Name: isolation and rna extraction of neurons, macrophages and microglia from larval zebrafish brains
Uploaded: 2018-04-27T14:00:00.000+00:00
Duration: 9 min 20 s
Description: Watch this Scientific Journal Video about Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains at JoVE.com

我们感谢克莱尔. 戴维斯博士和维罗尼克 Miron 博士 (爱丁堡, 联合王国的皇后医学研究所) 初步帮助和讨论实验方法和 QMRI 流式细胞仪和细胞分拣设施。这项工作得到了英国癌症研究职业创立奖 Sieger 博士的支持。

1. 样品和媒体准备
<ol><li>通过溶解6.4 毫米氯化钾、0.22 毫米氯化钠、0.33 毫米 CaCl2 2H2O、0.33 mm MgSO4 7H2o 在 H2o 中, 准备胚胎培养基 (E3)。</li>
	<li>受精后立即收集斑马鱼胚胎 (0 dpf)。<ol>
<li>将斑马鱼胚胎分裂成50每90毫米培养皿。在50毫升胚胎培养基 (E3) 中, 用200µM 1 苯基 2-硫脲 (PTU), 从发育的第一天结束 (0 dpf) 开始, 在实验期间抑制色素沉着, 以28.5 °c 的胚胎培养胚。</li>
		<li>在实验持续时间内每日更改 E3 + PTU 介质。</li>
		<li>屏幕 mpeg1:eGFP 和 NBT: DsRed 幼虫在 2 dpf 使用荧光灯显微镜为阳性转基因表达, GFP+巨噬细胞/小胶质和 DsRed+神经元。</li>
	</ol>
</li>
	<li>准备所有的媒体前一天的实验下的组织培养罩, 以避免污染, 然后储存在4摄氏度。<ol>
<li>在 HBSS 1x 中溶解15毫米 Hepes 和25毫米 d-葡萄糖, 准备培养基 A。</li>
		<li>在1体积的 HBSS 10x 中混合9体积的密度梯度介质, 制备密度梯度介质 (100%)。</li>
		<li>在88毫升的 DPBS 1x 中稀释22毫升的密度梯度介质 (100%), 制备密度梯度介质 (22%)。</li>
		<li>准备 DPBS 1x。</li>
		<li>将 E3 培养基与450微米 Tricaine 混合, 制备 E3 中 + Tricaine。</li>
	</ol>
</li>
</ol>2. 同质化
注意:所有步骤都执行4°c。
<ol><li>添加1.5 毫升 Tricaine (15 毫米) 到90毫米培养皿中含有50幼虫的50毫升的 E3 胚胎培养基治疗与200µM PTU 到末期麻醉他们。<ol>
<li>从培养皿中吸取10个被麻醉的幼虫, 用3毫升的巴斯德塑料散装吸管。</li>
		<li>将被麻醉的幼虫 10 10 转化成55毫米的培养皿, 里面装满冰冷的 E3 胚培养基 + Tricaine。</li>
		<li>在显微镜下, 将10幼虫对准培养皿的中心。然后用外科微剪刀 (排除游泳膀胱避免漂浮的头) 在蛋黄囊上方的样带幼虫头。</li>
		<li>从培养皿中吸取头, 用3毫升的巴斯德塑料散装吸管。等待, 直到所有的头聚集在吸管尖端, 然后转移到一个玻璃均质机包含1毫升冰冷介质 a (转移在极小的容量减少媒介稀释由 E3 + Tricaine)。把玻璃杯均质放在冰上。每个实验条件使用一个匀质机。</li>
		<li>用新的每30分钟更换每一个含有冰冷 E3 + Tricaine 的小培养皿, 以确保在冷 E3 + Tricaine 培养基中进行横断。</li>
		<li>当颜色开始褪色时, 更换玻璃匀质机中的冷介质 A。 注意:介质稀释可以改变头部组织由于温度的变化。</li>
		<li>一旦所有的头被收集 (600 头/条件), 删除的最大数量的媒体 A 从玻璃均质体, 并取代它与1毫升新鲜的冰冷介质 a。</li>
		<li>在冰上用紧的玻璃匀质机扰乱大脑组织。执行40轮粉碎和轮流 3-5 dpf 幼虫和50的7和 8 dpf 幼虫。</li>
	</ol>
</li>
	<li>增加2毫升的培养基 a 到细胞悬浮 (1 毫升培养基 a/200 头), 这将稀释细胞和减少其与髓鞘的团聚, 以方便他们的分离过程中的密度梯度介质离心。<ol>
<li>为了消除细胞团聚, 运行细胞悬浮通过40µm 细胞过滤器放置在一个寒冷的50毫升猎鹰管在冰上维持。重复此操作3次。</li>
		<li>将1毫升的细胞悬浮液转移到冷1.5 毫升管中, 在4摄氏度时将其旋转300克, 10 分钟。</li>
		<li>使用10毫升注射器 + 针 23G x 1 "删除上清液。</li>
	</ol>
</li>
