Name: fluorescence activated cell sorting (facs) and gene expression analysis of fos-expressing neurons from fresh and frozen rat brain tissue
Uploaded: 2016-08-27T14:00:00.000+00:00
Duration: 8 min 37 s
Description: Watch this Scientific Journal Video about Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue at JoVE.com

This work was supported by the NIDA Intramural Research Program (Bruce T. Hope, Yavin Shaham). F.J.R. was supported by an appointment to the NIDA Research Participation Program sponsored by the National Institutes of Health and administered by the Oak Ridge Institute for Science and Education, and received additional financial support from a Becas-Chile scholarship managed by CONICYT and the Universidad de los Andes, Santiago, Chile. The Johns Hopkins FACS Core facility was supported by Award P30AR053503 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institute of Health.

所有实验均按照健康指南全国学院的机构动物护理和使用委员会（IACUC）的护理和使用实验动物16进行。 注意:下面使用低结合离心管的所有步骤都保持在冰上，除非另有指定。 1.准备组织收集前<ol><li>离心机设置为4℃。 </li><li>火抛光具有大约1.3递减的直径，0.8和0.4毫米每个样品一组三个玻璃巴斯德移液管。 </li><li>准备含1毫升冷缓冲液A标1.7毫升管和保持管冰。 </li><li>制备含有脑切片矩阵，刮刀和其上以执行清扫玻璃板（或倒玻璃培养皿）的冰盘。预寒意两个或多个刀片上的玻璃板和使用组织纸晾干触及的T所有的仪器问题。 </li><li>解冻在室温（RT）处理30分钟组织收集前的酶溶液。 </li></ol> 2.组织收集和解剖<ol><li>发起组织收集前的行为和药物治疗90分钟。 注:对于这里示出的结果，5毫克/公斤的甲基苯丙胺对一种新的环境（测试条件）和幼稚大鼠大鼠急性腹膜内注射保持在它们的饲养笼（对照条件）进行。 </li><li>通过将其放置饱和异氟醚玻璃干燥器罐子麻醉大鼠和斩首大鼠30 - 60秒后用断头台。 用剪刀去除头部皮肤和肌肉，暴露颅骨。用咬骨钳切割颅骨和开拓枕骨大孔，取出头骨后面的部分。使用骨钳沿头骨的顶边切割以暴露脑。小心不要损伤大脑。使用小铲下轻轻舀Ð提高大脑。提高大脑和切断的神经，直到大脑是免费的17。 </li><li>使用刀片解剖组织。 注:获得使用以下两种方法的组织样本:（2.3.1）新鲜解剖组织; （2.3.2）解剖后冷冻的组织;或（2.3.3）组织冷冻冷冻全脑解剖。保持大脑切片矩阵，刀片和玻璃干板整个解剖和切碎流程作为的冷凝水可能会导致细胞的裂解低渗。使用放大镜，以确保准确的解剖。 <ol><li>对于新鲜解剖组织，将新鲜榨取的大脑已在冰上冷却大脑切片矩阵（大鼠脑形金属模具在1mm间隔的刀片插槽）。插入两个或更多个预冷刀片到槽切割包含所关注的脑区域（多个）冠状切片。放置在冷冻玻璃板切割片。 注:解剖大脑区域选择性对冷冻玻璃板兴趣纳秒。 </li><li>解剖后冷冻组织，从新鲜提取脑，类似于2.3.1上述切​​片解剖感兴趣的脑区域（多个）。放置解剖组织成微管并通过浸没在-40°异戊烷微管，持续20秒快速冷冻组织。保存在-80ºC冷冻，直到进一步的处理在任何时候都冻结解剖组织。 </li><li>对于冰冻组织，立即冻结在-40ºC异戊烷和储存在密封的袋子在-80ºC新鲜提取大脑长达6个月。 <ol><li>上解剖当天，将冷冻的脑在设定为约一低温恒温器-20℃（不超过华氏-18℃）约2小时，达到平衡的温度。 注:大脑可以很容易地在此温度下进行切割，而冷得多的温度使大脑太脆安全地切割。 </li><li>使用刀片切1 - 2毫米冠状切片冷冻大脑在低温恒温器。使用钝12至16号针头，以获得冷冻条件下，从这些切片组织拳。始终保持冷冻，直到进一步的处理解剖组织。 </li></ol></li></ol></li><li>添加1 - 缓冲液A 2滴切碎之前覆盖解剖组织。从使用两个刀片以防止磨碎处理的其余部分中的神经元损失的组织去除大部分的白质。如果与冷冻组织开始，然后允许组织向施加缓冲液A溶液之前在冷玻璃板解冻时间不超过1分钟。 </li><li>剁碎的组织在每个正交方向上的100倍以上的冷却玻璃板的剃刀刀片。切碎当握住刀片垂直于玻璃板。 注:彻底剁碎是所有协议的步骤至关重要。 </li><li>使用刀片的切碎的组织转移到含有1ml冷缓冲液A的倒置管3的离心管 - 5次使电机EP中的解决方案的所有组织糜。 </li></ol> 3.细胞分离注:使用温和的结束比上年末为以下所有步骤的混合。不要使用旋涡混合器，避免气泡造成的细胞损伤。 <ol><li>离心样品管在110 XG 2分钟，4ºC。弃上清。慢慢加入1毫升冷的刚解冻的酶溶液（含有蛋白水解酶的​​混合物）下微管的内壁。立即吸了整个颗粒和轻轻地用中等大小的尖端直径吸管吸取上下只有4次驱散组织糜颗粒。 </li><li>立即倒置微管，以防止粘附于底部的沉淀，并在4℃孵育与最终过端混合样品30分钟</li><li>酶组织消化后，离心机在4ºC960 XG 2分钟管子。除去上清液，加入0.6毫升冷的缓冲液A.立即出现的加入缓冲液A后Y，使用相同的枪头驱散颗粒通过吸了整个颗粒和吸管轻轻向上和向下的5倍。 </li><li>机械磨碎消化的组织，并在使用以下步骤15ml试管收集。对于每个样本，使用一套独立的3火抛光的玻璃巴斯德移液器降直径附着乳胶泡约1.3，0.8和0.4毫米。 <ol><li>轻轻磨碎每个样品与1.3毫米的玻璃吸移管的10倍。让样品静置2分钟在冰上（碎片和未离解的细胞将沉淀在底部）。收集上清液（〜0.6mL）中，并转移到15毫升锥形管（管＃1）。 </li><li>添加0.6毫升缓冲液中的溶液，以剩余的粒料。磨碎样品与0.8毫米的玻璃吸移管的10倍。让样品静置在冰上2分钟。收集上清液（〜0.6mL）中，并转移到同一的15毫升锥形管＃1。 </li><li>加入0.6毫升缓冲液的溶液将剩余PELLE吨。磨碎样品与0.4毫米的玻璃吸移管的10倍。让样品静置在冰上2分钟。收集上清液（〜0.6毫升;不接触与非解离的细胞沉淀），并转移到相同的15毫升锥形管＃1。 注意:保持0.4毫米直径的吸管在管＃1以下研磨步骤。 </li><li>重复步骤3.4.3三次用最小玻璃吸管（〜0.4mm直径），并且收集上清液到一个单独的15毫升锥形管（管＃2）。 </li><li>磨碎在管＃1和＃2所收集的细胞悬浮液，使用0.4毫米的玻璃吸移管10次以上，并保持在冰上。 </li></ol></li></ol> 4.细胞固定和透<ol><li>加入800微升100％的冷乙醇准备（从管＃2单元两个微管从管＃1单元和两个），每个样品4微管（储存在使用前-20℃）每管，并让他们在冰。注意:最终乙醇浓度为50％。 </li><li>吸取〜800微升细胞悬浮液（从管＃1和＃管2）到每个含冷乙醇的4管和颠倒管混匀样品。 </li><li>在冰上孵育管15分钟，同时反相，每5分钟的管中。 </li><li>固定/透化后，离心管1700 xg离心在4ºC4分钟并弃上清。离开50微升的溶液，以防止细胞的损失。 注:在此阶段的粒料是白色和粘比透以前少在管子的壁上。因此，小心地取出上清液通过触摸微管的管壁里没有颗粒和制定上清慢慢用微量。 </li></ol> 5.细胞过滤<ol><li>重悬来自步骤4.4用冷PBS将粒料如下: <ol><li>添加550微升冷PBS的两个微管，从管＃1来的一个，并使用适度大直径圆周佩特笔尖轻轻吸取细胞悬液上下5次。