Name: a novel in vitro live-imaging assay of astrocyte-mediated phagocytosis using ph indicator-conjugated synaptosomes
Uploaded: 2018-02-05T13:30:00
Description: Watch this Scientific Journal Video about A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes at JoVE.com

作者感谢妍珠荣格的实验支持, 在体纯化和 Jungjoo 公园的图像突与 PS 曝光。此外, 我们亦感谢锺众的实验室成员进行有益的讨论。这项工作得到了韩国国家研究基金会 (NRF) 资助的韩国政府 (MSIP) (NRF-2016M3C7A1905391 和 NRF-2016R1C1B3006969) (W。C)。

这里描述的所有方法都得到了韩国科学和技术研究所动物护理和使用委员会 (IACUC) 的批准, KA2016-08。
1. 体净化
注意: 这些过程从以前发布的纸张19中进行了修改, 并进行了一些改进, 以提高纯化突的收率 (图 1)。
<ol><li>准备不连续密度梯度介质。<ol>
<li>将以下内容放在冰上: 梯度缓冲液中的3%、10% 和23% 密度梯度解 (?...

<ol>
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在这篇文章中, 我们提出了一种长期活体成像体外的方法, 利用纯化的胶质细胞和 pH 指标-共轭突的吞噬试验。我们发现, 与小胶质细胞相比, 星形胶质在吞噬突时具有不同的吞没和降解能力。此外, 我们的数据表明, 星形胶质细胞分泌的因素, 其中包含桥接分子, 如 GAS6, 蛋白质, 和 MEGE8, 是必不可少的有效的 PS 依赖的吞噬神经细胞的大脑。此外, Megf10高星形胶质细胞显示有缺陷的吞噬突?...

胶质细胞是中枢神经系统中主要的细胞类型, 它指的是大脑中不可兴奋的细胞。以前, 胶质细胞被认为是单纯的支持细胞, 主要在维持神经元存活和基底突触特性方面扮演被动角色。然而, 新出现的证据表明, 胶质细胞在神经生物学的各个方面发挥更积极的作用, 如维持脑内稳态, 调停突触形成1,2,3和突触消除4、5和调制突触可塑性67。中枢神经系统中的胶质细胞包括星形胶质, 小胶质, 和突。在这些细胞中, 胶质细胞和小胶质蛋白通过吞噬突触来发挥吞噬作用4,5, 凋亡细胞8, 神经碎片9, 以及致病性蛋白质, 如淀粉样β斑块10,11。在发育中的大脑中, 星形胶质细胞通过 MERTK 和 MEGF10-dependent 吞噬功能消除了背侧膝状核 (dLGN) 的突触 (4。同样, 小胶质细胞也消除 C1q-coated 突触在发育阶段通过经典补充级联5。有趣的是, 有人认为, 突触修剪的缺陷可能是几种神经紊乱的引发器之一。例如, 它已经表明, 在补充成分 4 (C4) 的突变, 这增加了补充介导的突触修剪由小胶质细胞, 强烈与精神分裂症的患病率在人12。最近的一篇论文还表明, 经典的补体通路是 hyperactivated 在 AD 的起始阶段, 并诱导早期突触损失在这种疾病13。
与小胶质细胞介导的吞噬作用相比, 星形胶质细胞介导的吞噬作用是否有助于各种神经功能紊乱的发生和进展, 尚不清楚。然而, 最近的一篇论文指出, 改变细胞的正常突触修剪率的因素可能会扰乱大脑的稳态, 并导致 AD 易感性和病理14。星形胶质细胞的突触修剪率由载脂蛋白异构体强力控制, 具有 ad 的保护性等位基因 (ApoE2), 大大提高 ad (ApoE4) 的速率和风险等位基因, 显著降低率。此外, 表达ApoE4的转基因小鼠的突触 C1q 比对照组或ApoE2小鼠14大得多。这些数据表明, 受损的星形胶质细胞介导的吞噬在早期 AD 大脑可能会诱发衰老 C1q-coated 突触/突触碎片的积累, 激活补体介导的胶质吞噬, 驱动突触变性.在ApoE4载体中, 星形胶质细胞的吞噬能力受损也可能导致受影响的大脑中淀粉样蛋白β斑块的失控积累。
此外, 它已经表明, 在老年果蝇大脑中的胶质细胞失去了他们的吞噬能力, 由于减少翻译的德雷柏, 一个同源的Megf10 , 星形细胞用于吞噬突触。恢复德雷柏的水平挽救了胶质细胞的吞噬能力, 它有效地清除受损的大脑中的轴突碎片与年轻的大脑一样的程度, 表明老化诱导的吞噬能力的变化星形胶质细胞可能导致大脑稳态破坏15。
根据这些新发现, 调节星形胶质细胞的吞噬能力可能是预防和治疗各种神经紊乱的一种有吸引力的治疗策略。在这方面, 有几次试图提高星形胶质细胞的吞噬能力, 例如, 通过酸性纳米粒子诱导溶酶体的酸化16和表达转录因子 EB (TFEB), 可以提高溶生物17。尽管有这些尝试, 但目前仍不清楚的是, 星形胶质细胞和胶质在它们的吞噬动力学上有何不同, 我们是否应该增加或减少它们在各种疾病中的吞噬能力。
本文提出了一种新的体外检测星形胶质细胞吞噬能力的方法。数据显示星形胶质细胞和小胶质吞没和降解的不同动力学。星形胶质细胞被条件培养基 (ACM), 其中包含的分泌因素, 从胶质, 是必不可少的有效吞噬的星形胶质细胞和小胶质。此外, Megf10, 在星形胶质细胞的吞噬受体和Ced-1和德雷柏同源, 发挥关键的作用, 在胶质-介导吞噬8,18。