	<li>用1毫升冰冷的22% 密度梯度介质, 用0.5 毫升的冰冷 DPBS 1x (不要混合, 两种溶液间的界面) 重新悬浮细胞颗粒。<ol>
<li>旋转管在950克没有刹车和慢速加速30分钟在4摄氏度。 注意:这一步骤将髓鞘与其他细胞分离, 将其在 DPBS 1x 和22% 密度梯度介质的界面上进行隔离, 而细胞则会在试管底部颗粒状。当细胞浓度不太高时, 髓鞘去除效果更有效。</li>
		<li>使用10毫升注射器 + 针 23G x 1 "丢弃最大的 DPBS, 密度梯度介质和髓鞘被困在他们的界面。</li>
		<li>洗涤细胞与0.5 毫升培养基 A + 2% 正常山羊血清 (), 然后旋转管在300克10分钟在4°c。</li>
		<li>丢弃上清的最大值, 然后将所有细胞颗粒从相同的实验条件中汇集到1毫升的培养基 A + 2%。</li>
	</ol>
</li>
	<li>如果感兴趣的细胞表达一个荧光蛋白象巨噬细胞或小胶质从 mpeg1:eGFP 或神经元从 NBT: DsRed 转基因鱼 (筛查转基因鱼参见 1.1.3), 通过35µm 细胞过滤器盖子运行细胞悬浮并且转移他们成冷5毫升在冰上的管, 防止光。 注意:另外, 可以对小胶质细胞进行免疫染色。</li>
</ol>3. 小胶质细胞免疫染色
注意:所有步骤都在4摄氏度执行。
<ol><li>用0.3 毫升的培养基, 再将细胞颗粒重新悬浮2%。分裂他们在 3 x 1.5 毫升管子: 一个为无瑕细胞测量自动荧光从感兴趣的细胞, 其次为二次抗体 (1/200) 测量次要抗体的非特异的结合对小胶质和第三作为测试 (4C4 老鼠单克隆抗体 (小胶质细胞特异) (1/20) + 二次抗体 (1/200))。<ol>
<li>将低内毒素, 无叠氮 (叶) 在1% 到细胞 (所有管), 以阻止 CD16/CD32 相互作用的 Fc 领域的免疫球蛋白。每5分钟孵育细胞10分钟, 温和搅拌。</li>
		<li>添加4C4 抗体 (1/20) 到细胞 (管 3) 和孵育30分钟与温和搅拌每10分钟。</li>
		<li>旋转管在300克10分钟4摄氏度, 然后丢弃上清。</li>
		<li>洗一次与0.5 毫升的介质 A + 2%, 然后旋转管在300克10分钟在4°c。</li>
		<li>用0.5 毫升的培养基再悬浮细胞颗粒 A + 2%, 以1% 为10分钟, 每5分钟以温和搅拌的方法孵化细胞。</li>
		<li>将二次抗体 (1/200) 添加到细胞 (管2和 3)。每10分钟进行30分钟的孵育细胞和轻微的保护。</li>
		<li>旋转管在300克10分钟4摄氏度, 然后放弃上清。</li>
		<li>清洗两次与0.5 毫升的介质 a + 2%, 然后重新悬浮细胞颗粒1毫升的媒体 a + 2%。</li>
	</ol>
</li>
	<li>运行细胞悬浮通过一个35µm 细胞过滤器帽, 并将其转移到冷5毫升在冰上, 保护免受光。</li>
</ol>4. 细胞分选 (外地资产管制)
注意:执行所有步骤4°c。
<ol><li>利用外地资产管制仪对神经元、巨噬细胞/小胶质和小胶质进行排序。 注意:这一步骤通常由外地资产管制系统设施的工作人员执行, 而设置取决于所用设备的类型。<ol>
<li>添加 DAPI 在每一个1µg/毫升的浓度在每个流化管的标签死细胞。</li>
		<li>从所有脑细胞中建立和分类神经元, 巨噬细胞或小胶质。分离的细胞从碎片的大小和粒度的功能, 然后门单细胞的向前散射和侧面散射。通过 DAPI 标记从活细胞中排除死细胞。通过各自的阳性染色识别神经元、巨噬细胞/小胶质或小胶质。</li>
	</ol>
</li>
	<li>收集1.5 毫升管中含有1毫升冰介质的细胞 A + 2% 在冰上。对每个细胞类型使用不同的管。<ol>
<li>旋转管在300克10分钟4摄氏度, 然后丢弃上清。</li>
		<li>用0.5 毫升的介质冲洗一次, 然后丢弃最高的上清。</li>
	</ol>
</li>
</ol>5. RNA 提取
<ol><li>提取 RNA 从不同的细胞类型分离的外地资产管制。对于 RNA 提取使用一个特定的套件, 并遵循制造商的指导方针。确保在 RNase 自由的环境中工作, 清洁工作区和吸管与 RNase 净化产品和使用过滤器提示。<ol>
<li>新鲜准备1毫升溶解缓冲剂补充与50µM β巯基乙醇。 注意:在这里, β-巯基乙醇增加, 以减少 RNA 降解。</li>
		<li>用 RNase 自由水制备70% 和80% 乙醇溶液。</li>
		<li>溶解细胞与75µL 溶解缓冲剂补充50µM β巯基乙醇。然后使用碎纸机来改善细胞的破坏。</li>
		<li>根据制造商手册继续提取 RNA。</li>
		<li>在该协议的末尾, 洗脱 rna 与14µL RNase 游离水获得足够的 RNA 浓度。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

