We would like to acknowledge Dr. Michael Wagner (SUNY Downstate Medical Center) and Ben Thiede (Dr. Jeff Corwin lab, University of Virginia) for providing us with the F9 RARE-LacZ cells and technical support. This study is funded by the New Jersey Commission on Spinal Cord Research (NJCSCR) (Grantnumber:10-3092-SCR-E-0) and the New Jersey Commission on Brain Injury Research (NJCBIR) (Grant number: CBIR13FEL006).

所有的动物工作是按照批准罗格斯-RWJMS IACUC方法进行。 1.解决方案和媒体准备<ol><li>准备库存和缓冲溶液按表1。按表2准备工作的解决方案。 </li></ol> 2.文化和F9稀土的LacZ细胞的维持<ol><li> 解冻冷冻细胞 <ol><li>如表2中详述的制备F9稀土LacZ的细胞培养基。 </li><li>外套10厘米培养皿（处理过的细胞培养物）用10毫升的0.2％明胶（参照表2）进行30分钟，在室温。洗去多余的明胶用10毫升蒸馏水水两漂洗。立即加入9毫升F9稀土的LacZ细胞培养基到烧瓶中，以防止干燥的明胶。 </li><li> 3分钟-在37℃水浴中2解冻F9稀土的LacZ细胞14的小瓶。板的细胞（约1×10 6个细胞/ ml）到涂覆菜Containing9毫升培养基。在37℃下培养，5％ 的 CO 2。改变介质的第二天。 </li></ol></li><li> F9稀土的LacZ细胞的维持 <ol><li>以1:10的比为3天 - 保持有规律的通路，每2。 注意:不要让细胞长成汇合，因为这会导致其分化。 </li><li>分裂培养物在80-90％铺满。删除媒体并用10ml 1×磷酸盐缓冲盐水（PBS）洗涤细胞。移除PBS并加入胰蛋白酶1毫升，孵育5分钟。再加9毫升培养介质中，吸移管的上下和分裂细胞1:10进行传代。 </li></ol></li><li> 冷冻F9稀土LacZ的细胞 <ol><li> Trypsinize含有F9稀土的LacZ细胞在80 10 cm培养皿 - 90％汇合如在步骤2.2.2详述。转移离解的细胞入15ml离心管中并旋转，在200×g离心5分钟。 </li><li>在10毫升F9稀土的LacZ的F取出上清，悬浮细胞沉淀reezing培养基（ 见表2）。分装1毫升到标有冷冻管并转移到小瓶冷冻容器。将第二天-80℃冰箱O / N和转让小瓶液氮罐中冷冻集装箱。 </li></ol></li></ol> 3. E8.5胚胎解剖<ol><li> 笼交配设置和检查插头 <ol><li>设置含有一种交配对配合笼。第二天上午，检查阴道塞的存在和指定日的插头被发现为0.523天 。在E8.5天，收获的胚胎。 </li></ol></li><li> 胚胎解剖 <ol><li>牺牲通过颈脱位水坝和喷雾用70％乙醇腹部区域（ 见表 2）。使削减用手术剪在腹部上方的皮肤和横向拉开两片（前部和后部），露出腹部。作出类似的削减在腹膜以露出腹部。 </li><li>找到子宫和输卵管子宫释放，并使用剪刀23系膜及。放置在用10ml 6cm皿（处理过的非细胞培养物）的子宫1×PBS中洗掉多余的脂肪和血。转移到含有新鲜10毫升1X PBS另外6 cm培养皿。 </li><li>单独通过削减其关闭用手术剪刀包含一个胚胎中提取一个子宫。切除子宫及蜕膜和释放含有使用两对无胚胎卵黄囊。 5镊子23。 </li><li>从胚胎中分离卵黄囊和卵黄囊转移到1.5ml微量离心管中用于基因组DNA提取将用于基因分型。使用P20吸管用大口径尖，整个胚胎移植到含有10微升胰蛋白酶的1.5 ml离心管。放置在冰上的管中。 </li><li>重复步骤3.2.3 - 3.2.4，直到所有的胚胎都被解剖。 </li></ol></li><li> 胚胎离解<ol><li>孵育含有胰蛋白酶的胚胎1.5毫升管在37℃下5分钟，以促进酶解。 <ol><li>磨碎轻轻使用用于胚胎机械解离一个P20移液器，和板含有一个解离的胚胎在F9稀土的LacZ细胞的一个孔在96孔板在整个10微升体积（见步骤5.1电镀F9稀土的LacZ细胞在一个96孔板）。培养O / N在37℃和5％的CO 2。 </li></ol></li></ol></li></ol> 4.椎板切除术，成人的解剖腰脊髓神经球和文化<ol><li> 椎板切除术 <ol><li>通过颈椎脱臼牺牲一个成人（P60）鼠标。 </li><li>喷雾用70％乙醇的背侧，并用手术剪一个横向切割。拉活板（前部和后部）分开，露出背部和脊柱。 </li><li>用手术剪刀，剪掉覆盖脊椎的肌肉。定位脊髓的腰部区域，肋的下方。使用剪刀，做一个深深的伤口，以切断脊椎在这一点上暴露脊髓。 </li><li>拉起脊柱的腰部区没有。 5钳子使得脊髓的露出横截面朝向实验者。用细剪刀提示，剪断在露出端的3点钟和9点钟位置的脊椎和肌肉。抓住用钳子切断脊椎的背部皮瓣。直到脊髓腰段的长度露出尾部继续这些削减。 </li><li>不使用从椎骨释放脊髓。 5以及钝端钳。脊髓转移到装有3毫升的DMEM / F12培养基的15毫升管。保持冰，直到所有的腰椎脊髓已被隔离。 </li></ol></li><li> 脊髓解剖 <ol><li>倾与脊髓培养基成6cm皿（无细胞培养物处理的）。 </li><li>下一个立体显微镜，通过抓住使用两对无神经节和血管移除脊髓周围段背根神经节和血管。 5镊子轻轻地从脊髓拉着他们离开。转移脊髓段到一个直径6cm培养皿没有介质和细使用手术刀刀片剁碎的组织。 </li><li>转移组织糜到含有1ml的神经球的解离介质15毫升管（ 见表2）。把管冰，并重复步骤4.2.1 - 4.2.2，直到所有脊髓已被处理。 </li><li>孵育含离解培养基组织糜管在37℃下进行10分钟，轻弹的一侧的管进行混合，然后在37℃孵育另外10分钟。经过酶消化，用磨碎的直径减小机械解离火抛光移液器。 注:准备递减之前进行实验直径火抛光的吸管。以一个玻璃巴斯德吸管和插头广角端用干净的棉花。使用酒精灯，火抛光在火焰中移液器的提示，以创建不同直径:"常规"，"精"和"超等"。