Name: selective depletion of microglia from cerebellar granule cell cultures using l-leucine methyl ester
Uploaded: 2015-07-07T15:00:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

This research was support by an Aims2Cure, UK and a UCL Impact Award Ph.D. studentship to JMP and an MRC Capacity Building Ph.D. studentship in Dementia to JMP.

本文所述的所有实验均按照英国动物（科学程序）1986年法进行的。 1.准备仪器，文化传媒，和菜名<ol><li>准备两个不锈钢实验室解剖剪刀和两个不锈钢镊子实验室。高压灭菌器的所有仪器和地方文化引擎盖O / N在紫外光（UV）光灭菌。 </li><li>使500毫升最小必需培养基（MEM）培养基（10％胎牛血清[FBS]，20mM的氯化钾，25mM的碳酸氢钠 ，30mM的D-葡萄糖，2mM的L-谷氨酰胺，100U / ml青霉素，100 100μg/ ml链霉素和6微克/毫升氨苄青霉素），并过滤杀菌。储存在4℃数月。 </li><li>准备包含14个毫米，1号盖玻片厚度24孔培养板。高压灭菌的盖玻片和外衣在100毫克/升聚d-赖氨酸（PDL）如前所述3。紫外线消毒O / N。 </li></ol> 2.准备CGC培养液<ol><li>制备"溶液A"（100毫升）的高压灭菌，处理过的UV光，无菌的dh 2 O含有250mg葡萄糖，300毫克牛血清白蛋白[BSA]的，955毫克磷酸盐缓冲盐水[PBS]（ 钙离子和毫克+游离）和1毫升3.8％ 硫酸镁 ·7H 2 O. </li><li>制备含1毫升5毫克/毫升胰蛋白酶稀释于9ml溶液A的&#39;溶液B&#39;（10毫升）中</li><li>准备"溶液C"（10毫升）含1000单位的DNA酶，0.5毫克的大豆胰蛋白酶抑制剂，100微升3.8％硫酸镁4·7H 2 O，稀释至10毫升溶液A. </li><li>制备，含3.2 ml溶液的C，稀释至20ml用溶液A&#39;溶液D&#39;（20毫升）中</li><li>制备的BSA / Earle氏平衡盐溶液（EBSS）含有EBSS，25mM的碳酸氢钠 ，3mM的硫酸镁溶液·7H 2 O，和4％BSA，pH 7.4中，先前所描述的3。 </li><li>制备LME通过加入纯LME到5毫升的MEM培养基，得到150毫LME的终浓度，使用台式pH计返回溶液至pH 7.4，并过滤灭菌使用28mm的聚醚砜（PES）为0.2μm注射器过滤器。 </li></ol> 3.培养信用担保公司<ol><li>收集4-7日龄的幼鼠小脑如前所述3。含冰5毫升解决方案的培养皿立即到位。 </li><li>倒出从小脑和地方组织在培养皿盖过量的溶液。 </li><li>切碎组织，在至少三个不同方向的一个良好的火烧刀片。 </li><li>加入切碎的组织到溶液B（冲洗培养皿以溶液，以确保有限的组织丢失）并置于水浴中，在37℃下进行5分钟。轻轻摇动每两分钟。 </li><li>添加溶液D（20毫升）到管中以中和胰蛋白酶，抖动和占rifuge在65×g离心5分钟。 </li><li>倒掉上清。 </li><li>重悬在4毫升的溶液C.磨碎很好地减小直径，直到悬浮液是尽可能均匀的三个火烧玻璃吸管（约10每次）。 </li><li>慢慢地，轻轻地对BSA / EBSS顶部加几滴这种匀浆。如果它开始通过BSA / EBSS下沉，更磨碎和/或补充一点的解决方案C.将匀浆应该坐在一个层上的BSA / EBSS之上。不要摇晃。旋转，在100×g离心5分钟。 </li><li>除去上清，重悬沉淀（软）在1ml MEM培养基。 </li><li>计数使用血球和盘细胞为80万细胞/盖玻片在500μlMEM培养基。发生在一个培养箱中在37℃下用6％的CO 2。 </li><li>第二天（预习后24-36小时）改变介质。使含有10μM的细胞周期抑制剂阿糖呋喃胞苷（阿糖胞苷）的MEM培养基中的溶液。 <ol><li>对于每一个盖玻片，保留250微升旧培养基的新鲜24孔板，吸去休息，加入250微升正常的MEM培养基中，并吸出此以洗涤细胞，然后加入250μl的旧培养基的回细胞和顶部向上用250微升含阿糖胞苷介质中。 注:细胞可以来自6天内使用体外 （DIV）。 </li></ol></li></ol> 4.选择性地耗尽小胶质细胞（在演出6格） <ol><li>添加纯LME至5ml MEM培养基的单独的等分试样，得到150毫LME的终浓度。 </li><li>返回该溶液至pH 7.4，并过滤灭菌使用28mm的聚醚砜（PES）为0.2μm针筒过滤器。 </li><li>随着对于对照培养正常的MEM培养基稀释溶液至50mM的（2×LME）的MEM培养基中并置于水浴中的浓度在37℃下10分钟。 </li><li>除去一半（250微升）从CGC培养的MEM培养基中，并保持在37℃。 </li><li>处理细胞用含有2×LME（250微升）的MEM培养基， 即 1:1稀释得到的25mM的阱浓度，或与对照培养物预热MEM培养基而不LME（250微升）。 </li><li>孵育细胞在37℃下在湿润的气氛中，用6％CO 2下1小时。 </li><li>洗涤细胞在新鲜预热的MEM培养基两次以除去含LME介质，然后更换保留培养基和新鲜预热的MEM培养基等体积的相应孔中。 </li><li>返回培养至孵化器（加湿用6％的CO 2，37℃）和在任何进一步处理离开休息24小时。 </li></ol>