此处描述的实验性协议是一种健壮有效的方法, 可以将斑马鱼幼虫的脑细胞从3到8的 dpf 中分离出来。重要的是, 这是第一个允许特定的小胶质细胞与斑马鱼的大脑分离的协议。该协议的目的是保持细胞膜的完整性, 并尽量减少可能的修改在处理过程中发生的基因表达。这最后一点是关键的相关性的结果, 基于分析这些孤立的细胞基因组剖面。事实上, 小胶质细胞和巨噬细胞极化受到其微环境的强烈影响。在37摄氏度时, 这些细胞会改变他们的基因表达谱, 以应对实验条件 (损伤 (横断) 反应)。因此, 在 RNA 提取前4摄氏度进行这个实验是非常重要的, 以减缓细胞过程和新陈代谢活动。此外, 选择4°c 的机械脑组织, 而不是酶组织消化37°c, 以避免任何影响基因表达谱。
重要的是要强调, 这种方法是非常快的;在一天之内几个脑子细胞人口可以从至少二个不同的实验情况被隔绝和他们的 RNA 提取。该协议的总长度取决于每个条件使用的幼虫数量, 因为幼虫头的横断是限制步骤 (∼350头/小时)。一般而言, 要使用3、5和 7 dpf 的小胶质细胞, 建议将每个测试的样带∼600头, 以获得足够的 RNA 提取 (表 1)。由于小胶质细胞的表达率最低 (每头约112个细胞为7个 dpf), 所以可以减少其他细胞类型 (包括巨噬细胞) 的头数 (每头约170个单位为7个 dpf)。
该协议的另一个优点是, 一旦建立了外地资产管制分类器的设置, 相同的设置可以用于不同的实验。据观察, 细胞的数量完全适合从一个实验到另一个与盖茨以前设计, 显示的重复性的实验使用这种方法。
这种方法的一个缺点是收获的 RNA 量相对较低。然而, 这种限制比技术问题更像是一个生物学问题, 因为小胶质细胞的数量在大脑发育的早期阶段非常低 (表 1)。由于收集到的 RNA 数量较少, 需要考虑放大步骤来进行基因组广泛的基因表达分析。幸运的是, 这些放大步骤产生足够数量的高质量 cDNA。因此, 可以研究孤立细胞基因表达谱的全局变化。
最后, 该协议提供了一种健壮的方法, 以分离和研究各种中枢神经细胞类型的斑马鱼的大脑。这可用于在发展过程中加深对这些细胞的了解, 并研究它们在疾病中的作用。