对于"常规"抛光移液器，捻在火焰向圆边缘的移液管的端部不降低直径（约1毫米）。旋转中的火焰要略微较长的吸移管的端部以创建"精"（〜&gt;在1mm和&gt;直径5mm）和"超细"（〜0.5mm）的抛光移液器。不要用直径小于0.5mm使用吸管。 </li></ol></li><li> 神经干文化 <ol><li>含有降速管解离脊髓组织在200×g离心10分钟。在1ml神经球培养基除去上清，重悬细胞沉淀（ 见表2）。用P1,000吸管后跟P200移液管，以促进分解磨碎。 注意:避免产生气泡，这会降低细胞活力。 </li><li>转移10微升解离的细胞的成血球和获得细胞计数。板的细胞在用10毫升神经球培养基的T25烧瓶中的1×10 5/10 ml的密度。在37℃和5％CO 2的7天，直到神经球出现24。 </li></ol></li><li> 神经球离解 <ol><li>收集神经球文化和转移到15 mL管。旋转，在200×g离心10分钟。去除大部分上清留下1毫升后面。用P1,000吸管磨碎到重悬细胞沉淀并分离神经球成单个细胞。转移10微升解离的细胞的成血球并获得细胞计数。 </li><li>板1×10 5解离的神经球细胞过度F9 RARE-LacZ的的一个孔在96孔板（见5.1中96孔板电镀F9 RARE-LacZ的）。板3口井的重复。培养O / N在37℃和5％的CO 2。</li></ol></li></ol> 5. E8.5胚胎脊髓神经球的RA含量<ol><li> 电镀F9稀土LacZ的在96孔板 <ol><li>前一天收获E8.5胚或神经球的解离，涂布96孔板用100μl每孔的0.2％的明胶在室温30分钟。吸出明胶，并用200微升蒸馏水淋洗两次。 </li><li> Trypsinize F9稀土LacZ的细胞保持10 cm的培养皿如步骤2.2.2详细说明。板1×10 5细胞/孔的F9-RARE LacZ的细胞，但使用培养基不含G418这个时候。 </li><li>准备足够的井胚胎共培养（〜12 - 20，取决于产仔），神经球（3孔/基因型），以及用于在一式三份的标准曲线（27孔）。 </li></ol></li><li> F9稀土的LacZ和E8.5胚胎或脊髓神经球共培养 <ol><li>详见3.2节和板块上的F9顶部收获胚胎稀土的LacZ从5.1节的96孔板。以类似的方式，解离于在步骤4.4作为详细的96孔板F9 RARE-LacZ的顶部神经球和盘。培养O / N在37℃和5％的CO 2。 </li></ol></li><li> 生成标准曲线 <ol><li> 100纳米，10纳米，1纳米，100点，10点，下午1点，100 FM，由AT-的连续稀释:要生成一个标准曲线，具有以下浓度的准备全反式RA（AT-RA）解决方案RA股票（ 见表1）F9稀土LacZ的培养基。 </li><li> AT-RA浓度从5.1节加入100μl这些不同的到F9稀土LacZ的细胞。指定一个包含未处理F9稀土LacZ的细胞作为阴性对照井。制备各RA浓度和阴性对照孔的一式三份。培养O / N在37℃和5％的CO 2。 注意:F9 RARE-LacZ的细胞以剂量依赖的线性方式不同浓度的全 - 反式RA的响应。 </li></ol></li><li> RA含量 <ol><li>含有标准曲线和共培养的96孔板的O / N培养后，取出介质，并通过加入100微升2.5％的戊二醛固定的培养物（ 见表2）15分钟，室温。取出固定液，并用200微升1×PBS，每洗10分钟，洗两次澡。 </li><li>除去PBS中，并用200微升的LacZ洗涤溶液洗涤三次（ 见表2），每次洗涤10分钟。在第三次洗涤，准备在一个15毫升管包裹在箔的X-gal染色溶液（ 见表2）。计算多少染色液需要并准备适当的量（200微升/孔）。 注意:X-gal的染色液是光敏感。制备后用15分钟内的染色溶液。 </li><li>通过将用蒸馏水蘸纸巾移入塑料容器大到足以容纳一个96孔板制备的潮湿室中。加入200μl在X的每96孔板的半乳糖苷酶染色溶液并将板在湿润的腔室中。 </li><li>孵育加湿室含有96孔板在37℃，以允许的蓝色的沉淀物的发展。对于E8.5胚胎，孵育板O / N（12 - 16小时）。对于神经球文化，孵育4板 - 8小时。 </li></ol></li><li> 在OD 610和成像读取吸光度 <ol><li>显色反应后，取出的LacZ染色溶液和用200μl1×PBS中进行更换。使用标准ELISA板读数器来量化记者响应于RA的测量在610纳米的吸光度。图像单个孔使用装备有相机的标准立体显微镜。 </li></ol></li></ol> 6.亚克隆F9稀土的LacZ细胞<ol><li> 检查F9稀土的LacZ rResponse到RA <ol><li>在开始任何实验之前，先测试F9稀土LacZ的细胞对RA的响应。板F9稀土的LacZÇ在96孔板厄尔（参见5.1节），并添加1纳米全反式维甲酸（见步骤5.3）。在37℃CO / N孵育在湿润的腔室中。 </li><li>可视化使用显微镜蓝色沉淀的发展。如果颜色发展不贯穿孔中的细胞均匀（参照图1B，左），执行下面亚克隆详述。 </li></ol></li><li> F9稀土的LacZ细胞的亚克隆 <ol><li>斯普利特10 cm的培养皿中培养的F9稀土LacZ的细胞作为步骤2.2.2详细说明。代替1:10的比例的，板1×10 5个细胞/ 10毫升的低接种密度在10cm皿以允许分离的菌落的生长，使得每个集落产生从单个F9稀土的LacZ细胞。 </li><li>两天后，涂布24孔板用500μl每孔的0.2％明胶的30分钟。除去明胶溶液，并用500微升蒸馏水冲洗孔两次。用500μlF9稀土LacZ的细胞培养基的更换水。 </li><li>挑选使用无菌P10的枪头和转让到一个很好的殖民地;做24次，得到24个独立的殖民地。培养在37℃，3，5％CO 2 - 4天。 </li><li>拆分单个菌落成两个24孔板。后在培养2天后，添加的RA为1nm至之一24孔板和培养O / N各孔中。按照第5.4节详述，以测试高反应进行的LacZ染色。每个可视化的显色反应以及在显微镜下才能确定强和均匀的响应。 </li><li>一旦一个强亚克隆响应被确定，从另一24孔板展开相应的培养。进一步扩大在10cm皿的殖民地，冻结下来几个小瓶在2.3节详述，并放弃所有其他殖民地。 注意:只有关于原始菌落的四分之一导致高​​反应者。