重复相同的一个微管由管＃2的到来。 </li><li>从管＃1细胞，使用相同的移液管，所述第一细胞悬浮液转移到第二沉淀。轻轻吹打组合的细胞悬液上下5次重悬第二沉淀。 </li><li>从管＃2细胞，重悬，并结合在5.1.2中描述的颗粒（ 即，第三和第四微管）。 </li></ol></li><li>过滤来自分别使用两种不同的对细胞滤器（100微米和40微米孔径大小）如下步骤5.1.2和5.1.3两种细胞悬浮液: <ol><li>预湿通过加入PBS至过滤器的内侧的每个细胞过滤。使用移液管从过滤器的外侧除去过量的PBS中。 </li><li>移液器步骤5.1.2细胞悬浮液均匀涂抹于100微米孔径的第一个细胞过滤。收集在冰上的50ml管中的流通。使用PI佩特收集液流 - 通过附着在细胞过滤的外侧底部。 </li><li>使用相同的移液管的流动通从100微米的细胞滤网转移到40微米的细胞滤网。收集在冰上另外50毫升管此流过。当40微米的细胞过滤通过传输流，切换到新的枪头，以避免从100微米的细胞过滤污染。 </li><li>重复这些步骤步骤5.1.3细胞悬液采用一套新的过滤器。 </li></ol></li><li>从每个样品结合两种流过为每个样品约1ml的最终体积。 </li></ol> 6.孵化与抗体注:使用脑细胞悬液的小等分，为运行主样本之前，适当的流式细胞仪的设置设置准备多个对照样品。对于每个抗体标记，准备对照样品，其中包括没有抗体，只有本身condary抗体（或其他荧光标记），然后一次和二次抗体（或其他荧光标签）。使用这些控件设置单独与特定的流式细胞仪的非特异性标记的大门。如果可能的话，使用不要求赔偿的荧光。加入第一抗体之前加入的PBS增加选通控制样品的700微升最终体积的最终体积。 <ol><li>通过过滤的细胞的100微升样品转移至1.7毫升微管制备光散射和DAPI对照样品。添加600微升冷PBS与1微克/毫升的DAPI对细胞核染色。孵育10分钟终过端的混​​合，并通过流过流式细胞仪之前清洗（如在步骤6.4.2至6.4.5表示）。 注:此示例只用于设置之前在FACS机器分拣剩余细胞的细胞/细胞核栅极。 </li><li>通过转让100＆＃制备单免疫标记对照样品181;微升过滤细胞对一个1.7毫升微型管。添加600微升冷PBS与一种抗体（ 例如，的NeuN抗体）。添加600微升冷PBS到与另一种抗体（ 例如，Fos抗体）微管。添加相应的二级抗体，如果它不是一直接缀合的初级抗体。 注意:这些样品被用来确定相应的光电倍增管（PMT）的电压，并在必要时，以评估两种荧光信号的重叠。抗体浓度应该滴定，以达到最佳的信号 - 噪声比中的所有信道。重叠的荧光信号，也可以通过流式细胞仪内不同的荧光通道的内部校准补偿。 </li><li>通过将100μl过滤的细胞转移到一个1.7毫升微型管制备双荧光标记的阴性对照样品。添加600微升冷PBS单独用二抗或IgG直接缀合的初级抗体。 注:氏与S样品可以进行排序，以确定每个荧光背景荧光之前使用每一次。 </li><li>制备主要样本进行排序。 <ol><li>转移700微升过滤细胞成1.7毫升微型管。加7微升（1:100）主Fos抗体的直接缀合的Alexa Fluor 647（抗的Fos-AF647）和1.4微升（1:500）初级的NeuN抗体的直接缀合到藻红蛋白（抗的NeuN-PE）。在4ºC与最终过端混合孵育管30分钟。 </li><li>在孵育结束时，加入800微升冷PBS到每个微管，包括主样品和对照样品，并通过倒置混合。 </li><li>微管离心在4ºC1300 XG 3分钟。弃去上清液，留下50微升上清液，以防止细胞的损失。 </li><li> 1毫升冷PBS添加到使用移液管有一个中等大小的顶端直径的颗粒和重悬细胞。 </li><li>离心机在4ºC1300 XG 3分钟。 ðiscard上清液。 </li><li>重悬在500微升冷PBS丸粒（调节终体积取决于颗粒的大小）。 </li><li>转移和悬浮液用细胞过滤网帽（40微米）过滤到圆底FACS样品管中。保持冰的免疫标记细胞和立即执行FACS。注意:对于过滤，垂直触及滤网盖枪头，并通过它推动细胞悬液。 </li></ol></li><li>设置流式细胞仪使用对照样品门。 <ol><li>使用光散射和DAPI控制样品从事件总数和细胞碎片识别神经元细胞群。 注:本制剂细胞体只有1 - 总的事件（其余的都是碎片和破碎的细胞过程）的2％。基于DAPI的荧光信号（指示核）和前向散射（小区大小），可以找到向后基于所述前向和侧向细胞体和栅极光散射特性将细胞（ 如图1的A，B）。 </li><li>使用单免疫标记对照样品，以确定PMT电压设定，并分析排放溢出从一个特定的荧光染料进入另一个过滤器/通道（ 如的Alexa Fluor 488 / FITC或藻红蛋白）。如果荧光信号重叠，通过手动调节薪酬水平纠正重叠。 </li><li>使用双荧光标记的阴性对照来建立阈值的正的人口。 注意:从该样品来的信号被认为是由于自体荧光和位于高于该阈值的主要试样的细胞可以被认为是阳性。通常情况下，用于排序的阈值的参数是更严格的，并大致标记的细胞的阴性对照以上的顶部2/3实际上排序。 </li></ol></li><li>排序由FACS主要样本的神经元到1.7毫升微管。收集细胞于50微升RNA的提取液。排序，涡旋并离心管并通过上下抽吸10次来恢复所有分选的细胞，从该管的壁混合该悬浮液后。 </li><li>孵育在42ºC的分选的细胞悬浮液30分钟，以确保所有的细胞之前，提取RNA裂解，然后在2,800 xg离心在4ºC离心2分钟。 </li><li>将上清转移到长期存储的无RNase的管中，在-80ºC或立即处理用于RNA分离，cDNA合成，靶基因的预扩增和qPCR样品，如先前13-15所述。 </li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-induced+reinstatement+of+methamphetamine+seeking+is+associated+with+unique+molecular+alterations+in+Fos-expressing+dorsolateral+striatum+neurons.">Context-induced reinstatement of methamphetamine seeking is associated with unique molecular alterations in Fos-expressing dorsolateral striatum neurons.</a> J Neurosci. 35, 5625-5639 (2015).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incubation+of+methamphetamine+craving+is+associated+with+selective+increases+in+expression+of+Bdnf+and+trkb,+glutamate+receptors,+and+epigenetic+enzymes+in+cue-activated+fos-expressing+dorsal+striatal+neurons.">Incubation of methamphetamine craving is associated with selective increases in expression of Bdnf and trkb, glutamate receptors, and epigenetic enzymes in cue-activated fos-expressing dorsal striatal neurons.</a> J Neurosci. 35, 8232-8244 (2015).</li><li>US National Research Council. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the care and use of laboratory animals. , (2011).</li><li>Koya, E., Margetts-Smith, G., Hope, B. 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The authors declare that they have no competing financial interests.

