该项目由加拿大卫生研究所PG＃20R47867支持。

1.通过生物素化测定星形胶质细胞中的相对细胞表面蛋白表达注意:在这里，我们说明了这种生物素化技术在细胞外基质分子层粘连蛋白对透水通道水通道蛋白-4（AQP4）的细胞表面定位的影响的研究中的应用。该测定所需的专用材料包括磺基-NHS-LC-生物素和链霉亲和素 - 琼脂糖树脂（参见材料表 ）。 <ol><li>使用Noel 等人概述的方法12 ，在测定前约2周准备皮层星形胶质细胞培养物，并在75cm 2的通气培养瓶中培养。当星形胶质细胞为80-90％融合时，使用0.05％胰蛋白酶将其从培养物表面分离出来，然后通过1:3。 </li><li>在测定前48小时，当细胞再次融合80-90％时，通过星形胶质细胞1:3（占c以培养形式变化）到60mm细胞培养皿上，使得在实验发生的当天&gt; 70％汇合。确保细胞均匀分布在盘间。 </li><li>在测定前16小时，将层管蛋白移液到培养基中至最终浓度为24nM，并在37℃下孵育。 </li><li>在测定前，准备好以下物质，然后置于冰或冷藏中:将CM-PBS（100mg / L MgCl 2· 6H 2 O和100mg / L CaCl 2在1X PBS中，pH7.4），生物素缓冲液（0.5 （CM-PBS中的mg / mL磺基-NHS-LC-生物素），猝灭缓冲液（CM-PBS中的50mM NH 4 Cl），裂解缓冲液（25mM Tris，pH 7.4,25mM甘氨酸，150mM NaCl和5mM EDTA，1％triton X-100,1X蛋白酶抑制剂混合物），3X加载缓冲液（150mM Tris，pH 6.8,6％SDS，30％甘油，300mM DTT和0.01％溴酚蓝）和洗涤缓冲液Tris（pH 7.4），1.5mM EDTA，150mM NaCl，1％Triton X-100,1X蛋白酶抑制剂混合物）。 </li><li>雷姆从培养箱中拿着星形胶质细胞培养皿，然后弃去培养基。 </li><li>用4 mL冷冻的CM-PBS将细胞洗涤3次，并将其放在碎冰上。 </li><li>将2 mL生物素缓冲液吸入每个孔中，并轻轻地将菜肴往前倾几次，以确保完全覆盖。在冰上休息30分钟。 </li><li>用吸气器取出生物素缓冲液，并用4 mL淬灭缓冲液代替。在冰上离开10分钟。 </li><li>吸入淬火缓冲液，并用相同体积的相同体积代替。再次，在冰上离开10分钟。 </li><li>弃去淬灭缓冲液，用4 mL冷冻的CM-PBS洗涤细胞三次。 </li><li>使用细胞提升器将细胞切成1mL冷冻的CM-PBS，并将悬浮液转移到微量离心管中。 </li><li>通过在100×g离心3分钟来制备粒细胞。弃去上清液，并将细胞重新悬浮于500μL裂解缓冲液中。 </li><li>将样品放在冰上30分钟，每5分钟振荡一次在4°C的端端旋转器上。 </li><li>在4℃下以14,000xg离心裂解物10分钟，以沉淀任何不溶于洗涤剂的物质。将上清液转移到新的微量离心管中。 <ol><li>保存50μL此裂解液，加入缓冲液。然后在95℃下在干浴中加热变性;这是"输入"分数，含有生物素化的细胞表面蛋白质，以及非生物素化的胞质蛋白质。 </li></ol></li><li>使用一把锋利的剪刀从其末端切掉大约0.5厘米的材料，从而扩大移液器吸头的开口。使用该吸管头，将75μL链霉抗生物素蛋白 - 琼脂糖珠（通常在4°C下储存）转移到裂解物中，并在4℃下在振荡器/摇杆上孵育3小时。 <ol><li>作为链霉抗生物素蛋白 - 琼脂糖经常作为含有50体积％的珠粒的浆料出售，悬浮在抗微生物溶液中，研磨该浆料以确保珠粒均匀悬浮，然后将150μL悬浮液移液到每个样品中。 </li></ol></li><li>颗粒链霉亲和素 - 琼脂糖珠子，在4℃下以1500xg离心30秒。 <ol><li>保存50μL上清液（加入缓冲液，并在95℃在水浴或加热块中变性）;这代表"细胞内"级分，并且主要由非生物素化的胞质蛋白组成。 </li></ol></li><li>将沉淀的珠子重悬于1mL洗涤缓冲液中，并在4℃下摇匀3分钟。颗粒珠（如步骤1.16），并弃去上清液。重复此过程4x以最小化非生物素化胞质蛋白的非特异性结合。 </li><li>通过离心（1500xg，4℃，30s）离心沉淀珠粒，并丢弃上覆的洗涤缓冲液。加入50μL的1X加载缓冲液（用裂解缓冲液稀释）。通过在95℃下将其变性从珠中释放生物素和链霉亲和素;这个分数只能含有生物素化的细胞表面蛋白质（"细胞表面"部分）。 <ol><li>通过SDS-PAGE 13分离输入，细胞表面和细胞内级分，并通过蛋白质印迹分析14 。 注意:虽然我们在我们的实验中使用了4 - 20％的预制梯度凝胶，但是在本研究中，12-14％的分离凝胶与4％堆叠层（每个含有0.1％SDS）都足以满足感兴趣的蛋白质。还应使用适当尺寸范围的分子量标准。注意，有时可以观察到生物素化蛋白质的表观分子量升高。 </li></ol></li></ol> 2.通过生物素化测定星形胶质细胞中细胞表面蛋白的内在化率注意:在下文中，我们描述了在这种情况下用于跟踪星形胶质细胞中AQP4的内吞作用的典型脉冲追踪生物素化实验。这个方法基于马德里等人使用的方法15 。所需的专门材料包括磺基-NHS-SS生物素，链霉抗生物素蛋白 - 琼脂糖树脂，还原型谷胱甘肽和碘乙酰胺（参见材料表 ）。 <ol><li>使用上一节中概述的方法，在60 mm的培养皿中制备小鼠皮层星形胶质细胞的培养物。确保细胞在测定当天约70％汇合，并且每个培养皿含有相当数量的细胞。 </li><li>在测定之前，准备好以下物质，并置于冰或冷藏中:将CM-PBS（100mg / L MgCl 2· 6H 2 O和100mg / L CaCl 2在1X PBS，pH7.4中），生物素缓冲液（0.5在CM-PBS中的mg / mL磺基-NHS-SS生物素），还原缓冲液（50mM还原型谷胱甘肽，75mM NaCl和75mM NaOH），淬灭缓冲液（50mM碘乙酰胺，1％BSA，在CM-PBS中）裂解缓冲液（25mM Tris，pH 7.4,25mM甘氨酸，150mM NaCl和5mM EDTA，1％triton X-100,1X蛋白酶抑制剂混合物），3X加载缓冲液（150mM Tris，pH 6.8,6％SDS，30％甘油，300mM DTT和0.01％溴酚蓝）和洗涤缓冲液（10mM Tris，pH 7.4,1.5mM EDTA， 150mM NaCl，1％triton X-100,1X蛋白酶抑制剂混合物）。 </li><li>准备新鲜细胞培养基（补充有10％胎牛血清（FBS），1％青霉素/链霉素和1％L-谷氨酰胺的DMEM），并将其置于37℃水浴中。 </li><li>从培养箱中取出星形胶质细胞培养物，并用吸液器抽吸培养基。 </li><li>用4 mL冷冻的CM-PBS将细胞洗涤3次，并将其放在碎冰上。 </li><li>将3 mL生物素缓冲液吸入每个培养皿中，往前后摆动几次，以确保缓冲液分布均匀，并将其放在冰上30分钟。 </li><li>吸出生物素缓冲液，并用5 mL温培养基代替。在37℃培养培养皿15分钟，并在同一温度下培养30分钟。在4点再留个菜6; C为0分钟样品。 </li><li>在孵育期结束时，弃去培养基，用4mL冷冻的CM-PBS洗涤细胞三次。在细胞上吸取6 mL还原缓冲液，并在冰上放置15分钟。 </li><li>取出还原缓冲液，然后用6 mL新鲜还原缓冲液取代。在冰上再放置15分钟。 </li><li>除去还原溶液，并用6 mL淬灭缓冲液代替。在冰上离开15分钟。 </li><li>再次重复淬火步骤。 </li><li>丢弃淬灭缓冲液，用4 mL冷冻PBS洗涤细胞三次。 </li><li>使用细胞提升器将细胞切成1mL冷冻的PBS，并将悬浮液转移到微量离心管中。 </li><li>通过在100×g离心3分钟来制备粒细胞。弃去上清液，并将细胞重悬于500μL裂解缓冲液中。 </li><li>将其放在冰上30分钟，每5分钟旋涡一次。或者，将样品放置在4°C的终端旋转器上。 </li><li>离心机在4℃下以14,000xg的速度放置10分钟以沉淀洗涤剂不溶物质，然后将上清液转移到新的微量离心管中。保存50μL此裂解液，加入缓冲液，并在95℃在干燥浴中变性;这是"输入"分数，含有生物素化的内吞蛋白质，以及非生物素化的蛋白质。 </li><li>使用切割的吸头，将150μL的链亲和素 - 琼脂糖浆液加入到裂解物中，并在4℃下在振荡器/摇臂上孵育3小时。有关此步骤的更多详细信息，请参阅1.15。 </li><li>颗粒链亲和素 - 琼脂糖珠子，在4℃下以1500×g离心30秒。 </li><li>将珠重悬于1mL洗涤缓冲液中，并在4℃下摇动3分钟。颗粒珠（按照步骤2.18），弃去上清液。重复此过程4x以最小化非生物素化胞质蛋白的非特异性结合。 </li><li>通过在4℃下以1,500×g离心30秒，并弃去颗粒珠洗涤缓冲液。加入50μL1X加载缓冲液（用裂解缓冲液稀释）。通过在95℃变性从珠粒释放生物素和链霉亲和素;这个部分应该仅含有内在的细胞表面蛋白质（"胞吞"部分）。 </li><li>通过SDS-PAGE 13分离输入，细胞表面和未结合部分，并通过蛋白质印迹分析14 。 注意:虽然我们在我们的实验中使用了4 - 20％的预制梯度凝胶，但是在本研究中，12-14％的分离凝胶与4％堆叠层（每个含有0.1％SDS）都足以满足感兴趣的蛋白质。还应使用适当尺寸范围的分子量标准。注意，有时可以观察到生物素化蛋白质的表观分子量升高。 </li></ol>