典型地，第一轮亚克隆不导致均匀的LacZ染色，并需要随后的轮亚克隆需做达到均匀强烈反应。 </li></ol></li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+signaling+in+the+brain+marks+formation+of+optic+projections,+maturation+of+the+dorsal+telencephalon,+and+function+of+limbic+sites.">Retinoic acid signaling in the brain marks formation of optic projections, maturation of the dorsal telencephalon, and function of limbic sites.</a> J Comp Neurol. 470 (3), 297-316 (2004).</li><li>Kelley, M. W., Xu, X. M., Wagner, M. A., Warchol, M. E., Corwin, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developing+organ+of+Corti+contains+retinoic+acid+and+forms+supernumerary+hair+cells+in+response+to+exogenous+retinoic+acid+in+culture.">The developing organ of Corti contains retinoic acid and forms supernumerary hair cells in response to exogenous retinoic acid in culture.</a> Development. 119, 1041-1053 (1993).</li><li>McCaffery, P., Drager, U. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hot+spots+of+retinoic+acid+synthesis+in+the+developing+spinal+cord.">Hot spots of retinoic acid synthesis in the developing spinal cord.</a> Proc Natl Acad Sci. 91, 7194-7197 (1994).</li><li>Hu, L., Gudas, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+analogs+and+retinoic+acid+influence+the+expression+of+retinoic+acid+receptor+alpha,+beta,+and+gamma+mRNAs+in+F9+teratocarcinoma+cells.">Cyclic AMP analogs and retinoic acid influence the expression of retinoic acid receptor alpha, beta, and gamma mRNAs in F9 teratocarcinoma cells.</a> Mol Cell Biol. 10 (1), 391-396 (1990).</li><li>Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratocarcinomas+and+mammalian+embryogenesis.">Teratocarcinomas and mammalian embryogenesis.</a> Science. 209 (4458), 768-776 (1980).</li><li>Strickland, S., Mahdavi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+induction+of+differentiation+in+teratocarcinoma+stem+cells+by+retinoic+acid.">The induction of differentiation in teratocarcinoma stem cells by retinoic acid.</a> Cell. 15 (2), 393-403 (1978).</li><li>Li, B. I., 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=The+orphan+GPCR,+Gpr161,+regulates+the+retinoic+acid+and+canonical+Wnt+pathways+during+neurulation.">The orphan GPCR, Gpr161, regulates the retinoic acid and canonical Wnt pathways during neurulation.</a> Dev Biol. 402 (1), 17-31 (2015).</li><li>Hogan, B., Constantini, F., Lacy, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , (1986).</li><li>Deleyrolle, L. P., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+expansion,+and+differentiation+of+adult+Mammalian+neural+stem+and+progenitor+cells+using+the+neurosphere+assay.">Isolation, expansion, and differentiation of adult Mammalian neural stem and progenitor cells using the neurosphere assay.</a> Methods Mol Biol. 549, 91-101 (2009).</li></ol>