自从第一次量化老鼠脑 transcriptomes1以来, 在过去十年里, 关于大脑发育和脑部疾病的知识显著改善。事实上, 基因组广泛的基因表达分析给我们提供了关于脑组织和细胞的详细的基因信息, 可以补充和改进与其他技术和工具所做的观察。
斑马鱼是一种强有力的生物模型, 易于繁殖和基因修饰;它的光学透明度在幼虫阶段允许实况成像观察2。不幸的是, 与人和老鼠相比, 可用于免疫染色的抗体数量相当低。为了弥补这种情况, 转基因鱼鱼线很容易由转基因鱼类在细胞型特定促进剂下表达荧光蛋白。转基因斑马鱼线过去曾用于研究在中枢神经系统 (CNS) 发育和疾病中巨噬细胞和小胶质的作用3,4,5,6。然而, 为了深入了解这些过程, 我们需要了解基因表达在各自细胞类型中的变化。为了达到这个目的, 我们开发了一种实验方法, 专门从3到8天后受精 (dpf) 斑马鱼的大脑中分离出神经细胞, 巨噬细胞和小胶质。为建立该议定书, 我们与转基因鱼线合作, 表达绿色荧光蛋白 (GFP) 在巨噬细胞表达基因启动子 (mpeg1:eGFP) 和 DsRed 神经元在神经ß-蛋白启动子 (NBT: DsRed)7,8,9。此外, 我们进行免疫染色小胶质细胞使用 4C4, 一只老鼠单克隆抗体, 专门染色斑马鱼胶质细胞10,11。然后, 从这些细胞中提取核糖核酸 (RNA), 进一步定量聚合酶链反应 (qPCR) 或转录分析。本协议旨在有效地融汇斑马鱼幼虫的脑组织;收集神经元、巨噬细胞/小胶质细胞和小胶质, 而不改变其血浆膜完整性, 最后从高品质 (玲 > 7) 中提取 RNA 和数量进行基因组分析。不同于以前发表的研究, 使用胰蛋白酶治疗在37°c 消化脑组织12,13, 这个协议促进工作在4°c, 直到 RNA 提取步骤, 以减少基因表达谱的修改。这一步是至关重要的, 因为小胶质细胞和巨细胞是高度敏感的电池, 反应的变化, 他们的基因表达谱和极化14,15,16。
这里详细描述的协议显示了从斑马鱼幼虫大脑中分离出的神经元、巨噬细胞和小胶质细胞, 但实际上, 它可以适应大脑中的任何其他单元--要么使用转基因鱼线, 要么标记特定的抗体。这种方法可以通过基因组广泛的基因表达分析来更好地表征中枢神经系统细胞, 并有助于了解它们在发育和脑部疾病中的作用。

<table><tbody><tr><td>1-phenyl 2-thiourea (PTU)</td><td>Sigma</td><td>P7629</td><td></td></tr><tr><td>Hepes</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G8644-100ML</td><td></td></tr><tr><td>HBSS 1X</td><td>Gibco</td><td>14170-088</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-02</td><td></td></tr><tr><td>HBSS 10X</td><td>Gibco</td><td>14180-046</td><td></td></tr><tr><td>DPBS 1X</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Tricaine (MS222)</td><td>Sigma</td><td>A5040</td><td></td></tr><tr><td>Sterilin standard 90mm petri dishes</td><td>ThermoFisher</td><td>101VIRR</td><td></td></tr><tr><td>Surgical micro-scissors</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>3 mL Pasteur plastic bulk pipette</td><td>SLS</td><td>PIP4206</td><td></td></tr><tr><td>Glass homogenizer</td><td>Wheaton</td><td>357538</td><td></td></tr><tr><td>Sterilin standard 55mm petri dishes</td><td>ThermoFisher</td><td>P55V</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-02</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>50 ml polypropylen conical tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 R</td><td></td></tr><tr><td>10 mL syringe</td><td>BD</td><td>302188</td><td></td></tr><tr><td>Needle 23G x 1&#039;&#039;</td><td>BD</td><td>300800</td><td></td></tr><tr><td>Normal goat serum (NGS)</td><td>Cell Signalling</td><td>5425S</td><td></td></tr><tr><td>35 &amp;mu;m cell strainer cap</td><td>BD</td><td>352235</td><td></td></tr><tr><td>FACS tubes</td><td>BD</td><td>352063</td><td></td></tr><tr><td>Low Endotoxin, Azide-Free (LEAF)</td><td>Biolegend</td><td>101321</td><td></td></tr><tr><td>Alexa Fluor 647 Goat Anti-Mouse IgG (H+L)</td><td>Life Technologies</td><td>A11008</td><td></td></tr><tr><td>Anti-4C4</td><td>Courtesy of Catherina Becker (University of Edinburgh)</td><td></td><td></td></tr><tr><td>FACS sorter FACSAria II</td><td>BD</td><td>QMRI, FACS facility</td><td></td></tr><tr><td>RNeasy Plus Micro Kit</td><td>QIAGEN</td><td>74034</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Gibco</td><td>&amp;nbsp;31350-010</td><td></td></tr><tr><td>QIAshredder</td><td>QIAGEN</td><td>79654</td><td></td></tr><tr><td>SsoAdvanced Universal SYBR Green Supermix</td><td>Bio-Rad</td><td>1725271</td><td></td></tr><tr><td>SuperScript III First-Strand Synthesis System</td><td>Invitrogen</td><td>18080-051</td><td></td></tr><tr><td>LightCycler 96 Real-Time PCR System&amp;nbsp;</td><td>Roche</td><td></td><td></td></tr><tr><td>Ovation RNA-Seq System V2</td><td>NuGEN</td><td>7102-32</td><td></td></tr><tr><td>Agilent RNA 6000 Pico reagents</td><td>Agilent</td><td>5067-1513</td><td></td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent</td><td></td><td></td></tr><tr><td>RNaseZap</td><td>Ambion</td><td>AM9780</td><td></td></tr></tbody></table>