FACS可用于从新鲜或冷冻的成人脑组织的神经元和其他细胞类型进行排序。如在引言中提到的，使用冷冻组织的能力允许从已经历复杂和长期行为的程序，如在成瘾研究自我施用和复发的研究的动物样品的最佳利用。这些行为过程通常需要1 - 2小时或更长的时间，并且要求所有动物（10 - 20个）上13,18在同一天进行测试。它需要〜4小时处理4新鲜解剖脑组织样本，其包括脑解剖，细胞解离，透化和免疫标记。处理后，FACS每个样本排序之间需要20 - 30分钟。在裂解缓冲液中加热分选的细胞的最后步骤需要30分钟。总共，〜7小时，从脑解剖需要最终含RNA的细胞裂解液的步骤。然而，现在有可能冻结整个brai纳秒或新鲜解剖大脑区域的测试后，立即和以后处理。这极大地简化了测试，并且允许多个脑区在不同的一天进行排序。冷冻前的FACS组织也可能会提高效果。更多的NeuN阳性神经元是从冷冻的组织样品获得。这可能部分地通过增加的NeuN标签由于冷冻和其后的解冻时细胞膜的破坏，这将增加抗体获得胞内蛋白如的NeuN和Fos蛋白进行说明。类似的效果已被观察到的成纤维细胞，上皮细胞和HeLa细胞19。 细胞和神经元的产率可以通过下列步骤被最大化。在组织剥离，（分别为胼胝体和前联合的背侧纹状体和伏隔核，）去除大部分的白质。这可以防止组织附着在随后STE塑料提​​示和玻璃吸管PS。使用缓冲液A（见材料和​​试剂表规范），以保持覆盖的解剖组织。这提高了具有刀片（步骤2.5）的组织的切碎过程中的细胞的质量。额外的一组具有最小玻璃吸管（步骤3.9）和随后的滤波带的第二组细胞滤器（步骤5）的3研磨步骤加倍细胞和神经元的产率。重用细胞过滤器会阻塞过滤器和较低的细胞产量。再悬浮颗粒和研磨期间，当温柔与细胞。在这些步骤中使用了中等大枪头将减少由于剪切应力细胞损伤。用乙醇（步骤4.4）透后，除去上清液时仔细观察沉淀。沉淀是不紧凑，并且可以沿着所述的离心管的壁分布。粗略的计算表明Fos蛋白阳性神经元的产率是解剖纹状体组织样品中的起始数的大约10％。 </p&gt; 目前的协议可以被修改以适应其它实验目的。在当前的协议，组织收集甲基苯丙胺注射之后90分钟，因为Fos表达达到在这个时候，它是Fos蛋白表达的神经元的有效排序的关键最大水平。然而，组织可以在用于FACS的任何时间点被收集，这取决于实验的具体目的。初始组织样品存储，我们已成功地使用新鲜解剖组织，解剖后冷冻组织，或从冷冻全脑解剖分离使用FACS随后的基因表达的定量PCR分析的Fos表达神经元冷冻的组织。在这个协议（4-ºC15分钟）固定和透化，持续时间和温度进行了优化，以透既细胞质和细胞核膜，以及以固定组织。在持续时间和/或温度变化可改变最终结果。其他固定的解决方案，如paraformaldehyde和非离子型去污剂如皂角苷，已对胚胎和产后神经元20的工作，也可能适用于从成年脑的神经元。此外，最近的研究显示用于FACS从脑组织21髓鞘去除珠可以用作当前协议的修改。 目前的协议有两个重要的考虑限制。首先，该协议不适合用于检测表达主要在突触（ 例如 ，多巴胺或谷氨酸受体），因为这些细胞过程是从在研磨和离解步骤胞体取出细胞表型标志物。第二个可能的限制是，从FACS分选的细胞中获得的RNA的长度可能不足以为所有RNA分析方法。用于从FACS获得的RNA的RNA完整性数（RIN）分选的细胞是2.5-3.5，其对应于较短的平均的RNA长度之间。在此，更短的RNA尺寸通过补偿使用80的相对较短的定量PCR扩增- 100个基点，如前面所描述13-15。我们也使用这些长度较短的RNA成功用于微阵列分析11。然而，获得的短RNA的长度可能不足以为所有RNA分析方法。应当强调的是，使用特异性靶向的外显子 - 外显子连接处仅在完全剪接的mRNA发现在胞质溶胶中的PCR引物。这些引物不从细胞核检测含内含子的RNA。此外，我们已经先前使用这个协议与抗体和新鲜的脑组织，以检测酪氨酸羟化酶在细胞质和D1多巴胺受体在细胞膜10。这些胞质和细胞膜标记物的检测表明，离解过程产生基本完好的细胞，而不是简单地细胞核。 总体上，从冷冻样品神经细胞的FACS隔离打开其他应用程序。在分拣或基因expre无差异裂变观察（数据未示出），当样品在-80储存3天或3周 ºC。三个星期是一个合理的时间从一个特定的实验中所有样本进行排序（20 - 40个样本）和基因表达分离RNA。可用的RNA也已经从在-80℃（数据未在这里示出）保存6个月的脑组织得到的。因此，该协议可能是已存储数月甚至数年验尸冷冻人脑样品的FACS隔离是有用的。