The authors have nothing to disclose.

所述F9稀土LacZ的细胞系14是一个功能强大的系统，该系统可用于通过组织外植12,14,17,18产生的检测的RA。它是对RA的浓度低至100 FM敏感，与在未处理的RA 12,14,18细胞没有检测到的非特异性反应。此外，报道细胞向全反式RA响应以线性方式浓度的特定范围内，从而使LacZ的反应进行量化，并用于确定RA水平12,14,18的幅度。这里介绍的快速和容易的RA记者测定法利用对胚胎和培养的成体神经干细胞的可视化（ 图 2）和相对定量测量（ 图3）的F9 RARE-LacZ报道细胞系。记者系统允许单个样品之间的内源性的RA的水平，如在图2中所示的比较，它具有足够的灵敏度，以便能够检测的RA由培养的神经球（ 图2B）产生个别E8.5胚胎（ 图2A）以及RA产生。该细胞系的不同的RA浓度的剂量依赖性响应允许对RA的水平（ 图3A）在样品中的样本（ 图3B）之间的量化以及相对的RA水平的比较。值得注意的是，F9 RARE报道细胞系测定的RA由组织样品在培养时产生的总量是很重要的。这相当于RA合成和分解代谢的净平衡。 虽然F9稀土LacZ的细胞报告系统是高度敏感的，多功能的，有我们为了保证这个系统的可靠性已经把几个关键的故障排除步骤。首先， 如图1中 ，以维持这些细胞通过定期通道的健康没有让他们g是重要排至汇合。第二，我们观察到后面的通道趋向于导致不一致RA响应，即使该培养物都在不断下选择用G418保持。要解决此，建议经过20代放弃文化和解冻新瓶。此外，必须进行定期检查，以确保记者细胞依然强劲反应到RA在媒体的存在。第三，如果早期传代显示到RA（ 图1B，左侧 ），一个或多个轮亚克隆的差或不均匀反应，直到获得（ 图1B，右击 ）强烈反应可能是必要的。最后，共培养之前，建议组织样品如胚胎和神经球解离为单细胞。我们已观察到的样品的结果中更一致的色度测量的该离解。这可能是由于一个在培养孔更均匀分布的RA，作为离解的细胞是在单与记者的细胞形式的接触。 若干修改也向原始协议14进行。第一，F9 RARE-LacZ的细胞的RA响应被直接固定细胞，并在610nm处测定吸光度，而不是收集单元并分析β-半乳糖苷酶活性14裂解物进行定量。另一种修饰是与F9稀土LacZ的离解样品的直接共培养之前RA响应的定量。以前的方法分别汇报培养样品，并从这些文化只增加媒体F9稀土的LacZ 16,18。 尽管它是一个强大的系统中，F9稀土LacZ报告分析有几个限制。这个报道细胞系中的一个限制是，虽然它强烈响应全 - 反式RA的存在，它也响应于其他维甲酸的异构体，包括9-顺​​式RA和13-顺式-RA;然而，它们是100倍更敏感的全反式RA COMPA红色的其它异构体18。另一个限制该系统是线性的剂量依赖性反应仅在10 -13中号下降到10 -7 M在-RA 14,18;因此，如果这些范围之外的比较RA的水平，这可能是必要执行样品的系列稀释液，直到测量值落在线性范围内。最后，由于这是主要的比色测定法，发色的端点将样品和实验之间变化。因此，一个标准曲线始终平行，以便镀为每一个实验中使用它的报道细胞应答的定量是重要的。 总之，我们已经报道了使用F9稀土的LacZ RA记者细胞系的定量测量RA水平E8.5胚胎和，以前未在神经球。此报告系统的多功能性可允许在几乎任何细胞或组织类型的RA水平的定量，以及mAY导致在未来变化的应用的开发。