isolation and rna extraction of neurons, macrophages and microglia from larval zebrafish brains

为了深入了解不同中枢神经细胞在发育过程中的作用, 以及大脑病理的建立和进展, 在不改变基因表达谱的情况下, 分离这些细胞是很重要的。斑马鱼模型提供了大量的转基因鱼线, 其中特定的细胞类型被标记;例如神经元在 NBT: DsRed 线或巨噬细胞/小胶质在 mpeg1:eGFP 线。此外, 抗体已经发展到染色特定的细胞, 如小胶质与4C4 抗体。
这里, 我们描述了从斑马鱼脑中分离出的神经元, 巨噬细胞和小胶质。该协议的核心是避免酶组织消化37摄氏度, 这可能会改变细胞剖面。相反, 组织均匀化的机械系统在4°c 使用。该协议要求将大脑的同质化分为细胞悬浮、免疫染色和神经元、巨噬细胞和小胶质的分离。之后, 我们从这些细胞中提取 RNA, 并评估其质量/数量。我们设法获得高质量的 rna (rna 完整性数字 (qPCR) > 7) 来执行对巨噬细胞/小胶质和神经元的, 以及 transcriptomic 分析小胶质。这种方法能够更好地刻画这些细胞, 并更清楚地了解它们在发育和病理中的作用。

