在统一服务大学的校内计划资助。

1。新生大鼠脑组织解剖<ol><li>寒意莱博维茨的L-15条件培养基（莱博维茨的L-15 + 0.1％BSA + 1％笔/ STREP）至4°C。温暖的培养基（DMEM培养基+ 10％胎牛血清+ 1％青霉素/链霉素）至37°C。准备4-5毫升条件的L-15冰媒体4 60x15毫米培养皿。 100％的酒精消毒所有的手术工具。 </li><li>用70％乙醇冲洗P2新生儿说唱幼仔。 （P1〜P5之间的新生大鼠岁的幼崽，可用于在该协议）。 </li><li>迅速斩首老鼠幼犬用无菌锋利的剪刀，并立即下降到70％的乙醇头的。共有5幼鼠重复，然后转移到盐水首脑。 </li><li>整个大脑从元首和放入培养皿中，有4 - 5毫升L - 冰（15解决莱博维茨的L-15 + + 1％，0.1％BSA笔/链球菌）。 </li><li>重复程序1.2 - 1.4在垃圾其余幼仔。 </li><li>从脑脊膜和转移DESIRED脑组织进入一个新的Petri菜用4-5毫升的L-15的解决方案（ 即皮层，小脑，全脑等）（莱博维茨的L-15 + 0.1％BSA + 1％的PEN /链球菌）在冰上。 </li></ol> 2。制备混合胶质细胞群<ol><li>在不损害脑组织，用剪刀或刀片，10毫升吸管组织转移到无菌的50毫升锥形管。 </li><li>在4°C离心5分钟在2500离心力的锥形</li><li>吸上清。莱博维茨的L-15 +新鲜的L-15介质中加入4-5毫升（0.1％BSA + 1％笔/链球菌）。 </li><li>吸取组织上下用无菌10毫升的移液器10倍。 </li><li>放置到一个新的50毫升锥形管一个细胞过滤器（100微米的孔）。吸取组织上下无菌5毫升吸管和吸管冲洗细胞过滤一次，免除通过细胞进入锥形管过滤材料。 </li><li>细胞过滤冲洗4-5毫升新鲜的L-15媒体（莱博维茨的L-15 + 0.1％BS+ 1％笔/链球菌）。 </li><li> 5分钟紧张的细胞离心圆锥形在2500离心力4°C。 </li></ol> 3。混合胶质细胞培养的电镀和维护<ol><li>对于每个小狗大鼠脑处理，准备1无菌的T-75瓶，加入12毫升培养基（DMEM培养基+ 10％FBS + 1％青霉素/链霉素），将每个烧瓶。 </li><li>从颗粒细胞吸上清，加5-6毫升培养基细胞沉淀。吸取向上和向下10毫升吸管10倍。 </li><li>等量细胞悬液转移到每个T-75瓶。 </li><li>在37°C孵育为1-3周总在5％ 二氧化碳培养箱瓶。 </li><li>应首先培养瓶为5天，第五天，将在每个烧瓶文化媒体与新媒体的12毫升，并返回到孵化器。然后，每3天更换空调在每个烧瓶媒体12毫升新鲜媒体实现合流。这必须做NE非常仔细地在细胞附着在烧瓶底部不接触。 </li></ol> 4。初级小胶质细胞的分离和电镀<ol><li>后混合胶质文化的完全融合，从孵化器中删除瓶和瓶封口膜覆盖的上限，以防止气体交换与环境空气。 </li><li> 1小时摇匀瓶100 RPS（实验室助理，Jeio科技SI-600）在37°C。 </li><li>收集了10毫升吸管所有瓶瓶表面上不破坏胶质细胞层的媒体。媒体将在50 ml锥形管。添加新媒体的保温瓶和回报瓶孵化。 </li><li> 5分钟离心锥管在2500离心力4°C。 </li><li>所有锥形管吸出上清液。细胞颗粒高纯度的小胶质细胞。 1毫升的小胶质细胞电镀媒体（DMEM培养基+ 10％的FES + 1％青霉素/链霉素）重悬浮颗粒。 </li><li>在媒体使用的再悬浮细胞密度计数1血细胞计数。 </li><li>加入适当体积小胶质细胞的电镀媒体，以实现一个2×10e5细胞密度每毫升。板作为适当的实验分析。 </li><li>让小胶质细胞附着在一夜之间。 </li></ol> 5。代表结果上述结果，在高纯度的初级小胶质细胞培养协议。巨噬细胞/小胶质细胞特异性标志物（Iba1）免疫组化确定，镀金的小胶质细胞培养&gt; 90％纯（ 图2）。此外，星形胶质细胞，少突胶质细胞和神经细胞特异性标志物的染色表明最小的污染（ 图2）。建立混合胶质细胞培养后，小胶质细胞可以通过摇晃起来，以连续4次被隔离。最初的小胶质细胞的分离，将产生约2.2×106细胞/毫升或每10瓶和预期产量约900万细胞减少与每一个连续的沙柯。通过这种方法分离的小胶质细胞已成功地用于研究细胞的吞噬能力，如产生一氧化氮的功能，神经毒性（ 图3）13-14。 <img alt="图1" src="/files/ftp_upload/3814/3814fig1.jpg" /> 图1。准备混合胶质细胞培养和初级小胶质细胞的分离协议概述。 <img alt="图2" src="/files/ftp_upload/3814/3814fig2.jpg" /> 图2。使用（IBA-1，红色），小胶质细胞，星形胶质细胞（GFAP，绿色），神经元（NeuN染色，红色）和胶质细胞的免疫组织化学分析验证的高纯度的小胶质细胞的分离（CC1的，红色）的特异性标志。还表现在培养细胞用DAPI（蓝色）的DNA染色。 <img alt="图3" src="/files/ftp_upload/3814/3814fig3.jpg" /> 图3。小胶质细胞分离方法descri已用在床的检测，包括但不限于，吞噬功能检测，功能检测和神经毒性研究。在这些研究中，我们发现，小胶质细胞（IBA-1阳性，绿色），当暴露控制（A）或脂多糖（二）对待媒体，提高他们的荧光标记的乳胶珠（红色），可量化的吞噬功能（ C）此外，LPS（1ng/ml）治疗可以增加小胶质细胞的一氧化氮（四）生产。最后，通过同时Transwell小插入分隔或直接的小胶质细胞，神经元的文化，我们已经发现，小胶质细胞培养与LPS可以诱导神经细胞死亡，乳酸脱氢酶（LDH）释放（，E，F）。