视黄酸（RA）是视黄醇或维生素A的代谢物衍生，并通过几个蜂窝脱氢酶1的连续活动产生的。因为它的两亲化学性质的，它很容易地跨越脂质膜，并且可以作为可溶性形态形成因子1行动。这是通过作为核视黄酸受体配体和激活的基因表达2-10调控众多发育和成年细胞过程的基本分子。如果没有RA，这些RA受体（维甲酸受体）的DNA，并作为阻遏绑定拉雷什; RA结合这些受体把他们变成导致靶基因的表达1,5,6转录激活。 虽然RA信号通路是很好的特点，小尺寸（〜300 DA）和RA的两亲性使得直接组织可视化和RA的测量目前在技术上是不可行的11。对于目录的唯一方法RA水平等的测量是通过使用高效液相色谱（HPLC）11组织样品的RA隔离。这种方法通常需要大量的组织，通过集中不同的样品12便利。因此，该技术是适合于在单个胚胎或少量样品/组织的RA水平的测定不良。 为了研究RA的细胞功能，几种方法已被开发来间接推断的RA的存在。这些方法利用含有高度响应RAR-βRARE驱动的β-半乳糖苷酶或萤光素酶11,13,14的表达记者系统。通过Rossant 等人 13产生的转基因的稀土LacZ报告鼠标是用于原位 11,13,15,16可视RA信号的时空区域的理想的系统中，但不适合于定量测量。该F9稀土LacZ的细胞系14，在T他另一方面，为在组织外植体11,12,14,17,18检测的RA和RA水平的定量测量是有用的。 F9畸胎瘤细胞表达内源性α-，β-和γ-维甲酸受体19，和暴露于RA 19-21之后发起分化成顶叶内胚层。 F9细胞已长期被确立为RA诱导的细胞分化的模型，这就是为什么它被选定为一随机存取报告测定的理想细胞系。该F9衍生SIL-RA 15报告细胞瓦格纳等人产生14行包含一个E. coli&#39;LacZ基因的64 bp的维甲酸反应元件人β-维甲酸受体（RAR-β）基因（RARE）的一个副本下游。为了选择并维持稳定的克隆，该构建体也包含氨基糖苷磷酸转移酶（ 新霉素抗性 ）基因作为在G418存在的选择标记。这种结构合作nfers在RA的存在，这可以通过的LacZ染色进行可视化β-半乳糖苷的诱导表达和这个响应可以是随后使用比色法11,12,14,18定量。 这个非常通用报道细胞系已被广泛地穿过组织样本的共培养与记者细胞，如耳蜗17和胚胎底板植14用于检测内源性的RA生产。此外，记者线已通过培养分别胚胎脊髓的汇集部分和从这些培养至F9稀土的LacZ细胞18加入条件培养基用于在显影脊髓RA水平的定量。定量分析的LacZ染色后通过比色读数使用标准ELISA板读数器11,12,18进行。最后，此报道细胞系已经在检测由monitori存在RA代谢酶的使用NG改变RA水平12,18。 此处我们报告，此报道细胞系的敏感性也允许从单独共培养E8.5胚胎产生的RA水平的测量。这使得不同基因型的胚胎个体之间的比较。作为一个具体的例子，Gpr161是孤儿GPCR穿过RA信号通路22调节神经胚部分，和我们报告使用此报道细胞系来调查隐性突变在Gpr161（Gpr161 VL）上的整体的RA水平的影响胚胎。除了这一点，通用性和易于使用该报告系统的允许测量和不同组织样品中，比较，即使在从成年脊髓干细胞获得的神经球培养RA水平的自由。在这里，我们描述了通过直接共培养胚胎和神经球文化相对RA水平的定量测定与F9稀土的LacZ RA代表orter细胞系。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C3H/HeSn-Gpr161vl/J mouse strain</td>
			<td>Jackson Laboratory</td>
			<td>000316</td>
			<td></td>
		</tr>
		<tr>
			<td>F9 RARE-LacZ reporter cell line</td>
			<td></td>
			<td></td>
			<td>from Dr. Michael Wagner (SUNY Downstate Medical Center) and Ben Thiede (Dr. Jeff Corwin lab, University of Virginia)</td>
		</tr>
		<tr>
			<td>DMEM/F-12 (with L-glutamine and HEPES)</td>
			<td>ThermoFisher Scientific</td>
			<td>11330-057</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), qualified</td>
			<td>ThermoFisher Scientific</td>
			<td>26140-079</td>
			<td>Store at -20 &#176;C in 10-ml aliquots to avoid repeated freeze-thaw; warm in 37 C for media preparation</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140-122</td>
			<td>Store at -20 &#176;C in 10-ml aliquots to avoid repeated freeze-thaw; light-sensitive</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>ThermoFisher Scientific</td>
			<td>10131-027</td>
			<td>Store at 4 &#176;C; light-sensitive</td>
		</tr>
		<tr>
			<td>2% Gelatin</td>
			<td>Sigma Aldrich</td>
			<td>G1393</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>B-27 supplement (2 % stock solution)</td>
			<td>ThermoFisher Scientific</td>
			<td>17504-044</td>
			<td>Store at -20 &#176;C in 1-ml aliquots to avoid repeated freeze-thaw; warm at room temperature for media preparation</td>
		</tr>
		<tr>
			<td>Murine Epidermal Growth Factor (EGF)</td>
			<td>PeproTech</td>
			<td>315-09</td>
			<td>Prepare 100 &#956;g/ml stock solution in 0.1% bovine serum albumin (BSA) in 1X PBS; prepare working concentrations by diluting with 1X PBS; store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Murine Fibroblast Growth Factor (FGF)-basic</td>
			<td>PeproTech</td>
			<td>450-33</td>
			<td>Prepare 100 &#956;g/ml stock solution in 0.1% bovine serum albumin (BSA) in 1X PBS; prepare working concentrations by diluting with 1X PBS; store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>50% glutaraldehyde</td>
			<td>Amresco</td>
			<td>M155</td>
			<td>Store at 4 C</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate monohydrate (C24H39NaO4 &#183; H2O)</td>
			<td>Sigma Aldrich</td>
			<td>D5670</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>potassium ferrocyanide (K&#8324;[Fe(CN)&#8326;] &#183; 3H&#8322;O)</td>
			<td>Sigma Aldrich</td>
			<td>P-9387</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>potassium ferricyanide (K&#8323;[Fe(CN)&#8326;])</td>
			<td>Sigma Aldrich</td>
			<td>P-8131</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>X-gal (5-bromo-4-chloro-3-indolyl-&#946;-d-galactopyranoside)</td>
			<td>Promega</td>
			<td>V3941</td>
			<td>Store stock at -20 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>41639</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>all-trans Retinoic Acid (at-RA)</td>
			<td>Sigma Aldrich</td>
			<td>R-2625</td>
			<td>Store 1 ml aliquots of stock in -80 &#176;C; extremely sensitive to light</td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>ThermoFisher Scientific</td>
			<td>12605-010</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>25200-056</td>
			<td>alternative to TrypLE Express; aliquot and store at -20 C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>ThermoFisher Scientific</td>
			<td>14170-112</td>
			<td>Store at 4 C</td>
		</tr>
		<tr>
			<td>10X phosphate buffered solution (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>10010-023</td>
			<td>Store at 4 C</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LK003176</td>
			<td>Store at 4 &#176;C.&#160;</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Worthington</td>
			<td>LS003703</td>
			<td>Store in 1 ml aliquots at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Hyaluronic acid&#160;</td>
			<td>Calbiochem</td>
			<td>385908</td>
			<td>Store 200 &#956;l aliquots at -20 &#176;C.&#160;</td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>Worthington</td>
			<td>LS002139</td>
			<td>Store in 200 &#956;l aliquots at -20 C</td>
		</tr>
		<tr>
			<td>Kynurenic acid</td>
			<td>Sigma Aldrich</td>
			<td>K3375</td>
			<td>Store in 200 &#956;l aliquots at -20 C</td>
		</tr>
		<tr>
			<td>95% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing container</td>
			<td>ThermoFisher Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish (non-cell culture treated)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish (cell culture treated)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm culture dish (non-cell culture treated)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well cell culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml amber microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml cryovials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>surgical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fine surgical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>no. 5 forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>blunt-ended forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel blade</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass pasteur pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>alcohol lamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plate reader</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative measurement of relative retinoic acid levels in e8.5 embryos and neurosphere cultures using the f9 rare-lacz cell-based reporter assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

