下。 我们采用的协议，在实验室中立方米lture新生儿小胶质细胞是根据最近的方法，利用摇动混合胶质细胞培养在努力消除小神经胶质细胞的细胞培养瓶12从表面的变化。我们也给出了基于业， 等[13]首次描述了一个协议，从成年小鼠脊髓小胶质细胞培养方法。这种方法提供了更快的方法来培养成年细胞相比其他可用的协议14。将所得的制剂是70％的小胶质细胞，剩余的百分比组成的星形胶质细胞。虽然我们的文化的纯度较其他已发表 ​​的方法13，这种文化系统是有益的探索小胶质细胞在培养应对各种活化刺激，以及为研究疾病，主要影响脊髓强势炎症反应的一个主要特征。 所有描述的协议已获石溪分校学报JOURNALTY IACUC。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>EDTA, 5mM</td>
			<td>Invitrogen</td>
			<td>15567-028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lidocaine, 60mM</td>
			<td>Sigma</td>
			<td>L-5647</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin/EDTA</td>
			<td>Cellgro</td>
			<td>25-052-CI</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM, 1X</td>
			<td>Cellgro</td>
			<td>10-017</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium Pyruvate</td>
			<td>Cellgro</td>
			<td>25-000-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamycin Sulfate</td>
			<td>Biowittaker</td>
			<td>17-518Z</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 x 10mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Poly-D-Lysine, 100 &mu;g/ml</td>
			<td></td>
			<td></td>
			<td>Dilute 1:20</td>
		 </tr>
		 <tr>
			<td>HBSS, 1X</td>
			<td>Cellgro</td>
			<td>20-023-CV</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

culturing microglia from the neonatal and adult central nervous system

小胶质细胞是驻地的中枢神经系统（CNS）和巨噬细胞样的细胞，因此，极为重要的作用，在生理和病理过程，如中枢神经系统的成熟发展，多发性硬化，脊髓损伤。小胶质细胞可以激活并招募行动由神经元的损伤或刺激，如性轴索损伤看出，在MS或缺血性脑外伤导致中风。这些免疫功能的中枢神经系统成员也被认为在非病理条件下在突触可塑性的角色。我们采用从新生儿和成人组织的目的是最大限度地存活细胞数，同时最大限度地减少混淆的变量，如其他中枢神经系统的细胞类型和细胞培养碎片的存在下培养的小胶质细胞的协议。我们利用大和容易辨别的中枢神经系统组件（ 如皮质，脊髓节段），这使得整个过程可行，重复性好。成体细胞的使用s是一个合适的替代使用新生儿脑小胶质细胞，，许多病理研究主要影响产后脊髓。这些培养系统也可用于直接测试化合物的效果，可以抑制或促进小胶质细胞活化。由于小胶质细胞活化可以塑造成人中枢神经系统疾病的结果，需要在体外​​系统，在该系统中，新生儿和成人的小胶质细胞可以培养研究。

<html>静息和活化的小胶质细胞的一个例子是图1中所示。小胶质细胞可视化电镀后24小时（ 图1a），表现出分枝形态（休息）。曝光吸试剂，细菌脂多糖（LPS）被激活小胶质细胞形态变化细胞（ 图1b）。 电镀细胞计数的一个例子如图2所示。由于细胞碎片的存在下，DAPI Fluoromount（而不是台盼蓝）溶液中加入等体积的细胞悬浮液?...

Watch this Scientific Journal Video about Culturing Microglia from the Neonatal and Adult Central Nervous System at JoVE.com

Culturing Microglia from the Neonatal and Adult Central Nervous System

我们概述方法从新生儿大脑皮层和脊髓成人的可行的小胶质细胞的高效和快速隔离/文化。解剖和电镀皮质小胶质细胞可以在90分钟内完成，与随后发生〜10天的初始剥离后的小胶质细胞的收获。

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

culturing-microglia-from-the-neonatal-and-adult-central-nervous-system

Research

JoVE Journal

Immunology and Infection

27.7K 次观看.  Stony Brook University. 我们概述方法从新生儿大脑皮层和脊髓成人的可行的小胶质细胞的高效和快速隔离/文化。解剖和电镀皮质小胶质细胞可以在90分钟内完成，与随后发生〜10天的初始剥离后的小胶质细胞的收获。

视频: Culturing Microglia from the Neonatal and Adult Central Nervous System

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

表面等离子体共振检测到宿主细胞质的毒素易位

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

科研

免疫与感染

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

热酚水提取纯化及革兰氏阴性菌脂多糖的可视化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

皮层小胶质细胞的分离与保存免疫表型和功能从新生儿鼠

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

在鼻内siRNA输送到中枢神经系统期间放置小鼠的定位装置

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

分离中枢神经系统组织和相关脑膜，用于免疫细胞的下游分析

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...