学习过程中，动物形成复台高度特异性的刺激之间的关联。此高分辨率信息被认为是由稀疏分布的神经元称为神经合奏特定图案内的改变进行编码。神经乐团最近已确定了的立即早期基因（即早基因）如Fos的 ， 圆弧和Zif268及其蛋白质产物在神经元的行为或线索暴露在强烈激活的诱导。 fos基因表达的神经元尤其已经示出在背景和线索特定习得行为1-4发挥因果作用。因此，这些被激活的Fos表达神经元内独特的分子neuroadaptations是编码了解到，在正常的学习和正常的学习障碍形成的协会，如成瘾和创伤后应激障碍（PTSD）5的神经机制的热门人选。 荧光李宗红激活细胞分选（FACS）最近允许的Fos蛋白表达的神经元内独特的分子neuroadaptations分析。流式细胞术和细胞分选在1960 6,7被开发根据它们的光散射和免疫特征来表征和分离细胞，并且早已在免疫学和癌症研究中使用。然而流式细胞仪和FACS需要解离的单细胞难以从成人脑组织得到。 FACS最早是用来分离和分析绿色荧光蛋白（GFP）-expressing从没有要求抗体标记8,9转基因小鼠纹状体神经元。我们开发了一种基于抗体的FACS方法10来隔离并在的Fos表达由药物和/或暗示在野生型动物中11-15激活的神经元评估分子改变。在该方法中，神经元被标以对一般的神经元标记物的NeuN的抗体，而强烈激活的神经元标记与抗Fos蛋白的抗体。虽然我们最初的方法所要求达到10只，每个样品新鲜组织池，该协议的后续修改允许从单个老鼠13-15的Fos表达神经元和定量聚合酶链反应（定量PCR）分立的大脑区域的分析，流式细胞仪分离。总体而言，在Fos蛋白表达的成瘾研究12,14,15各种上下文和提示激活行为的过程中激活的神经元中发现独特的分子变化。 与新鲜组织进行的FACS的一个主要的后勤问题是，它需要一整天以解离通过FACS组织和处理。此外，只有大约四个样品每天可处理。这通常意味着只有一个大脑区域可从各脑进行评估和剩余的脑区域都被丢弃。这对于低吞吐量行为程序，如自我施用和消光TRAI一个主要问题宁认为需要手术治疗和强化训练几个星期。此外，在测试当天长期和复杂的行为过程使得难以在同一天进行的FACS。这将是一个显著优点是能够从动物行为测试后，立即冻结的大脑，然后在研究者的选择的不同的时间从一个或多个脑区域隔离的Fos表达神经元。 在这里，我们证明，我们的FACS协议可以用于分离从新鲜和冷冻的脑组织的Fos表达神经元（和其它细胞类型）。作为一个例子，我们分离的Fos表达后急性注射甲基苯丙胺，并从幼稚大鼠不打针（对照条件）从纹状体神经元。然而，该FACS协议可以按照任何行为或药物治疗中使用。我们的样品的后续的qPCR分析表明，从这些类型的细胞的基因表达可以与西米进行评估从新鲜和冷冻组织征地拆迁的效率。