所述F9稀土LacZ报道是生长在明胶包被的表面的粘附胚胎癌细胞系。的报道构建允许细胞与β-半乳糖苷酶。11,12,14,18的比例感应定量响应的RA。这些细胞显示上皮形态（ 图1A）。因为它们的高倍增率的，则建议这些细胞每2分割 - 以1:10的比为3天。在我们的与此报道细胞系的工作，我们已经观察到，即使在加入G418到培养基中，这些细胞对RA的响应减弱经过多次传代（ 图1B， 左 ），而这可能会影响的记者容量该细胞系。响应这种损失可能是由于在后面的段落转基因的部分损失。这样，周期性亚克隆有必要确保强有力和均匀的响应者RA（ 图1B，右）。 虽然该细胞系的各种用途，已经出版12,14-18，报道这里是使用该细胞系来衡量个别E8.5胚胎不同基因型的内源性RA水平。在Gpr161 VL / VL突变导致胚胎降低RA信号22。如由解离胚胎与F9稀土的LacZ细胞共培养中，这些Gpr161 VL / VL胚胎已相对于野生型同窝（ 图2A）降低内源性的RA。因为此报告细胞系，在这里也报道的通用性的是一种新型的用这些细胞以检测由从成人Gpr161 重量/重量的小鼠（ 图2B）中得到的神经球培养物产生的RA水平。记者细胞系能够证明从成年脊髓干细胞神经球文化产生内源性RA。伟大的差异erence在染色强度指示相对于E8.5胚胎脊髓神经球更大记者细胞反应。这可能是由于由神经球随时间产生的大得多的RA水平相比E8.5胚胎，这可能是由于这样的事实，神经球文化比E8.5胚胎更均匀，因此含有较多的RA生产细胞。 此外在报道细胞的剂量依赖性线性响应不同浓度在-RA的结果的。这个反应可以用比色法通过在610nm处读取吸光度来测量。然后所产生的标准曲线可用于定量样品中的RA的水平，如在野生型神经球培养物（ 图3A）或E8.5胚胎（ 图3B）。 <img alt="图1" src="/files/ftp_upload/54443/54443fig1.jpg" /> 图1. MAINTenance和F9稀土的LacZ细胞的亚克隆。 （A）F9稀土的LacZ细胞的相衬图像显示。这些细胞具有〜10小时的倍增时间，而且必须每3天通过。 -以1:10的比例（约70 80％汇合）（B）和加入1纳米的在-RA和随后的LacZ文化的周期检测染色必须完成，以确保文化保持强劲而均匀的响应者RA（右）。在贫穷和非均匀反应（左）的情况下，必须进行亚克隆。比例尺为100μm。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54443/54443fig1large.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/54443/54443fig2.jpg" /> 图2 中的LacZ染色共培养样品（游离E8.5胚胎/神经球）和F9稀土LacZ的细胞。（A）E8.5小鼠胚胎<html>不同基因型第解离并接种在F9稀土的LacZ细胞的顶部。 LacZ的染色24小时后可通过共培养样品产生的F9稀土LacZ的细胞内源性RA响应的可视化。所述Gpr161 VL突变导致降低内源性的RA作为可视化的报道细胞的降低的反应。（B）的左。从成人脊髓的神经干细胞培养的神经球的相衬图像被示出。对。从成年Gpr161 重量/重量脊髓衍生神经球解离并接种在F9稀土的LacZ细胞的顶部。蓝色沉淀之后的LacZ染色的存在表明内源性RA在这些神经球的存在。在神经球和胚胎E8.5共培养之间染色强度的差异表明神经球比E8.5胚胎目前RA的更高水平。比例尺为100μm。目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54443/54443fig3.jpg" /> 图3. 标准曲线和RA楼层比色定量。在F9稀土的LacZ报道细胞系的响应可以比色在610nm处使用标准ELISA板读数器来测量。（A）中使用的量化的相对的RA水平代表标准曲线野生型神经球文化秒。从共培养E8.5胚胎相对RA水平（B）的定量。 N = 3; * P &lt;0.05，学生t检验。误差棒SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54443/54443fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 申通CK缓冲器/解决方案</td><td> 组件 </td><td> 储存条件 </td></tr><tr><td> 10％的脱氧胆酸钠水合物（C 24 H 39的NaO 4·H 2 O） </td><td> 10毫升100毫升卫生署2 O </td><td> 4ºC </td></tr><tr><td>的1M氯化镁（MgCl 2的） </td><td></td><td> RT </td></tr><tr><td> 100X亚铁氰化钾（K 4 [铁（CN）6]·3H 6 O） </td><td> 2.12克在10毫升卫生署2 O </td><td>室温在黑暗中</td></tr><tr><td> 100×铁氰化钾（K 3的[Fe（CN）6]） </td><td> 1.64克在10毫升卫生署2 O </td><td>室温在黑暗中</td></tr><tr><td> 20毫克/毫升X-gal的（5-溴-4-氯-3-吲哚基β-D-吡喃半乳糖苷） </td><td>加入5毫升二甲基甲酰胺，使20毫克/毫升储液</td><td> -20ºC </td></tr><tr><td> 10毫米（3毫克/毫升）的所有-TRANS维甲酸</td><td> 16.67毫升无水乙醇溶解50毫克。