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?...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Forceps&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td>The curved end facilitates removal of the cerebellum&#160;</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3146</td>
			<td>Straight, sharp point facilitates rodent P4-7 dissection&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6414</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-ED1 antibody&#160;</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...

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

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. 