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The authors declare that they have no competing financial interests.

最重要的步骤，以确保成功选择性消除小胶质细胞来自CGC和/或混合培养物有:1）保持无菌，健康的文化CGC; 2）过滤灭菌的含LME介质并返回该溶液至pH 7.4; 3）保持保留CGC媒体和LME-含37媒体 °C，这样可以避免热冲击;和4）迅速工作，以减少单元保持在培养箱外的时间。 我们使用25毫米LME从我们的CGC文化耗尽胶质 - 以前在我们的实验室进行了优化的浓度。尽管如此，一些小胶质细胞仍留在培养之后，LME治疗，其中有一些相信可能反映分化，非增殖的小胶质细胞的群体。事实上，汉比等人（2006）已发现，50-75毫LME被要求以消除来自高密度星形胶质细胞单层13的小胶质细胞。我们也曾经在纯小胶质细胞C中所示ultures，10毫米LME足以杀死所有的小胶质细胞，但只有一个24小时的培养时间14。 替代方法来消除污染小胶质细胞培养模型最近被曝光。克罗克等人（2008）证实了一种新方法通过促 ​​进神经干细胞（NSC）分化为星形胶质细胞在出生后（P0-P2）小鼠15，以消除从星形细胞培养的小胶质细胞。他们发现，这种技术完全消除小胶质细胞从培养，虽然该现象的基本机制是未知的。此外，熊丸等人，（2012）中使用的脂质体氯膦酸盐-已知诱导凋亡的巨噬细胞一biophosphonate -消除小胶质细胞原代星形细胞培养从P3小鼠16。该化合物适用于以类似的方式和在功效LME的相同水平;但作者认为，由于LME可能是有毒的在特定条件下的星形胶质细胞由于它的自由扩散进入细胞-星形胶质细胞破坏能力的附着力13 -脂质体氯膦酸二钠代表的改进方法，从文化的小胶质细胞耗尽。然而，我们发现，25毫米LME一直没有对星形胶质细胞活力和增殖的影响。 本文所描述的协议使用LME的特定浓度，以选择性消除从信用担保公司的神经元富集培养物的小胶质细胞。这代表了一个有力的实验方法来确定的小胶质细胞在各种条件下的神经元的影响，并继续提供重要的数据，以研究人员研究神经炎症的神经性疾病中的作用。