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没有利益冲突的声明。

虽然此协议经常用来生产纯净和健康的小胶质细胞的研究实验，在程序的关键部件的技术方面的慎重考虑，将尽量减少在孤立的小胶质细胞的变化。首先，是要尽量减少缺氧和缺血性脑损伤的组织，在新生幼鼠脑组织工作，及时清扫。然而，同样重要的是从大脑中彻底清除脑膜覆盖在解剖，因为在场的脑膜将有助于显着的成纤维细胞污染，由于这些培养条件下的快速增殖率。 <p class="jove_content"...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 60毫米×15毫米培养皿</td><td>费希尔品牌</td><td> 0875713A </td><td></td></tr><tr><td>夏普解剖剪刀</td><td>罚款科学工具</td><td> 14094-11 </td><td></td></tr><tr><td>杜蒙＃7B钳标准提示，弯曲，11厘米</td><td>罚款科学工具</td><td> 11270-20 </td><td></td></tr><tr><td>杜蒙＃5镊子标准的提示，直，11厘米</td><td>罚款科学工具</td><td> 11251-10 </td><td></td></tr><tr><td> 50毫升锥形离心管</td><td>厂商VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5毫升血清移液器</td><td>格雷尼尔生物一</td><td> 606180 </td><td></td></tr><tr><td> 10毫升血清移液器</td><td>格雷尼尔生物一</td><td> 607180 </td><td></td></tr><tr><td> 100微米无菌尼龙细胞过滤器</td><td>鹘</td><td> 35-2360 </td><td></td></tr><tr><td> 75厘米2组织培养瓶</td><td>康宁</td><td> 430641 </td><td></td></tr><tr><td> Dulbecco的最低基本培养基（DMEM培养基） </td><td> Gibco公司（Invitrogen公司） </td><td> 31053-028 </td><td></td></tr><tr><td>莱博维茨的L-15培养基</td><td> Gibco公司（Invitrogen公司） </td><td> 11415064 </td><td></td></tr><tr><td>胎牛血清</td><td> Gibco公司（Invitrogen公司） </td><td> 16000-036 </td><td></td></tr><tr><td>胎儿马血清</td><td>费舍尔</td><td> SH3007402 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Gibco公司（Invitrogen公司） </td><td> 15140163 </td><td></td></tr><tr><td> 100％的乙醇</td><td>华纳格雷厄姆公司</td><td> 64-17-5 </td><td></td></tr><tr><td>磷酸盐缓冲盐溶液，10X，pH值7.4 </td><td>质量生物学，INC。 </td><td> 119-069-131 </td><td> 1X在无菌蒸馏水</td></tr><tr><td>生化危机袋</td><td>厂商VWR </td><td> 14220-028 </td><td></td></tr><tr><td>血球</td><td>豪塞尔科学</td><td> 1492 </td><td></td></tr><tr><td> 6井细胞与cellBIND表面培养皿</td><td>康宁</td><td> 3335 </td><td></td></tr></tbody></table>

primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue

小胶质细胞在哺乳动物的脑细胞的总人口约12％的帐户。虽然神经元和神经胶质细胞被认为是神经系统的主要类型的细胞，小胶质细胞发挥 ​​显著作用，在正常脑组织的生理组织碎片和病原体监测和维持动态平衡通过吞噬活性1,2实质。小胶质细胞被激活过程中的损伤和疾病的条件，包括神经退行性疾病，脑外伤，中枢神经系统感染3。这些活化条件下，增加小胶质细胞的吞噬活性，经过morpohological和增殖的变化，积极分泌活性氧和氮物种，促炎性趋化因子和细胞因子，往往激活旁分泌或自分泌环4-6。由于这些小胶质细胞反应在神经系统疾病的发病机制的研究主要集中在microglia保证。 由于大脑细胞的异质性，这是技术上的困难，具有纯度高，在体内实验中获得足够的小胶质细胞的样品材料。小胶质细胞的神经保护和神经功能的研究现状，需要常规的技术方法始终产生足够的产量为研究的纯洁和健康的小胶质细胞。我们提出，在文字和视频，从混合胶质文化的隔离纯初级小胶质细胞的各种下游应用协议。简单地说，这种技术采用分离新生幼鼠脑组织生产混合胶质细胞培养。混合胶质细胞培养达到汇合后，小胶质细胞主要是机械分离的文化由摇晃的时间很短。小胶质细胞，然后镀上高纯度的实验研究。 这种方法的原理和协议在文献7,8所述。此外，隔离初级小胶质细胞的替代方法有很好的描述9-12。匀浆脑组织可通过密度梯度离心分离，产生初级小胶质细胞12。然而，离心是适中的长度（45分钟），并可能会导致细胞损伤和激活，以及，导致富含胶质细胞和其他细胞群。另一个协议已动用隔离初级小胶质细胞的各种生物通过长时间的晃动，而在文化9-11（16小时）。摇动后，媒体上清离心分离小胶质细胞。这不再两步分离方法也可能干扰小胶质细胞的功能和激活。我们主要是利用以下的小胶质细胞的分离协议在我们的实验室有许多原因：（1）初级小胶质细胞在体内的生物学模拟更忠实，比永生的灭鼠小胶质细胞株（2）额定机械破坏，最大限度地减少潜在的细胞功能障碍或激活，（3）没有足够的产量可以通过混合胶质细胞培养获得。 重要的是要注意该协议使用新生幼鼠脑组织小胶质细胞分离和使用旧的大鼠，分离小胶质细胞可以显着影响产量，激活状态，孤立的小胶质细胞的功能特性。有证据表明，老龄化与小胶质细胞功能障碍，增加神经发炎和神经退行性病变的联系，所以以前的研究都采用体外成人小胶质细胞小胶质细胞在神经退行性疾病中的作用更好地了解地方老龄化是一个重要参数。但是，小胶质细胞体外不能被保存在长时间的文化。因此，虽然该协议扩展了初级小胶质细胞中的文化生活，也应注意到，小胶质细胞的行为ðifferently从成人的胶质细胞和体外研究时，应慎重考虑翻译体内设置。

Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

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

隔离初级小胶质细胞的大脑细胞的异质性是必不可少的，以调查他们在生理和病理条件下的作用。本协议描述了机械隔离和混合细胞培养技术，提供高产量和高纯度的，可行的初级小胶质细胞<em&gt;在体外</em&gt;学习和下游应用。

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 ...

primary-microglia-isolation-from-mixed-glial-cell-cultures-neonatal

Research

JoVE Journal

Immunology and Infection

39.1K 次观看.  Uniformed Services University. 隔离初级小胶质细胞的大脑细胞的异质性是必不可少的，以调查他们在生理和病理条件下的作用。本协议描述了机械隔离和混合细胞培养技术，提供高产量和高纯度的，可行的初级小胶质细胞在体外学习和下游应用。

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

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 ...

科研

免疫与感染

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

从小学的人角质形成细胞器官筏文化的代

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

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 ...

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 ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

从大鼠睾丸支持细胞的分离和管周细胞

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

从小鼠在原代肝细胞培养物的炎性反应的诱导

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

新生儿初乳喂养前后离散催产素分泌脑核细胞应激与炎症的评估

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

原生动物寄生虫感染原发性星细胞和微胶质的伴随分离

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...