所描述的协议是一种简单的方法, 以分离的神经元, 巨噬细胞和小胶质鱼的幼虫大脑。从这些孤立的细胞中提取出大量的高质量 (> 7) RNA。本协议的目的是从中枢神经系统中分离出不同类型的细胞, 对其基因表达谱进行最小的修饰, 以分析和表征细胞的特性和功能。因此, 整个协议执行在4°c 与机械脑组织均匀化。该方法已成功地用于实验室进行的两项研究。在第一项研究中, 神经元和巨噬细胞/小胶质体从 8 dpf mpeg1:eGFP+/NBT: DsRed+幼虫 (图 1) 中分离出来。在其大小 (FSC a) 和粒度 (SSC a) (图 1a) 的作用下, 该资产管制署允许从碎片中分离出细胞。然后从双峰或细胞团聚中分离出单个细胞 (图 1B)。从单个单元格填充中, 绘制了一个门以消除死单元格 (DAPI+)。相应的点图显示, 该实验协议保留了细胞的细胞膜完整性, 因为死细胞的速率只有 26.7% (图 1C)。最后, 神经元 (DsRed+) 和巨噬细胞/小胶质 (GFP+) 很容易与活细胞群门隔离。神经元的数量 (23.1%) 似乎比脑内的巨噬细胞/小胶质种群 (1.56%) 更突出 (图 1D)。该协议允许将 RNA 从这些细胞中分离出来进行后续的 qPCR 分析, 比较神经元和巨噬细胞/小胶质的特异基因的表达。图 2显示了增殖细胞核抗原 (pcna)对β肌动蛋白保留基因的神经元和巨噬细胞/小胶质细胞基因表达水平。
第二项研究的重点是小胶质细胞从3、5和7的 dpf 幼虫大脑中分离出来。与上述实验相比, 细胞被免疫染色隔离使用 4C4, 这种抗体专门标记小胶质 (图 3 A D)。如前所述, 从活单元格中选择小胶质细胞 (4C4+) 并收集 (图 3D)。斑马鱼幼虫大脑中的小胶质细胞数是可变的 (表 1), 在 3 dpf (每条鱼的∼ 25) 中非常低。用微毛细管电泳系统测定了这些细胞中提取的 RNA 的质量和数量。从 5 dpf 斑马鱼的小胶质细胞中提取的 rna 得到的结果提供了一个 rna 分析的例子 (表 1 (5 dpf; 实验 4))。图 4显示了为本示例获得的电泳迹线及其图形表示, 其中核糖体 RNA 的清晰可视化 (28s 和 18s)。这些数据是必要的计算样本, 并确定 RNA 浓度。表 1总结了每条鱼的孤立小胶质细胞数量、每个小胶质细胞的 RNA 数量和每个不同实验在3、5和 7 dpf 中获得的玲评分。用这种方法提取的小胶质细胞中 rna 的数量和质量, 使我们能够使用试剂盒将 rna 放大成 cDNA。爱丁堡基因组学提供的质量和数量测试证实, 扩增 cDNA 对库准备和后续测序有足够的质量。图 5显示了 cDNA 片段的大小分布及其使用电泳系统测量的量。在这个样本中, 该 cDNA 的平均大小为 299bp, 浓度为 36100 pmol.表 2分别说明了从 RNA 样品中扩增 cDNA 进行的质量和数量测试 (表 1 (5 dpf; 实验 4))。扩增 cDNA 已成功地用于测序。
在实验室进行的几项研究证实, 从神经元、巨噬细胞和小胶质中提取的 RNA 的质量和数量可以用于随后的 qPCR 和基因组广泛的基因表达分析。因此, 该实验协议可用于可靠地隔离不同类型的 CNS 细胞, 而不改变其细胞膜完整性和限制其基因表达谱的修改。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57431/57431fig1.jpg"> 图 1: 对 mpeg1:GFP 中的神经元和巨噬细胞/小胶质进行分类的操作+/NBT: DsRed+ 8 dpf 斑马鱼幼虫.(丙)连续的门控显示所有脑细胞 (A) 的顺序选择, 单细胞的正向散射和侧散射 (B)。(C) DAPI 标签排除死细胞。(D) DsRed 和 GFP 阳性染色分别鉴定神经元和巨噬细胞/小胶质。<a href="https://cloudflare.jove.com/files/ftp_upload/57431/57431fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57431/57431fig2.jpg"> 图 2: 基因表达分析的pcna和β肌动蛋白神经元和巨噬细胞/小胶质.RNA 从孤立的神经元和巨噬细胞/小胶质可以转录成 cDNA 用于定量 PCR 分析。由 qPCR (N = 3) 确定的孤立神经元和巨噬细胞/小胶质体中的β肌动蛋白保留住基因的pcna mRNA 表达水平。用比较 (ΔΔCT) 法测量褶皱变化。误差线表示平均值.<a href="https://cloudflare.jove.com/files/ftp_upload/57431/57431fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57431/57431fig3.jpg"> 图 3: 对3个 dpf 斑马鱼幼虫的小胶质细胞进行分类.(丙)连续浇口显示所有脑细胞 (A) 的顺序选择, 单细胞的正向散射和侧散射 (B)。(C) DAPI 标签排除死细胞。(D) 4C4 阳性染色鉴别小胶质细胞。<a href="https://cloudflare.jove.com/files/ftp_upload/57431/57431fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57431/57431fig4.jpg"> 图 4: 5 只斑马鱼小胶质细胞提取 RNA 的微毛细管电泳结果.两个高峰是18S 和28S 核糖体 RNA。使用18S 和28S 核糖体亚基的生成比率和整个电泳痕迹的分析, 自动计算出 bioanalyzer 软件的 RNA 完整性数。小胶质细胞 RNA 有8.6。<a href="https://cloudflare.jove.com/files/ftp_upload/57431/57431fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57431/57431fig5.jpg"> 图 5: 5 个 dpf 斑马鱼小胶质细胞 RNA 样品的扩增 cDNA 质量和数量测试.图像显示了 299 bp 的平均大小的样本的 cDNA 片段大小分布.<a href="https://cloudflare.jove.com/files/ftp_upload/57431/57431fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>条件</td>
			<td>实验</td>
			<td>鱼号</td>
			<td>单元格编号</td>
			<td>每条鱼的细胞数</td>
			<td>RNA 浓度 (pg/ul)</td>
			<td>总 RNA (pg)</td>
			<td>每细胞 RNA 量 (pg)</td>
			<td>玲得分</td>
		</tr>
<tr>
<td>3dpf</td>
			<td>1</td>
			<td>700</td>
			<td>11922</td>
			<td>17.03</td>
			<td>126</td>
			<td>1512</td>
			<td>0.13</td>
			<td>8</td>
		</tr>
<tr>
<td>3dpf</td>
			<td>2</td>
			<td>600</td>
			<td>22527</td>
			<td>37.55</td>
			<td>253</td>
			<td>3036</td>
			<td>0.13</td>
			<td>7。9</td>
		</tr>
<tr>
<td>3dpf</td>
			<td>3</td>
			<td>600</td>
			<td>18688</td>