制备1ml等份。 </td><td> -80ºC在黑暗中，在1.5ml琥珀色微量离心管</td></tr><tr><td>木瓜蛋白酶</td><td>溶解一小瓶7毫升HBSS </td><td> 4ºC </td></tr></tbody></table> 表1.缓冲器和解决方案组成。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 工作缓冲器/解决方案 </td><td> 组件 </td><td> 储存条件 </td></tr><tr><td> F9稀土LacZ的细胞培养基</td><td> 50毫升胎牛血清（FBS）（终浓度10％） </td><td>过滤消毒。 </td></tr><tr><td></td><td> 5毫升100X青霉素链霉素（终浓度1×） </td><td>保存在4ºC。 </td></tr><tr><td></td><td> 4毫升50毫克/毫升G418（终浓度0.4毫克/毫升） </td><td></td></tr><tr><td></td><td>至500ml DMEM / F-12（有L-谷氨酰胺和HEPES） </td><td></td></tr><tr><td> 0.2％的明胶</td><td> 25毫升2.0％明胶</td><td>保存在4ºC。 </td></tr><tr><td></td><td> 80毫升蒸馏水</td><td></td></tr><tr><td></td><td>拌匀</td><td></td></tr><tr><td> 70％的乙醇</td><td> 30毫升95％乙醇</td><td>在喷雾瓶中放置。 </td></tr><tr><td></td><td> 70毫升蒸馏水</td><td>存储在室温</td></tr><tr><td>神经干分离介质</td><td> 1毫升20U木瓜蛋白酶</td><td>准备新鲜的每一次</td></tr><tr><td></td><td> 100微升13毫克/毫升胰蛋白酶</td><td></td></tr><tr><td></td><td> 100微升7毫克/毫升透明质酸</td><td></td></tr><tr><td></td><td> 28微升10毫克/毫升DNA酶I </td><td></td></tr><tr><td></td><td>50微升4毫克/毫升犬尿酸</td><td></td></tr><tr><td>神经球培养基</td><td> DMEM / F-12（有L-谷氨酰胺和HEPES） </td><td>准备新鲜的每一次</td></tr><tr><td></td><td> 2％B-27的补充</td><td></td></tr><tr><td></td><td> 20纳克/ ml成纤维细胞生长因子（FGF） </td><td></td></tr><tr><td></td><td> 20ng的表皮生长因子（EGF） </td><td></td></tr><tr><td> 2.5％戊二醛</td><td> 250微升50％的戊二醛</td><td>准备新鲜的每一次</td></tr><tr><td></td><td> 4750微升1X PBS </td><td></td></tr><tr><td> LacZ的洗涤液</td><td> 0.5毫升10％脱氧胆酸盐（终浓度0.1％） </td><td>保存在4ºC。 </td></tr><tr><td></td><td> 1.0毫升100％NP-40（终浓度0.2％） </td><td></td></tr><tr><td></td><td> 50毫升10X PBS </td><td></td></tr><tr><td></td><td>蒸馏水至500ml </td><td></td></tr><tr><td>的X-gal染色溶液</td><td> 1个亚铁氰化钾</td><td>准备新鲜的每一次</td></tr><tr><td></td><td> 1X铁氰化钾</td><td></td></tr><tr><td></td><td>为1mg / ml的X-gal </td><td></td></tr><tr><td></td><td>以适当的音量使用LacZ的洗涤液</td><td></td></tr><tr><td> F9稀土LacZ的冷冻介质</td><td> F9稀土LacZ的培养基+ 5％DMSO </td><td>准备新鲜的每一次</td></tr></tbody></table> 表2.工作缓冲器和解决方案组成。