<table><tbody><tr><td>Brain matrix</td><td>CellPoint Scientific</td><td>69-2160-1</td><td>to obtain coronal brain slices</td></tr><tr><td>Hibernate A low fluorescence</td><td>Brain Bits</td><td>HA-lf</td><td>Buffer A in the protocol is used for processing tissue and cells from the dissociation to fixation steps</td></tr><tr><td>Accutase</td><td>Millipore</td><td>SCR005</td><td>Enzyme solution in the protocol is used for enzymatic digestion of tissue prior to trituration</td></tr><tr><td>Protein LoBind Tube 1.5 ml, PCR clean</td><td>Eppendorf</td><td>22431081</td><td>to prevent cell lost during the protocol</td></tr><tr><td>Cell Strainer, 40 &amp;micro;m</td><td>BD Falcon</td><td>352340</td><td>to filter cell suspension</td></tr><tr><td>Cell Strainer, 100 &amp;micro;m</td><td>BD Falcon</td><td>352360</td><td>to filter cell suspension</td></tr><tr><td>Falcon 5 ml round-bottom polystyrene test tube with cell strainer snap cap</td><td>BD Bioscience</td><td>352235</td><td>to filter cell suspension before passing though the flow cytometer</td></tr><tr><td>Pasteur Pipet, Glass</td><td>NIH supply</td><td>6640-00-782-6008</td><td>to do tissue trituration</td></tr><tr><td>Milli-Mark Anti-NeuN-PE, Clone A60 (mouse monoclonal) antibody</td><td>EMD Millipore</td><td>FCMAB317PE</td><td>antibody to detect neurons</td></tr><tr><td>Phospho-c-Fos (Ser32) (D82C12) XP Rabbit (Alexa Fluor 647 Conjugate)&amp;nbsp;</td><td>Cell Signaling Technology&amp;nbsp;</td><td>8677</td><td>antibody to detect Fos-expressing cells</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D8417</td><td>to label nuclei</td></tr><tr><td>PicoPure RNA Isolation Kit</td><td>Applied Biosystems.</td><td>KIT0204</td><td>The kit includes the RNA extraction buffer for step 6.14. It is used to collect sorted cells</td></tr><tr><td>Superscript III first strand cDNA synthesis system</td><td>Invitrogen</td><td>18080-051</td><td>to synthesize cDNA from RNA</td></tr><tr><td>TaqMan PreAmp Master Mix</td><td>Applied Biosystems.</td><td>4391128</td><td>to do targeted-preamplification from cDNA</td></tr><tr><td>TaqMan Advance Fast Master&amp;nbsp;</td><td>Applied Biosystems.</td><td>4444963</td><td>to do PCR using TaqMan probes</td></tr><tr><td>Fos TaqMan probe</td><td>Applied Biosystems.</td><td>Rn00487426_g1</td><td>TaqMan probe/primers</td></tr><tr><td>&lt;em&gt;NeuN&lt;/em&gt; TaqMan probe</td><td>Applied Biosystems.</td><td>CACTCCAACAGCGTGAC</td><td>nucleotide sequence for neuronal gene marker</td></tr><tr><td>&lt;em&gt;NeuN&lt;/em&gt; Forward primer</td><td>Applied Biosystems.</td><td>GGCCCCTGGCAGAAAGTAG</td><td>nucleotide sequence for neuronal gene marker</td></tr><tr><td>&lt;em&gt;NeuN&lt;/em&gt; Reverse primer</td><td>Applied Biosystems.</td><td>TTCCCCCTGGTCCTTCTGA</td><td>nucleotide sequence for neuronal gene marker</td></tr><tr><td>&lt;em&gt;Gfap&lt;/em&gt; TaqMan probe</td><td>Applied Biosystems.</td><td>Rn00566603_m1</td><td>TaqMan probe/primers for astrocityc gene marker</td></tr><tr><td>&lt;em&gt;iba-1&lt;/em&gt; TaqMan probe</td><td>Applied Biosystems.</td><td>Rn00574125_g1</td><td>TaqMan probe/primers for microglya gene marker</td></tr><tr><td>&lt;em&gt;Gapdh&lt;/em&gt; TaqMan probe</td><td>Applied Biosystems.</td><td>CTCATGACCACAGTCCA</td><td>nucleotide sequence for reference/housekeeping gene</td></tr><tr><td>&lt;em&gt;Gapdh&lt;/em&gt; Forward primer</td><td>Applied Biosystems.</td><td>GACAACTTTGGCATCGTGGAA</td><td>nucleotide sequence for reference/housekeeping gene</td></tr><tr><td>&lt;em&gt;Gapdh&lt;/em&gt; Reverse primer</td><td>Applied Biosystems.</td><td>CACAGTCTTCTGAGTGGCAGTGA</td><td>nucleotide sequence for reference/housekeeping gene</td></tr><tr><td>&lt;em&gt;Oligo2&lt;/em&gt; TaqMan probe</td><td>Applied Biosystems.</td><td>Rn01767116_m1&amp;nbsp;</td><td>TaqMan probe/primers for oligodendrocytic gene marker</td></tr><tr><td>P1000 pipettor</td><td>Rainin</td><td>17014382</td><td>It is refered to as the pipette with a large tip diameter in steps 3.1 and 3.3 for mild tissue trituration and step 6.7 to resuspend cells</td></tr><tr><td>7500 Fast Real-time PCR system</td><td>Applied Biosystems.</td><td>446985</td><td>for quantitative PCR</td></tr><tr><td>FACSAria I Cell Sorter</td><td>BD Biosciences</td><td></td><td>for FACS sorting</td></tr></tbody></table>