			<td>31.15</td>
			<td>255</td>
			<td>3060</td>
			<td>0.16</td>
			<td>7。7</td>
		</tr>
<tr>
<td>3dpf</td>
			<td>4</td>
			<td>600</td>
			<td>11121</td>
			<td>18.54</td>
			<td>189</td>
			<td>2268</td>
			<td>0.20</td>
			<td>7。8</td>
		</tr>
<tr>
<td>3dpf</td>
			<td>5</td>
			<td>600</td>
			<td>15581</td>
			<td>25.97</td>
			<td>131</td>
			<td>1572</td>
			<td>0.10</td>
			<td>8。4</td>
		</tr>
<tr>
<td>3dpf</td>
			<td>6</td>
			<td>600</td>
			<td>11965</td>
			<td>19.94</td>
			<td>256</td>
			<td>3072</td>
			<td>0.26</td>
			<td>8。2</td>
		</tr>
<tr>
<td>5dpf</td>
			<td>1</td>
			<td>600</td>
			<td>58629</td>
			<td>97.72</td>
			<td>362</td>
			<td>4344</td>
			<td>0.07</td>
			<td>7。4</td>
		</tr>
<tr>
<td>5dpf</td>
			<td>2</td>
			<td>600</td>
			<td>32510</td>
			<td>54.18</td>
			<td>348</td>
			<td>4176</td>
			<td>0.13</td>
			<td>8。1</td>
		</tr>
<tr>
<td>5dpf</td>
			<td>3</td>
			<td>600</td>
			<td>77884</td>
			<td>129.81</td>
			<td>594</td>
			<td>7128</td>
			<td>0.09</td>
			<td>8。3</td>
		</tr>
<tr>
<td>5dpf</td>
			<td>4</td>
			<td>600</td>
			<td>50755</td>
			<td>84.59</td>
			<td>305</td>
			<td>3660</td>
			<td>0.07</td>
			<td>8。6</td>
		</tr>
<tr>
<td>5dpf</td>
			<td>5</td>
			<td>600</td>
			<td>44967</td>
			<td>74.95</td>
			<td>134</td>
			<td>1608</td>
			<td>0.04</td>
			<td>7。6</td>
		</tr>
<tr>
<td>5dpf</td>
			<td>6</td>
			<td>600</td>
			<td>51031</td>
			<td>85.05</td>
			<td>163</td>
			<td>1956</td>
			<td>0.04</td>
			<td>7。9</td>
		</tr>
<tr>
<td>7dpf</td>
			<td>1</td>
			<td>600</td>
			<td>60496</td>
			<td>100.83</td>
			<td>183</td>
			<td>2196</td>
			<td>0.04</td>
			<td>7。6</td>
		</tr>
<tr>
<td>7dpf</td>
			<td>2</td>
			<td>450</td>
			<td>55517</td>
			<td>123.37</td>
			<td>183</td>
			<td>2196</td>
			<td>0.04</td>
			<td>7。8</td>
		</tr>
<tr>
<td>7dpf</td>
			<td>3</td>
			<td>600</td>
			<td>88897</td>
			<td>148.16</td>
			<td>465</td>
			<td>5580</td>
			<td>0.06</td>
			<td>8。1</td>
		</tr>
<tr>
<td>7dpf</td>
			<td>4</td>
			<td>600</td>
			<td>63008</td>
			<td>105.01</td>
			<td>356</td>
			<td>4272</td>
			<td>0.07</td>
			<td>8。4</td>
		</tr>
<tr>
<td>7dpf</td>
			<td>5</td>
			<td>350</td>
			<td>34956</td>
			<td>99.87</td>
			<td>245</td>
			<td>2940</td>
			<td>0.08</td>
			<td>8。1</td>
		</tr>
<tr>
<td>7dpf</td>
			<td>6</td>
			<td>600</td>
			<td>63887</td>
			<td>106.48</td>
			<td>341</td>
			<td>4092</td>
			<td>0.06</td>
			<td>7。8</td>
		</tr>
</tbody></table>表 1: 小胶质细胞分离和 RNA 提取数据从 3, 5 和7的 dpf 斑马鱼幼虫的总结。
<table fo:font-size="7" fo:keep-together.within-page="1"><tbody>
<tr>
<td>内部示例 ID</td>
			<td>外部示例 ID</td>
			<td>量子比特 (ng/ul)</td>
			<td>量子比特 (ng/ul)</td>
			<td>平均浓度 (ng/ul)</td>
			<td>容量 (ul)</td>
			<td>ug 收到</td>
			<td>最小浓度通过/失败</td>
			<td>通过/失败 minimim 数量</td>
			<td>建议数量的传递/失败</td>
		</tr>
<tr>
<td>10907SD0010</td>
			<td>5 dpf (实验 4)</td>
			<td>58。8</td>
			<td>58。4</td>
			<td>58。6</td>
			<td>30</td>
			<td>1.76</td>
			<td>通过</td>
			<td>通过</td>
			<td>通过</td>
		</tr>
<tr>
<td colspan="10">库准备的示例要求:</td>
		</tr>
<tr>
<td>库准备</td>
			<td>最小数量 (ng)</td>
			<td>建议数量 (ng)</td>
			<td>最小浓度</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>TruSeq 纳米凝胶游离 350 bp 从 cDNA 中插入文库</td>
			<td>600</td>
			<td>1100</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>表 2: 5 例斑马鱼小胶质细胞 RNA 样品中扩增 cDNA 的数量测试。

