Name: culturing in vivo-like murine astrocytes using the fast, simple, and inexpensive awesam protocol
Uploaded: 2018-01-10T13:00:00
Description: Watch this Scientific Journal Video about Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol at JoVE.com

我们承认来自欧洲研究理事会 (FP7/260916)、亚历山大·洪堡-基金会 (Sofja Kovalevskaja) 和德意志 Forschungsgemeinschaft (DE1951 和 SFB889) 的资助。

这里描述的所有动物实验都是按照德国动物福利指南进行的。
注意: 协议的时间轴和主要步骤如图 1所示。
1. 准备工作
<ol><li>
准备以下解决方案。
	<ol>
<li>准备 DMEM + 为培养多边形 MD 星形胶质细胞: DMEM 10% FCS, 100 U 青霉素和100µg/毫升链霉素。</li>
		<li>为培养星状胶质细胞制备 NB + H: neurobasal 培养基, 5 ng/毫升 HBEGF, 1x B-27 ?...

<ol>
	<li>Puschmann, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+3D+cell+culture+system+minimizes+cellular+stress+and+maintains+the+in+vivo-like+morphological+complexity+of+astroglial+cells.">Bioactive 3D cell culture system minimizes cellular stress and maintains the in vivo-like morphological complexity of astroglial cells.</a> Glia. 61 (3), 432-440 (2013).</li><li>Puschmann, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HB-EGF+affects+astrocyte+morphology,+proliferation,+differentiation,+and+the+expression+of+intermediate+filament+proteins.">HB-EGF affects astrocyte morphology, proliferation, differentiation, and the expression of intermediate filament proteins.</a> J Neurochem. 128 (6), 878-889 (2014).</li><li>Foo, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+method+for+the+purification+and+culture+of+rodent+astrocytes.">Development of a method for the purification and culture of rodent astrocytes.</a> Neuron. 71 (5), 799-811 (2011).</li><li>Wolfes, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+culturing+stellate+astrocytes+reveals+spatially+distinct+Ca2++signaling+and+vesicle+recycling+in+astrocytic+processes.">A novel method for culturing stellate astrocytes reveals spatially distinct Ca2+ signaling and vesicle recycling in astrocytic processes.</a> J Gen Physiol. 149 (4), (2016).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J Cell Biol. 85 (3), 890-902 (1980).</li><li>Krencik, R., Zhang, S. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+functional+astroglial+subtypes+from+human+pluripotent+stem+cells.">Directed differentiation of functional astroglial subtypes from human pluripotent stem cells.</a> Nat Protoc. 6 (11), 1710-1717 (2011).</li><li>Morita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+regulation+of+calcium+oscillation+in+astrocytes+by+growth+factors+and+pro-inflammatory+cytokines+via+the+mitogen-activated+protein+kinase+cascade.">Dual regulation of calcium oscillation in astrocytes by growth factors and pro-inflammatory cytokines via the mitogen-activated protein kinase cascade.</a> J Neurosci. 23 (34), 10944-10952 (2003).</li><li>Saura, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cells+in+astroglial+cultures:+a+cautionary+note.">Microglial cells in astroglial cultures: a cautionary note.</a> J Neuroinflammation. 4, 26 (2007).</li><li>Raponi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S100B+expression+defines+a+state+in+which+GFAP-expressing+cells+lose+their+neural+stem+cell+potential+and+acquire+a+more+mature+developmental+stage.">S100B expression defines a state in which GFAP-expressing cells lose their neural stem cell potential and acquire a more mature developmental stage.</a> Glia. 55 (2), 165-177 (2007).</li><li>Royo, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+AAV+serotypes+stably+transduce+primary+hippocampal+and+cortical+cultures+with+high+efficiency+and+low+toxicity.">Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity.</a> Brain Res. 1190, 15-22 (2008).</li><li>Malarkey, E. B., Parpura, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+characteristics+of+vesicular+fusion+in+astrocytes:+examination+of+synaptobrevin+2-laden+vesicles+at+single+vesicle+resolution.">Temporal characteristics of vesicular fusion in astrocytes: examination of synaptobrevin 2-laden vesicles at single vesicle resolution.</a> J Physiol. 589, 4271-4300 (2011).</li><li>Zamanian, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+reactive+astrogliosis.">Genomic analysis of reactive astrogliosis.</a> J Neurosci. 2 (18), 6391-6410 (2012).</li><li>Srinivasan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca(2+)+signaling+in+astrocytes+from+Ip3r2(-/-)+mice+in+brain+slices+and+during+startle+responses+in+vivo.">Ca(2+) signaling in astrocytes from Ip3r2(-/-) mice in brain slices and during startle responses in vivo.</a> Nat Neurosci. 18 (5), 708-717 (2015).</li><li>Shigetomi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+calcium+microdomains+within+entire+astrocyte+territories+and+endfeet+with+GCaMPs+expressed+using+adeno-associated+viruses.">Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.</a> J Gen Physiol. 141 (5), 633-647 (2013).</li><li>Srinivasan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Transgenic+Mouse+Lines+for+Selectively+Targeting+Astrocytes+and+Studying+Calcium+Signals+in+Astrocyte+Processes+In+Situ+and+In+Vivo.">New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.</a> Neuron. 92 (6), 1181-1195 (2016).</li><li>Otsu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+dynamics+in+astrocyte+processes+during+neurovascular+coupling.">Calcium dynamics in astrocyte processes during neurovascular coupling.</a> Nat Neurosci. 18 (2), 210-218 (2015).</li><li>Agarwal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+Opening+of+the+Mitochondrial+Permeability+Transition+Pore+Induces+Microdomain+Calcium+Transients+in+Astrocyte+Processes.">Transient Opening of the Mitochondrial Permeability Transition Pore Induces Microdomain Calcium Transients in Astrocyte Processes.</a> Neuron. 93 (3), 587-605 (2017).</li></ol>

