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 border="0" cellpadding="0" cellspacing="0" style="width:840px;" width="840">
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			<td height="21" style="height:21px;width:252px;">1-phenyl 2-thiourea (PTU)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Sterilin standard 90mm petri dishes</td>
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			<td height="21" style="height:21px;width:252px;">Surgical micro-scissors</td>
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			<td style="width:103px;">357538</td>
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			<td height="21" style="height:21px;width:252px;">Sterilin standard 55mm petri dishes</td>
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			<td height="21" style="height:21px;width:252px;">40 &#181;m cell strainer</td>
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			<td style="width:103px;">352340</td>
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			<td height="21" style="height:21px;width:252px;">50 ml polypropylen conical tube</td>
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			<td style="width:103px;">352070</td>
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			<td height="21" style="height:21px;width:252px;">Centrifuge</td>
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			<td height="21" style="height:21px;width:252px;">10 mL syringe</td>
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			<td style="width:103px;">302188</td>
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			<td height="21" style="height:21px;width:252px;">Needle 23G x 1&#39;&#39;</td>
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			<td style="width:103px;">300800</td>
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			<td style="width:319px;">Cell Signalling</td>
			<td style="width:103px;">5425S</td>
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			<td height="21" style="height:21px;width:252px;">35 &#956;m cell strainer cap</td>
			<td style="width:319px;">BD</td>
			<td style="width:103px;">352235</td>
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			<td height="21" style="height:21px;width:252px;">FACS tubes</td>
			<td style="width:319px;">BD</td>
			<td style="width:103px;">352063</td>
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			<td height="21" style="height:21px;">Anti-4C4</td>
			<td colspan="2">Courtesy of Catherina Becker (University of Edinburgh)</td>
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			<td style="width:103px;">&#160;31350-010</td>
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			<td height="21" style="height:21px;width:252px;">QIAshredder</td>
			<td style="width:319px;">QIAGEN</td>
			<td style="width:103px;">79654</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:252px;">SsoAdvanced Universal SYBR Green Supermix</td>
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			<td style="width:103px;">1725271</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:252px;">SuperScript III First-Strand Synthesis System</td>
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			<td style="width:103px;">18080-051</td>
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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">LightCycler 96 Real-Time PCR System&#160;</td>
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			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Ovation RNA-Seq System V2</td>
			<td style="width:319px;">NuGEN</td>
			<td style="width:103px;">7102-32</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Agilent RNA 6000 Pico reagents</td>
			<td style="width:319px;">Agilent</td>
			<td style="width:103px;">5067-1513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2100 Bioanalyzer</td>
			<td style="width:319px;">Agilent</td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNaseZap</td>
			<td style="width:319px;">Ambion</td>
			<td style="width:103px;">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

Universidad de Cartagena UDEC

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 Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

科研

神经科学

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

斑马鱼幼虫的前脑的电生理记录

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

分离和游离感觉神经元的文化活动从鸡胚

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

可溶和不可溶α-突触核蛋白从帕金森脑连续提取

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

去除<em&gt;果蝇</em从幼虫鱼片的感官神经元和表皮细胞的免疫荧光分析&gt;肌肉组织

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

密度梯度离心法分离小鼠原发胶质细胞的研究

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

多尾根结鱼巨噬细胞的快速隔离

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...