Watch this Scientific Journal Video about Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains at JoVE.com

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

我们提出了一种在生理和病理条件下, 从斑马鱼脑中分离出神经元、巨噬细胞和小胶质的协议。分离后, 从这些细胞中提取 RNA, 分析其基因表达谱。该协议允许收集高质量的 RNA 来执行下游分析, 如 qPCR 和转录组学。

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

isolation-rna-extraction-neurons-macrophages-microglia-from-larval

Research

JoVE Journal

Neuroscience

13.6K 次观看.  University of Edinburgh. 我们提出了一种在生理和病理条件下, 从斑马鱼脑中分离出神经元、巨噬细胞和小胶质的协议。分离后, 从这些细胞中提取 RNA, 分析其基因表达谱。该协议允许收集高质量的 RNA 来执行下游分析, 如 qPCR 和转录组学。

视频: Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

分离培养成年斑马鱼脑源性神经球

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

科研

发育生物学

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

斑马鱼原代细胞的培养与转染

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

处理斑马鱼幼虫的肠道细胞以进行单细胞 RNA 测序

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

作者聚焦:分离和分析斑马鱼幼虫的肠道细胞以研究胃肠道发育的转录组学方面 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric ...

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 ...

神经科学

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

脑室微注射脂多糖到斑马鱼幼虫中以评估神经炎症和神经毒性

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

使用洗涤剂和酶无酶方法从整个组织进行核分离

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

生物学

Brain Cell Isolation from Zebrafish Larvae: A Technique to Obtain Intact Neurons, Macrophages, and Microglia from Zebrafish Brain Tissue

Source: Mazzolini, J. et al. <a href="https://www.jove.com/v/57431/isolation-rna-extraction-neurons-macrophages-microglia-from-larval">Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains.</a> J. Vis. Exp. (2018)
This video describes a protocol to isolate neurons, macrophages, and microglia from larval zebrafish brains by mechanically disrupting the brain tissue. The purified cell populations can further be used for genomic analysis.

从斑马鱼幼虫中分离脑细胞:一种从斑马鱼脑组织中获得完整神经元、巨噬细胞和小胶质细胞的技术

Source: Mazzolini, J. et al. Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains. J. Vis. Exp. (2018)
This ...

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

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此视频中的章节

分离培养成年斑马鱼脑源性神经球

分离和斑马鱼胚胎单细胞的表征

斑马鱼原代细胞的培养与转染

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

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

脑室微注射脂多糖到斑马鱼幼虫中以评估神经炎症和神经毒性

使用洗涤剂和酶无酶方法从整个组织进行核分离

从斑马鱼幼虫中分离脑细胞:一种从斑马鱼脑组织中获得完整神经元、巨噬细胞和小胶质细胞的技术