四步在协议之内是重要的: 1) 准备 HBEGF 浓度精确地 5 ng/毫升, 因为小增量在集中可能导致不成熟的文化,例如, 10 ng/ml HBEGF 将 de-differentiate 星形胶质细胞4;2) 在含有组织或细胞的溶液中避免气泡, 这会改变 pH 值, 阻碍星形胶质星的健康;3) pre-equilibrating 培养基, 达到生长健康的星形胶质细胞的最佳 pH 值和温度;4) 每周只交换一半培养基, 因为任何残留的小胶质细胞在存在足够的养...

星形胶质细胞通过营养支持、血流、突触信号和可塑性以及细胞间通讯来影响大脑功能, 所有这些都是围绕着薄星过程的机制。这里描述的 AWESAM 协议允许对这些过程的研究, 不受神经元和其他神经胶质的干扰, 这是有用的例如, 因为许多蛋白质的表达, 甚至 Ca2 +信号重叠在不同的脑细胞类型.此外, 此方法克服了以前可用技术的局限性。特别是, 我们的协议提供了在体内样的形态学 (星形胶质细胞与薄的过程) 和基因表达快速, 方便, 便宜的方式。
细胞形态、基因表达以及许多其他的调控过程都受到环境的直接影响。在培养皿中, 这指的是周围细胞释放的因子, 也是细胞生长的媒介。对于星形胶质细胞, HBEGF 以前报告诱导一种星状形态学1 , 但也发现 de-differentiate 星形胶质细胞2。然而, 较低浓度的 HBEGF 后来用于产生更多的在体内样星形胶质细胞通过 RNA 测序在两个不同的协议3,4。此外, 星形胶质细胞的形态随培养基成分的变化而改变, 不管是什么协议4: 低浓度 HBEGF 的 Neurobasal 培养基都是生长中的正晶状胶质星, 而其他培养基成分 (如如,含血清的 DMEM) 产生多边形细胞 (即使是在 immunopanning 新分离的星形胶质)。
AWESAM 协议克服了以前技术的缺点生长星形胶质细胞单一4。以前的技术为生长星形胶质细胞单一有以下缺点: 多边形形态学, 这是不典型的在体状胶质星型,在体内(MD 方法)5;协议的长度和成本 (从诱导多能干细胞制备星形胶质细胞需要三月, 需要许多试剂, 包括昂贵的生长因子)6;低量的材料 (immunopanning 方法)3;星形胶质细胞的 de-differentiation 和3D 矩阵的要求 (Puschmann et al)1,2. 相比之下, AWESAM 协议提供:在体内样的形态学 (星形胶质细胞与薄的过程);快速、简便、廉价的方法;大量的材料;与 immunopanned 和 MD 星形胶质细胞相比, 最体内样的基因表达。此外, 2D 文化允许研究的 Ca2 +信号和囊泡回收在薄的过程中, 并在事件接近膜 (例如, TIRF 显微镜是不可能在3D 文化)。
MD 方法自 1980年5发布以来已被广泛使用, 为多边形星形胶质细胞单一提供了一种简单快捷的技术。简言之, MD 方法需要在胎儿小牛血清 (FCS) 含有 DMEM 的混合脑细胞, 其次是为星形胶质细胞提供丰富的震动步骤 (就像所有其他的脑单元类型从碟中分离出来) 和在同一培养基中的进一步培养。DMEM 补充与 FCS 是用于许多其他细胞类型, 从成纤维细胞和脂肪细胞到不同的癌细胞系, 所有这些共享的多边形形态学显示由 MD 星形胶质细胞在文化。直到早期的 2000s7, 几乎没有考虑到适合于星形胶质细胞的介质, 特别是有利于其典型的星状形态学发现的体内。2011年发布的一项协议确实实现了这种星状形态学: 称为 immunopanning 方法, 它采用了最优化的无血清培养基培养星形胶质细胞3。使用这种方法, 新的孤立的脑细胞暴露在一系列的菜肴的抗体, 靶向细胞类型特定的细胞表面蛋白, 以丰富的星形胶质。尽管 immunopanned 星形胶质细胞的形态学和表达谱有更多的在体内, 但大多数的体外研究仍依赖于 MD 型胶质蛋白的方法。MD 方法是简单和快速的, 而 immunopanning 方法包括更复杂和耗时的步骤在第一天的文化 (如较长的酶消化周期, 仔细分层的解决方案的不同密度, immunopanning 本身,几个离心步骤-所有之前电镀)。但是, 通过使用更专门的介质, AWESAM 方法提供了 MD 方法的速度和 immunopanning 协议中的在体内样貌。
总的来说, AWESAM 协议是有用的研究更多的在体内状的星形胶质细胞在2D 单一从神经元和其他神经胶质分离 (如以前的特点4由 immunostainings, immunoblots, 和 RNA 测序)。它允许研究薄的星过程, 并为可视化自发的 Ca2 +信号和接近膜的事件提供了很大的可访问性 (如, 由 TIRF 显微镜成像)。