人类大脑包含估计为85十亿神经元和另一个85十亿非神经元细胞，包括神经胶质1。对于过去的100年神经科学家主要集中在神经元细胞群的大部分时间里，相信神经胶质细胞比被动的支持细胞为神经元提供结构支持更小 - "神经胶质细胞"翻译成英语的，因此希腊词源"胶水"。然而，最近它已成为日益明显的是，神经细胞 - 胶质细胞的相互作用可能是更为基本的神经生物学，神经生理学和许多神经变性疾病的发生和进展的基本方面。小脑颗粒细胞（信用担保公司），最丰富的神经元的同质人口在人的大脑，支配小脑和弥补90％以上，其细胞成分。因此，这些细胞已被广泛用于在体外作为吨的模型系统他研究神经发育，功能和病理2-6。 不过，CGC文化还含有小胶质细胞和神经胶质细胞等在争议显著的比例。其结果是，CGC数据推定显示直接神经元对不同的细胞治疗可能实际上出现 - 在部分或全部 - 从在培养相邻神经胶质的间接二次响应。为了评估这一点，我们选择的除去从CGC神经元培养小胶质用的L-亮氨酸甲酯（LME）的助剂。 LME是一个lysomotropic剂原本用于选择性地破坏巨噬细胞7，并一直使用也选择性地从神经元，星形胶质细胞和神经胶质混合培养8,9,10消耗小胶质细胞。 LME由巨噬细胞和小胶质细胞内化，其中它引起溶酶体破裂和随后的细胞凋亡13,14。巨噬细胞和小胶质细胞是典型丰富的溶酶体，使他们成为particula暴露于LME治疗RLY脆弱。该协议提供了一个功能强大，简单易用的方式，以确定小胶质细胞在利用CGC和其他神经元/胶质细胞培养系统实验的贡献。

<table><tbody><tr><td>Forceps&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4142</td><td>The curved end facilitates removal of the cerebellum&amp;nbsp;</td></tr><tr><td>Micro-dissecting scissors</td><td>Sigma-Aldrich</td><td>S3146</td><td>Straight, sharp point facilitates rodent P4-7 dissection&amp;nbsp;</td></tr><tr><td>L-leucine methyl ester hydrochloride</td><td>Sigma-Aldrich</td><td>7517-19-3</td><td></td></tr><tr><td>EBSS solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E7510-500 ml</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>27964-99-4</td><td>Coat coverslips 1 day before use&amp;nbsp;</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025</td><td></td></tr><tr><td>Soybean trypsin inhibitor&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T6414</td><td></td></tr><tr><td>Mouse anti-ED1 antibody&amp;nbsp;</td><td>Abcam</td><td>ab31630</td><td></td></tr></tbody></table>

selective depletion of microglia from cerebellar granule cell cultures using l-leucine methyl ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