fluorescence activated cell sorting (facs) and gene expression analysis of fos-expressing neurons from fresh and frozen rat brain tissue

神经可塑性和习得行为的分子变化的研究是从全脑区的研究转向的稀疏分布激活的神经元称为介导学协会神经歌舞团特定集合的研究。荧光活化细胞分选（FACS）最近已经成年大鼠脑组织进行了优化，并允许使用抗神经元的NeuN标记和Fos蛋白，强烈地激活的神经元的标志物激活的神经元的隔离。到现在为止，Fos蛋白表达的神经元和其他细胞类型，从新鲜组织，从而享有很长的处理天脑样品的允许号码非常有限的冗长和复杂的行为过程之后进行评估隔离。在这里，我们发现，Fos蛋白表达来自背侧纹状体神经元和Fos蛋白的mRNA产率分别为新鲜解剖组织和组织在-80ºC冷冻3之间相似- 21天。另外，我们证实了表型所述的NeuN阳性和的NeuN阴性分选的细胞通过神经元的评估基因表达的（ 的NeuN），星形细胞（GFAP），少突胶质细胞（Oligo2）和microgial（Iba1）标记物，这表明冷冻组织也可用于的FACS分离的神经胶质细胞类型。总体而言，它是可以收集，解剖和冷冻多个FACS会话脑组织。这最大化从有价值动物受试者已经经常发生长和复杂的行为过程获得的数据量。