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">0.05% trypsin-EDTA</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:344px;">25300054</td>
			<td style="width:167px;">warm in 37 &#186;C waterbath before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.25% trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>warm in 37 &#186;C waterbath before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B-27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>50X stock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>100X stock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin / streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>50 stock; penicillin: 5000 U/ml; streptomycin: 5000 &#181;g/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>without L-glutamine</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM</td>
			<td>Gibco</td>
			<td>41966029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HBEGF</td>
			<td>Sigma</td>
			<td>4643</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HBSS</td>
			<td>Gibco</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FCS</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td>1X</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100 &#956;m nylon cell strainer</td>
			<td>BD</td>
			<td style="width:344px;">352360</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Hera Cell 240i cell culture incubator</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laboratory shaker</td>
			<td>Heidolph</td>
			<td>Rotamax 120&#160;</td>
			<td>use inside cell culture incubator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

culturing in vivo-like murine astrocytes using the fast, simple, and inexpensive awesam protocol

AWESAM (a low-成本easy stellate strocyte m方法) 协议需要一种快速、简单且廉价的方法来生成大量的在体内像老鼠和大鼠星形胶质细胞单一: 脑细胞可以从不同的脑区分离出来, 经过一周的细胞培养, non-astrocytic 细胞通过将培养皿放在孵化器中6小时的振动器上而被动摇。其余的星形胶质细胞被传代成新的板块, 并有一个特定的特殊介质 (称为 NB + H)。含低浓度肝素结合 EGF 样生长因子 (HBEGF), 用于代替血清在培养基中。AWESAM 星形胶质细胞生长于 NB + H 后, 具有星状形态, 并具有优良的工艺特点。此外, 这些星形胶质细胞比以前发表的方法产生的星形胶质细胞有更多的体内样的基因表达。Ca2 +成像, 囊泡动力学, 和其他事件接近的膜可以因此研究的精细星过程体外,例如, 使用活细胞共聚焦或 TIRF 显微镜。值得注意的是, AWESAM 星形胶质细胞也表现出类似于星形胶质细胞体内的自发 Ca2 +信号。

星形胶质细胞与神经元共, 生长在 NB + 培养基上, 在2周的培养后呈星状 (图 2)。除了铌 + 培养基, 共星形胶质细胞还暴露在未知的神经元衍生因素, 可能有助于他们的生存和形态学。相比之下, MD 星形胶质细胞生长在 DMEM + 作为单一 (在摆脱其他细胞类型之前, 通过) 出现多边形后2周。AWESAM 星形胶质细胞在震动后生长, 在2周的培养后出现星状与许多精细?...

Watch this Scientific Journal Video about Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol at JoVE.com

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

这里描述的 AWESAM 协议是最理想的培养小鼠星形胶质细胞与其他脑神经干细胞的快速, 简单, 便宜的方式。AWESAM 星形胶质细胞展示自发 Ca2 +信号, 形态学和基因表达谱类似于星形胶质细胞在体内。

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Research

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Neuroscience

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

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Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

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As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes

耦合星形胶质细胞网络的大小、形状和方向性分析

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

An Emerging Target Paradigm to Evoke Fast Visuomotor Responses on Human Upper Limb Muscles

唤起人类上肢肌肉快速视觉运动反应的新兴目标范式

To reach towards a seen object, visual information has to be transformed into motor commands. Visual information such as the object&#8217;s color, shape, ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

使用从纤维细胞直接转换为神经元祖先细胞和分化为星形细胞的神经系统疾病体外建模

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...