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作者宣称没有利益冲突。

修改: 由于这些方法设计用于贴壁细胞，我们已经规定了使用含有100mg / L MgCl 2· 6H 2 O的PBS和 100mg / L的CaCl 2 （CM-PBS）洗涤步骤，并作为某些缓冲液的碱基，以确保细胞保持附着于培养物表面，并且细胞 - 细胞结不被破坏。然而，如果细胞在程序的每个步骤之间沉淀，则该协议也可以应用于非贴壁细胞类型。在这些情况下，可以用PBS代?...

细胞表面的蛋白质起着维持细胞功能的重要作用。在许多情况下，它们的活性依赖于或者通过内吞过程调节，这些内吞过程暂时将其封闭在胞内位点，或者将它们引导到降解途径1,2,3,4,5 。在这里，我们强调了2种基于生物素化的方法，旨在允许用户专门标记和分离在质膜上表达的蛋白质，以及新内化的蛋白质。通过这些方法，可以量化任何目的蛋白质的细胞表面表达和内吞速率，从而允许更清楚地评估其调节。 通过生物素化测定相对细胞表面蛋白的表达 生物素或维生素B7（以前称为维生素H 6 ）是一种小水溶性分子，可用于化学修饰生物分子的活性胺，巯基和羧基。目前的细胞表面生物素化试剂主要由生物素或其衍生物的膜 - 不渗水磺化N-羟基琥珀酰亚胺（磺基-NHS）酯组成，其与设在蛋白质赖氨酸残基侧链上的胺反应，当细胞表面在碱性条件下脱质子时，后者与生物素部分7形成酰胺键。因此，可以通过使用抗生物素蛋白（一种对生物素具有极大亲和力的66-69kDa四聚体蛋白）来分离细胞表面蛋白，以约10-15的解离常数结合后者，将其标记为最强非共价相互作用已知8 </sup&gt; ， 9 。 在以前的研究中已经使用了许多量化蛋白质在细胞表面的方法。使用针对目的蛋白质特异性的荧光标记抗体进行标记，然后通过荧光显微镜观察是常用的方法，但是严重依赖于可结合细胞外表位的抗体的可用性。最近，已经成功地使用涉及使用与暴露于酸性介质反应的具有pH敏感性荧光团的嵌合蛋白质的方法。然而，这样的测定通常涉及这些构建体在其中未被天然存在的目的蛋白质的细胞系中的外源表达。这些方法仍然能够提供关于亚细胞定位和胞外步行的有价值的信息因此如果这些工具可用，则应与本文所述的基于生物素化的方法结合使用。 在典型的生物素化测定中，首先在4℃PBS中彻底洗涤细胞。这样可以消除由培养基引入的任何痕量的血清蛋白，从而确保在下一步中这些不会消耗过量的生物素。更重要的是，降低温度会导致内吞作用显着减速。然后加入生物素化试剂。接下来，再次洗涤细胞，然后与含有甘氨酸或NH 4 Cl的淬灭缓冲液一起温育，其目的是灭活所有剩余痕量的未反应的生物素。然后将细胞裂解，随后加入琼脂糖固定的链霉抗生物素蛋白以沉淀生物素化的蛋白质。分析通常通过western印迹进行，允许各种pr的相对细胞表面表达oteins要量化。 由于该测定的基础，它仅适用于具有暴露于细胞外环境的部分的蛋白质。可能在其环区内可能具有许多反应性赖氨酸的多巴跨膜蛋白是该方法中最容易的，而单程蛋白往往不太容易被生物素化。即使在这些情况下，仍然存在构象变化或分子间相互作用可能堵塞某些活性位点的可能性，导致低于预期的生物素化产率。 通过生物素化测定细胞表面蛋白的内在化率 该测定的原理与细胞表面生物素化的原理大致相同，除了一些例外，其中最重要的是使用可逆生物素化试剂。生物素组（其中的）具有disu在其结构内的易受还原剂影响的粘合剂;这被开发以确保仅在测定期间摄入细胞内部位的细胞表面蛋白质将被遗传生物素化。测定通常以以下方式进行。首先用冷试剂洗涤细胞并生物素化，然后重新引入37℃的细胞培养基，将细胞返回培养箱;这导致标记的细胞表面蛋白质经历内吞。然后加入不能穿透膜的还原剂谷胱甘肽，以破坏与残留在细胞表面上的蛋白质连接的生物素部分的二硫键。最后，破坏的二硫键与碘乙酰胺反应，消耗不稳定的硫醇基并防止键重组。如前所述，然后将细胞裂解，并使用链霉抗生物素蛋白 - 琼脂糖沉淀标记的蛋白质。 限制光盘由于在方法之间共享的相似之处，上一节中所使用的内容也适用于此。此外，值得铭记的是，该测定中涉及的温度变化阻止了对于每次增量的时间内含量多少蛋白质的确切测定，特别是在迅速内化或快速回收蛋白质的情况下。因此，该测定仅提供了内吞率的半定量估计。全内反射荧光显微镜可用于跟踪每个加载的囊泡的摄取，并提供更精确的内吞动力学测量。因此，假设目标蛋白的荧光标记嵌合构建体可用，因此可以提供非常有用的补体。