急性注射甲基苯丙胺后排序从单只新鲜和冷冻背侧纹状体组织的Fos阳性和Fos蛋白阴性神经元。 上述方案用于甲基苯丙胺（5毫克/千克）的腹膜内注射后从单个的大鼠背侧纹状体90分钟的Fos阳性和Fos蛋白阴性的神经元进行排序。在他们的家笼幼稚大鼠用作对照。背其集合（新鲜组织）或被冷冻并储存在-80ºC为1,7或21天之后进行处理后，立即进行处理纹状体组织任一。从这些冷冻时间点的结果不是彼此显著不同，所以该数据汇集。 要设置在仪器上的分选条件下，对​​照样品无论从家笼控制或methampheta（如上所述）用于矿山注入集团。当这些样品中的每个粒子或事件穿过所述流式细胞仪的细胞，它们被赋予与该事件的光散射和荧光特性相关联，并记录在电子表格中的识别号码。在此表中的事件可以然后在散点图或密度图，这些特性（ 图1）来组织。具有相似特征的事件可以被分组或由不同的成对的特性，以确定门如那些含有细胞，神经元，或Fos蛋白阳性神经元"门控"。一旦栅极使用上述的对照样品确定的，主样品按照这些门注入流式细胞仪和排序。细胞群体是从基于其正向光散射的所有事件（细胞和碎片）选通（FSC;粒子的大小的指示）和侧向光散射（SSC;粒子的克的指示ranularity）。如图中的FSC相对于SSC散点图（ 图1A和1B）中，所有事件的密度图揭示了具有相似尺寸和粒度小同质种群中，除了大的异质群体（ 图1A和1B）。包含细胞体小的同质群体被确定和门为"细胞"的基础上，FSC和SSC特征与以往研究11,13-15和DAPI标签。从比新鲜组织（1.9％），冷冻的组织（2.6％）获得细胞的比例略高。在大型异构人口主要是碎片，推测为树突和轴突的进程。 接着，从"细胞"门单个细胞进行鉴定根据它们的大小，这是通过对在x轴上的每个事件的FSC信号的宽度来表示。这比单细胞的大型活动被认为是细胞聚集<html>并排除在"单细胞门，所有的后续分析步骤，此范围内进行的"单个单元"门（ 图1C和1D）。此单细胞群的大部分（&gt; 92％）是阳性的DAPI染色（数据未示出）。 首先，神经元基于该指示的NeuN免疫反应（ 图1E和1F）他们的体育荧光强度确定。神经元从新鲜组织和冷冻组织38％的"单细胞"闸门包括所有事件的大约24％。几乎所有的在这个"神经元"门的事件（在新鲜组织为98％，在冷冻组织99％）呈阳性的细胞的细胞核（ 图1G和1H）的DNA的DAPI染色。在单细胞栅极剩余DAPI标记的事件的NeuN阴性和包括神经胶质细胞，少突胶质细胞和小胶质细胞，如通过qPCR在图3中证实。 ove_content"FO:保持-together.within页="1"&gt;然后神经元被列为的Fos阳性或Fos蛋白阴性的神经元基于它们的Alexa Fluor 647的荧光信号（Fos蛋白免疫反应）不同细胞类型的标记，Fos蛋白。在神经元的增加，由于不同的神经活动和时间过程的水平表达水平逐渐，因此，不会有对Fos蛋白阴性的神经元的Fos阳性之间的明确的阈值，在第一步骤（只用于离线分析，以计算的Fos阳性细胞）的百分比，我们定义）从幼稚家笼控制大鼠的NeuN阴性人口基于最大的Alexa-647荧光（对于Fos蛋白的Fos阳性神经元的阈值。Fos蛋白阳性神经元被表示在从图2中不同的实验条件下，蓝色正方形在新鲜和冷冻的组织中，从甲基苯丙胺注射的大鼠的百分比的Fos阳性神经元。（1.9 - 2.2％， 图2C和2D）是两次作为共mpared至家笼大鼠（0.7 - 0.8％， 图2A和2B）。 使用靶基因扩增前和RT-PCR的FACS分选的细胞，确认细胞类型特异性基因。 在第二步骤中，以确保从用于随后的mRNA主要样本仅Fos蛋白阳性神经元的排序分析和减少的Fos阴性细胞的夹杂物，Alexa的-647荧光阈值升高，使得Fos蛋白的仅上部三分之二在蓝色正方形阳性事件被分类收集。该阈值具有至少10倍大于上述用于计算样本中的Fos阳性神经元的百分比阈值更高的荧光（每个细胞更高Fos表达）。细胞的四群，从一个单一的组织样品，包括的NeuN阴性+ Fos蛋白阴性（〜5,000个事件），的NeuN阴性+的Fos阳性排序（介于25 - 83事件），的NeuN阳性+的Fos负ative（5000〜事件）和的NeuN阳性+ Fos蛋白阳性（44不等 - 为家庭组的133事件，185 - 450事件为甲基苯丙胺组）。第一，细胞类型特异性基因的NeuN阳性（包括的Fos阳性和Fos蛋白阴性）和的NeuN阴性群体（包括的Fos阳性和Fos蛋白阴性）的表达被证实（ 图3）。GAPDH为作为参照/看家基因的基础上，从我们以前的研究结果13。为了控制在PCR反应中的样品和cDNA在不同层次的RNA，使用两种靶基因和GAPDH cDNA的引物进行双链的qPCR反应。对于GAPDH看家基因的Ct值保持在15至31之间，用于精确测量。对新鲜和冷冻组织， 的NeuN mRNA水平在的NeuN阳性人群比的NeuN阴性群体（P &lt;0.05显著较大（约8倍）; 图3A </stroNG&gt;;数据来自家庭和甲基苯丙胺类相结合）。相反，对于新鲜和冷冻组织，GFAP（胶质细胞标记; 图3B）和Oligo2（少突胶质细胞标记物; 图3C）的mRNA水平比在的NeuN阳性人群（对更大的的NeuN阴性人口&lt;0.05 ）。在的NeuN阴性群体中观察到的mRNA水平，但这没有统计学显著;更高Iba1的类似的趋势（ 图3D小胶质细胞标记物）。载有无法在特定细胞类型来精确地测量特定mRNA的极低水平一些样品（ 例如 ，GFAP mRNA表达的NeuN标记的神经元）。最大的Ct值被定义为35，以排除过低被精确的检测值。较高的Ct值超过了我们的qPCR实验的置信度。 FOS mRNA表达从接收到的甲基苯丙胺的单次注射（ 图4）的大鼠中的NeuN阳性的神经元群检测。对于新鲜和冷冻组织，Fos的 mRNA水平比的Fos-负更大的神经元中的Fos阳性神经元（P &lt;0.05）。此外，虽然有在Fos蛋白阳性神经元从新鲜组织Fos蛋白 mRNA的3倍的增加，Fos蛋白 mRNA的Fos蛋白阳性神经元从冷冻的组织中检测到了11倍的增长。两者合计，这些mRNA的结果证实了Fos蛋白阳性神经元的身份，从新鲜和冷冻组织的Fos阴性的神经元和非神经元群体。此外，细胞类型特异性基因和感兴趣的其它基因也可从新鲜和冷冻的条件下，分选的细胞进行分析。 <img alt="图1" src="/files/ftp_upload/54358/54358fig1.jpg" /> 图1. 派息浇注从新鲜和冷冻鼠背侧纹状体sociated和标记的细胞。在&#39;细胞&#39;门被前向和侧向散射特性（AD）确定，由核DAPI染色（GH）阳性标记证实。在"细胞"人口中的"神经元"门是由荧光的NeuN为（; EF PE）来确定。 （A - B）的细胞门:所有事件的线性图，根据它们的前向散射（X轴，细胞大小）和侧向散射（Y轴，粒度）。 （C - D）单细胞闸门:AB中所示的电池门内前向散射高度（Y轴）和宽度（X轴）的线性图。 （E - F）的神经元栅极:免疫荧光对在光盘中所示的单电池内浇口PE-标记的NeuN（Y轴）的对数曲线图揭示了低级集群中的活动和非神经元细胞的上部集群中的神经元。 （G </stroNG&gt; - 高 ）核染色:对神经细胞内浇口DAPI标记的核（Y轴）荧光的对数曲线。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54358/54358fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54358/54358fig2.jpg" /> 图2. 基于双标签的的NeuN和Fos蛋白的新鲜和冷冻鼠背侧纹状体的Fos阳性和Fos蛋白阴性神经元的门控，从大鼠新鲜和冷冻背侧纹状体（单后直接从他们的家笼或90分钟取甲基苯丙胺的腹膜内注射）进行解离，并用抗的NeuN和Fos蛋白直接缀合的抗体标记，并使用FACS机器排序。排序是基于藻红蛋白（PE）标记的NeuN免疫荧光（X轴）和Alexa 647标记的Fos immunofluorescence（Y轴）。的Fos阳性（蓝点）和FOS阴性（红点）的神经元分别位于上和下右象限，分别。点图显示的NeuN阳性细胞（神经元）和的NeuN阴性细胞（灰色点）;蓝色的方块表示上述阈值Fos表达的神经元。 （A - B）家庭组:直接从他们的笼子采取天真老鼠。 （C - D）甲基苯丙胺类:接受甲基苯丙胺单一注射鼠。为FOS阳性神经元的阈值被选定为略高于最大的Alexa-647荧光（FOS-IR）的家笼对照组的NeuN阴性细胞中观察到。虽然0.7 - 所有神经元的0.8％的家庭组中的Fos阳性，1.9 - 所有神经元的2.2％的甲基苯丙胺类中的Fos阳性。 <img alt="图3" src="/files/ftp_upload/54358/54358fig3.jpg" /> 图3。 </在强&gt; 细胞类型特异性基因表达流式细胞分选，从新鲜和冷冻鼠背侧纹状体细胞。的NeuN阳性神经元（FOS阳性和Fos蛋白阴性）和的NeuN阴性细胞（FOS阳性和Fos蛋白阴性）进行了排序使用所描述的协议和细胞类型特异性基因的mRNA表达水平被用来确认细胞分选（A） 的NeuN为神经细胞的标记物。（B）的 GFAP是胶质细胞的标记物（C）Oligo2是标记为少突胶质细胞（D）Iba1为小神经胶质细胞的标记物。为的NeuN的mRNA，数据表示为平均值±SEM从新鲜组织相对表达水平倍值的NeuN阴性细胞（9 - 14）。对于GFAP（6 - 12），Oligo2（N = 9 - 11）和Iba1（N = 5 - 8）的mRNA，数据相对表现为平均倍数值的±SEM来表达水平在Neu从新鲜组织的N-阳性细胞。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54358/54358fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54358/54358fig4.jpg" /> 图4. 在苯丙胺注射。Fos蛋白阳性和Fos蛋白阴性的神经元之后，从新鲜或冷冻的大鼠背纹状体的FACS分选的神经元 的Fos mRNA水平如上述协议描述和Fos蛋白的mRNA水平证实了排序。数据以平均值±从新鲜组织相对表达水平折值的Fos阴性神经元（N = 3 - 4）SEM。