这种技术的以选择性地消除从CGC和/或混合培养物的小胶质细胞的能力依赖于研究者的准确识别，并从它们的周围细胞分化的小胶质细胞的后续能力。这可以使用一个小胶质特异性细胞制造者，如植物凝集素B4中实现， 如图1所示。作为表现出在图2中，没有观察到改变星形细胞和神经元密度和形貌进行记录，对于各主要的治疗组。重要的是，观察到的神经元或星形细胞形态无显著变化时培养物用LME单独治疗，支撑所述化合物的一个小胶质特定的操作。 图3A示出了在星形细胞密度，并从主啮齿类星形细胞培养12形态没有可观察的变化，但不显示污染小胶质细胞的损失。在图3B中 ，如所述的，未进行任何变更astrocyTE密度和形态均可见;然而，观察到污染小胶质数目和反应性的损失。对于在本手稿中使用的免疫细胞化学（ICC）协议的简要说明，请参见参考11。 <img alt="图1" src="/files/ftp_upload/52983/52983fig1.jpg" /> 图1:LME治疗信用担保公司的表征 。 （A）中从免疫细胞化学代表性图像上CGC培养进行（ICC）的实验治疗后25-75毫LME 1小时，随后洗掉。 IC卡被用DAPI（蓝色），总细胞数的定量和那些显示凋亡形态学和植物凝集素-B4（ⅠB4）（绿色），用于小胶质鉴定和定量进行。比例尺= 30微米。细胞凋亡的显示形态定义为核染色质凝结（灰条（二）定量;％的在现场总细胞数），总的小胶质细胞数，作为对照水平（绿色棒）的百分比。不成对学生t -tests进行相关的控制和LME的具体治疗之间进行; * P &lt;0.05，** P &lt;0.01，*** P &lt;0.001; N = 3（ 即 3个独立重复）。 <img alt="图2" src="/files/ftp_upload/52983/52983fig2.jpg" /> 图2:25mM的LME治疗对神经元和星形胶质细胞中的CGC培养的影响表征 。小组代表图像从CGC培养1小时进行处理后，25毫米LME ICC实验。 IC卡用DAPI（蓝色），抗β-III微管蛋白（绿色），用于识别改变的神经元密度和抗GFAP（红色）的识别变化星形细胞密度和形态的执行。阴性对照组（-ve CTR）图像代表CGC文化，其中初级antibodies被省略。比例尺= 30微米。 <img alt="图3" src="/files/ftp_upload/52983/52983fig3.jpg" /> 图3:25毫LME治疗在从星形细胞培养物中除去小胶质细胞的有效性的进一步表征 。 （A） 的面板代表性图像从初级星形细胞培养1小时进行预处理后用25mM LME ICC实验。 IC卡用DAPI（蓝色），抗GFAP（红色），用于识别改变星形细胞密度和形态，以及抗ED1（绿色），用于识别的小胶质细胞数的执行。 （B）从用LPS（1微克/毫升）处理24小时的初级星形细胞培养进行治疗后的ICC实验以下预处理用25mM LME 1小时代表性图像。 IC卡被作为A，但用抗GFAP（绿色）和抗iNOS的（红色）描述，以确定激活微克进行LIA。阴性对照组（-ve CTR）图像代表CGC文化，其中省略了初级抗体。比例尺= 30微米。

Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

小胶质细胞的小脑颗粒细胞使用L-亮氨酸甲酯选择性耗尽

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

selective-depletion-microglia-from-cerebellar-granule-cell-cultures

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.6K 次观看.  University of Washington. Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature. 

视频: Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

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 ...

科研

免疫与感染

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 ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

神经科学

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

密度梯度离心法分离小鼠原发胶质细胞的研究

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

从一个成年小鼠脑半球分离区域特异性微胶质，进行深度单细胞RNA测序

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

通过磁活化细胞分选在脱髓鞘动物模型中分离小鼠原代小胶质细胞

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...

Modeling Reactive Oxygen Species-Induced Neuronal Death in Mouse Cerebellar Granule Neurons

Source: Laaper, M. et al., <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a>&#160;J. Vis. Exp. (2017).
This video outlines the generation of a neuronal injury model using reactive oxygen species (ROS) in mouse primary cerebellar granule neurons. It details the process of inducing oxidative stress by adding high concentrations of hydrogen peroxide. ROS exposure causes DNA strand breaks and genomic instability. Additionally, the oxidation of cellular proteins impairs their functions, while lipid peroxidation compromises membrane integrity. This cascade leads to cytochrome c release and caspase activation, resulting in neuronal apoptosis.

小鼠小脑颗粒神经元活性氧诱导的神经元死亡建模

1. Experimental Preparation
NOTE: The following stock solutions can be prepared and maintained until use.

	Dissection Solution
	
		Dissolve 3.62 g of ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://app.jove.com/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

从成年小鼠大脑的皮层中分离小胶质细胞

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...

Neuronal Culture Techniques

Primary Neuronal Culture

Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M., et al. <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a> J. Vis. Exp. (2017)
This video demonstrates the technique for isolating and culturing primary mouse cerebellar granule neurons. Cells are treated with an antimetabolite to eliminate proliferating glial cells and produce a pure culture of cerebellar granule neurons.

小鼠原代小脑颗粒神经元的分离和培养

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Preparation
NOTE: ...

Selectively Removing Microglia from a Rat Cerebellar Granule Neuron Culture

Source: Jebelli, J., et al. <a href="https://app.jove.com/v/52983">Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for the selective removal of microglia from a primary rat neuronal culture. Cerebellar granule neurons are treated with an antimetabolite to eliminate proliferating glial cells. Additionally, they are treated with lysosome-targeting agents to deplete microglia from the culture.

从大鼠小脑颗粒神经元培养物中选择性去除小胶质细胞

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Instruments, ...