<table>
	<tbody>
		<tr>
			<td>Synaptosome purification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE healthcare life sciences</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Start Bradford Protein Assay Kit 2</td>
			<td>BIO-RAD</td>
			<td>5000202</td>
			<td></td>
		</tr>
		<tr>
			<td>pH indicator conjugation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>LPS solution</td>
			<td>DMSO100</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo red, succinimidyl ester</td>
			<td>Molecula probes</td>
			<td>P36600</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunopanning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X Earle&#8217;s balanced salt solution (EBSS)</td>
			<td>Sigma</td>
			<td>E7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Bovogen</td>
			<td>BSA025</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyrebonuclease 1 (DNase)</td>
			<td>Worthington</td>
			<td>Is002007</td>
			<td></td>
		</tr>
		<tr>
			<td>(DMEM)</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>(dPBS)</td>
			<td>Welgene</td>
			<td>LB001-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Griffonia Simplicifolia Lectin(BSL-1)</td>
			<td>Vector Labs</td>
			<td>L-1100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG+IgM(H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgM (&#956;-chain)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin-binding epidermal growth factor</td>
			<td>Sigma</td>
			<td>E4643</td>
			<td></td>
		</tr>
		<tr>
			<td>Human HepaCAM antibody</td>
			<td>R&#38;D systems</td>
			<td>MAB4108</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrin beta 5 monoclonal antibody (KN52)</td>
			<td>eBioscience</td>
			<td>14-0497-82</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma</td>
			<td>C7880</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamate</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetly-L-cyteine (NAC)</td>
			<td>Sigma</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>O4 hybridoma supernatant(mouse IgM)</td>
			<td>Bansal et al.23</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>Is003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluristrainer 20 &#956;m</td>
			<td>PluriSelect</td>
			<td>43-50020-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified rat anti-mouse CD45</td>
			<td>BD Pharmingen</td>
			<td>550539</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified mouse anti-rat CD45</td>
			<td>BD Pharmingen</td>
			<td>554875</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T9935</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Worthington</td>
			<td>LS003086</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tube (Tube, Thinwall, Ultra-Clear)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Collect IP-ACM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosep Advance Centrifugal Devices with Omega Membrane (10k)</td>
			<td>PALL</td>
			<td>MAP010C37</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosep Advance Centrifugal Devices with Omega Membrane (30k)</td>
			<td>PALL</td>
			<td>MAP030C37</td>
			<td></td>
		</tr>
		<tr>
			<td>Phagocytosis live imaging assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Juli stage</td>
			<td>NanoEntek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Time Series Analyzer V3 plugins</td>
			<td>https://imagej.nih.gov/ij/plugins/time-series.html</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel in vitro live-imaging assay of astrocyte-mediated phagocytosis using ph indicator-conjugated synaptosomes

星形胶质细胞是大脑中的主要类型, 直接接触突触和血管。虽然胶质细胞已被认为是主要的免疫细胞, 只有吞噬在大脑中, 最近的研究表明, 星形细胞也参与了各种吞噬过程, 如发育突触消除和清除阿尔茨海默病 (AD) 的淀粉样β斑块。尽管有这些发现, 但与小胶质细胞相比, 星形胶质细胞的吞没和靶向降解的效率还不清楚。这种缺乏信息的主要原因是缺乏一个分析系统, 其中星形胶质细胞和小胶质细胞介导的吞噬动力学是很容易比较的。为了实现这一目标, 我们开发了一个长期的活体成像体外吞噬试验, 以评估纯化的星形胶质细胞和小胶质的吞噬能力。在这种测定中, 吞没和降解的实时检测是可能的使用 pH 指示物共轭突, 它发出明亮的红色荧光在酸性细胞器, 如溶酶体。我们的新的检测方法提供了简单和有效的活体成像探测的吞噬。此外, 这种体外吞噬试验可以作为一个筛选平台, 用来识别能增强或抑制星形胶质细胞吞噬能力的化学物质和化合物。由于突触修剪故障和致病蛋白积累已被证明是导致精神疾病或神经退行性疾病, 化学物质和化合物, 调节吞噬能力的胶质细胞应有助于治疗各种神经紊乱

在这体外吞噬试验长期活成像, 我们使用突从成年小鼠脑匀, 这是分离的梯度解决方案之间的23% 梯度溶液和10% 梯度溶液由离心 (图 3)。在制备后, 突暴露 ps 在他们的外膜 (图 4), 表明他们失去了他们的功能, 可以识别的 ps 受体在星形胶质细胞和小胶质。如图 5所示, pH 值指示器-共轭突在被星形胶质细?...

Watch this Scientific Journal Video about A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes at JoVE.com

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

该协议提供了一个体外活体成像吞噬试验来测量星形胶质细胞的吞噬能力。纯化大鼠星形胶质细胞和小胶质和 pH 指标-共轭突。该方法可检测实时吞没和降解动力学, 为确定影响星形胶质细胞吞噬能力的因素提供了一个合适的筛选平台。

一种新的体外活体成像法测定星形胶质细胞的吞噬作用 pH 指标-共轭突

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

Research

JoVE Journal

Neuroscience

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

科研

神经科学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Live Imaging of Drosophila Larval Neuroblasts

Live Imaging of Drosophila Larval Neuroblasts

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

使用新颖的方法在自由移动的新生大鼠中记录脑电图

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Novel Assay for Cold Nociception in Drosophila Larvae

Novel Assay for Cold Nociception in Drosophila Larvae

新的分析冷痛觉在<em&gt;果蝇</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

体外骨髓源性巨噬细胞对髓鞘碎片的吞噬

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...