Watch this Scientific Journal Video about Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue at JoVE.com

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

在这里，我们提出了一个荧光活化细胞分选（FACS）协议来研究分子改建的Fos表达来自新鲜和冷冻的脑组织神经元歌舞团。使用冷冻组织可在多个会话许多脑区的FACS隔离，最大限度地利用宝贵的动物主题。

荧光活化细胞分选（FACS）和新鲜和冷冻大鼠脑组织的Fos表达神经元基因表达分析

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

fluorescence-activated-cell-sorting-facs-gene-expression-analysis-fos

Universidad San Sebastian

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Neuroscience

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

29.1K 次观看.  NIDA, NIH, DHHS. 在这里，我们提出了一个荧光活化细胞分选（FACS）协议来研究分子改建的Fos表达来自新鲜和冷冻的脑组织神经元歌舞团。使用冷冻组织可在多个会话许多脑区的FACS隔离，最大限度地利用宝贵的动物主题。 

视频: Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). 
The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine1 and TH is also present in a subset of adult pelvic ganglia neurons2-4 . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc1. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. 
To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine2-6. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (&gt;70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

An optimized procedure to purify neural crest-derived neuronal progenitors from fetal mouse tissues is described. This method takes advantage of expression from fluorescent reporter alleles to isolate discrete populations by fluorescence-activated cell sorting (FACS). The technique can be applied to isolate neuronal subpopulations throughout development or from adult tissues.

荧光激活细胞分选（FACS）自主神经祖细胞的分离，从胎鼠的内脏器官的优化程序

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

科研

神经科学

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

荧光钙成像及其后续原位杂交，用于异种性前体特征

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

发育生物学

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

生物学

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

活体影像随访单细胞追踪监测细胞生物学及多神经种群的谱系进展

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性<em&gt; KCNJ10</em&gt;（Kir4.1）

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the sub-granular zone (SGZ) of the dentate gyrus (DG) and their differentiation from newly born neurons into granule cells in the granule cell layer, is well validated across numerous studies. Using genetically modified animals, particularly rodents, is a valuable tool to investigate signaling pathways regulating AHN and to study the role of each cell type that compose the hippocampal neurogenic niche. To address the latter, methods combining single nuclei isolation with next generation sequencing have had a significant impact in the field of AHN to identify gene signatures for each cell population. Further refinement of these techniques is however needed to phenotypically profile rarer cell populations within the DG. Here, we present a method that utilizes Fluorescence Activated Nuclei Sorting (FANS) to exclude most neuronal populations from a single nuclei suspension isolated from freshly dissected DG, by selecting unstained nuclei for the NeuN antigen, in order to perform single nuclei RNA sequencing (snRNA-seq). This method is a potential steppingstone to further investigate intercellular regulation of the AHN and to uncover novel cellular markers and mechanisms across species.

Presented here is a method to sequence single nuclei isolated from the mouse dentate gyrus that excludes most neurons through fluorescence-activated nuclei (FAN)-sorting. This approach generates high-quality expression profiles and facilitates the study of most other cell types represented in the niche, including scarce populations such as neural stem cells.

荧光活化细胞分选（FACS）和新鲜和冷冻大鼠脑组织的Fos表达神经元基因表达分析

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荧光激活细胞分选（FACS）自主神经祖细胞的分离，从胎鼠的内脏器官的优化程序

荧光钙成像及其后续原位杂交，用于异种性前体特征

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

成像蛋白质 - 蛋白质相互作用在体内

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

活体影像随访单细胞追踪监测细胞生物学及多神经种群的谱系进展

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性

荧光激活神经元核负分选结合单核RNA测序研究海马神经源性生态位