<table border="0" cellpadding="0" cellspacing="0" width="773">
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			<td height="40" style="height:40px;width:280px;">Ammonium chloride (NH4Cl)</td>
			<td style="width:219px;">Fisher Scientific</td>
			<td style="width:129px;">A661-500</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-50</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Bromophenol blue</td>
			<td>Bio-Rad</td>
			<td>#1610404</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">cOmplete protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Disodium ethylenediaminetetraacetate dihydrate (EDTA)</td>
			<td>Bio-Rad</td>
			<td style="width:129px;">#1610729</td>
			<td></td>
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			<td height="40" style="height:40px;width:280px;">Dithiothreitol (DTT)</td>
			<td>Bio-Rad</td>
			<td>#1610611</td>
			<td></td>
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			<td height="40" style="height:40px;width:280px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>11960-044</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:280px;">EZ-Link Sulfo-NHS-LC-Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>#21335</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">EZ-Link Sulfo-NHS-SS-Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>#21331</td>
			<td></td>
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			<td height="40" style="height:40px;width:280px;">Fetal bovine serum</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td></td>
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			<td height="40" style="height:40px;width:280px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898&#160;</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Iodoacetamide</td>
			<td>Bio-Rad</td>
			<td>#163-2109</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Thaw on ice.</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:280px;">L-glutamine</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>25030-081</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Mouse monoclonal anti-&#946;-actin antibody (AC-15)</td>
			<td>Sigma-Aldrich</td>
			<td>A5441</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Mouse monoclonal anti-&#946;-dystroglycan antibody (43DAG1/8D5)</td>
			<td>Leica Biosystems</td>
			<td>B-DG-CE</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Penicillin/streptomycin</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Peroxidase AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td style="width:219px;">Jackson ImmunoResearch Laboratories</td>
			<td>715-035-150</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td style="width:219px;">Jackson ImmunoResearch Laboratories</td>
			<td>111-035-045</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Phosphate buffer saline</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Reduced glutathione</td>
			<td>Sigma-Aldrich</td>
			<td>G6529</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>862010&#160;</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Sodium hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>S318-100</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Streptavidin agarose resin</td>
			<td>Thermo Fisher Scientific</td>
			<td>#20347</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Rabbit polyclonal anti-AQP4 antibody</td>
			<td>Alomone</td>
			<td>AQP-004</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Tris base (Trizma base)</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td></td>
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			<td height="40" style="height:40px;width:280px;">Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td>BP153-1</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
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</table>

determining cell-surface expression and endocytic rate of proteins in primary astrocyte cultures using biotinylation

细胞表面蛋白介导各种功能。在许多情况下，它们的活性受调节其在质膜上的水平的内吞过程的调节。在这里，我们提供了有助于研究这些过程的两种方法的详细方案，这两种方法均基于细胞表面蛋白的生物素化原理。第一个被设计为允许半定量测定细胞表面上特定蛋白质的相对水平。其中，细胞的质膜蛋白的赖氨酸残基首先用生物素部分标记。一旦细胞被裂解，然后可以通过利用琼脂糖固定的链霉亲和素通过利用后者对于生物素的天然亲和力来特异性沉淀这些蛋白质。然后可以通过标准的Western印迹方法分析以这种方式分离的蛋白质。第二种方法提供了确定特定细胞吞噬率的方法ar细胞表面目标在一段时间内。首先用含有可切割二硫键的生物素衍生物修饰细胞表面蛋白。然后将细胞转移回正常培养条件，这导致一部分生物素化蛋白质的内吞摄取。接下来，使用膜不可渗透的还原剂谷胱甘肽使非内化生物素基团的二硫键减少。通过这种方法，内吞的蛋白质因此可以以高度的特异性被分离和定量。

使用细胞表面生物素化来评估星形胶质细胞中AQP4的质膜表达 使用所述方法将层粘连蛋白处理的星形胶质细胞培养物和未处理的对照细胞进行细胞表面生物素化。用琼脂糖缀合的链霉亲和素沉淀生物素化的蛋白质，然后通过SDS-PAGE分离。探测细胞表面级分作为细胞表面加载对照的AQP4和β-dystroglycan（β-DG），同时将输入和细胞内级分吸入AQP4和β-肌动蛋白（ <...

Watch this Scientific Journal Video about Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation at JoVE.com

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

本报告提供了两种基于生物素化的方法，设计用于测定在质膜上表达的蛋白质的细胞表面表达和内吞率。

使用生物素化测定原代星形细胞培养物中蛋白质的细胞表面表达和吞噬率

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

determining-cell-surface-expression-endocytic-rate-proteins-primary

Research

JoVE Journal

Neuroscience

9.4K 次观看.  University of British Columbia. 本报告提供了两种基于生物素化的方法，设计用于测定在质膜上表达的蛋白质的细胞表面表达和内吞率。 

视频: Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

嵌入后突触蛋白的免疫标记海马切片培养

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

科研

神经科学

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

细胞表面和内蛋白质的现场培养神经细胞的抗体后喂养差异化标记

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

大脑切片生物素化:一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

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

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

生活多巴胺能神经元可靠的鉴定中脑培养使用RNA测序和TH-启动子驱动的EGFP表达

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

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

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

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

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

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 ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

小鼠初级少突胶质的快速特异磁分离

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

在 vivosilk/aav 薄膜在光基因表达中的靶向表达

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...