Watch this Scientific Journal Video about Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay at JoVE.co

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

使用F9稀土LACZ基于Cell的报告基因检测在E8.5胚胎和神经球文化相对维甲酸水平的定量测量

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

quantitative-measurement-relative-retinoic-acid-levels-e85-embryos

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.8K 次观看.  Rutgers University. Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line. 

视频: Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

科研

神经科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

人类胶质母细胞瘤肿瘤细胞的分离和扩大使用的神经球含量

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

评估主要神经再生<em&gt;爪</em&gt;胚胎使用免疫染色

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

使用电动和一维和二维文化磁场神经元的外部激励

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor &#947;-secretase-mediated cleavage of either APP-C99 or Notch, using a Gal4 promoter-driven firefly luciferase reporter system. These assays were established by stably co-transfecting HEK293 cells with the Gal4-driven luciferase reporter gene and either the Gal4/VP16-tagged C-terminal fragment of APP (APP-C99; CG cells), or the Gal4/VP16-tagged Notch-&#916;E (N&#916;E; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress &#947;-secretase cleavage of APP-C99 in CG cells, but not N&#916;E in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for &#947;-secretase modulators, and these responses are in stark contrast to the pan-inhibition of &#947;-secretase induced by DAPT. Our studies provide direct evidence that &#947;-secretase activities toward different substrates can be differentiated in a cellular context. These new assays may therefore be useful tools in drug discovery for improved AD therapies.

We have successfully generated two substrate-specific &#947;-secretase assays. Both cell-based assays presented here are designed to quantify &#947;-secretase enzymatic activities via the output of firefly luciferase reporters.

基于细胞荧光检测平台的γ-分泌介导的淀粉样前体蛋白和缺口裂解的定量测定

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

微胶质细胞抗体介导的陶氏清除研究

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

小鼠体内合成蛋白的定量测量

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...