这项工作是支持DK065872（ENP），一个卓越的史密斯家庭基金会在生物医学研究奖（ENP），F31 DA023760（BMG，ENP）和P30 NS047243（塔夫茨大学神经科学研究中心）。

制备前文化 注 ：**神经胶质细胞，必须提前做好，让他们有时间，增殖和覆盖的菜肴底部镀。对于非典型的或实验的遗传背景的小鼠，确保胶质细胞和神经元的文化相匹配的基因型。 注 ：** 1-7天前清扫，准备好新鲜的神经介质，并取代在用2ml的菜的胶质介质。 注 ：**在清扫前的一天，以下项目需要紫外光下过夜左： <ul><li> 4夹层板</li><li> 2解离样品瓶与一个微型磁力搅拌棒</li><li> 2小瓶的瓶盖（2小孔，顶部探出） </li><li>摊开幻灯片环（和70％乙醇外套） - 离开盒也开放</li><li>至少2黄（10 -200μL）枪头盒</li><li> 1盒蓝色（100 -1000μl）移液器</li></ul>文化日 注 ：*千万不要碰任何东西离开紫外线在外过夜！ 引擎盖下保持无菌的环境是非常重要的的。 1）设置： 注 ：**抛开所有冻结的成分，使木瓜蛋白酶溶液。 <ol><li>用70％乙醇的清洁钳。盖，用无菌的黄色吸管尖每个提示。使用放置在无菌幻灯片戒指的盒子。 </li><li>热点板块： <ol><li>将下一个千毫升烧杯装500 -600毫升DH 2 O和一个大的磁力搅拌棒的引擎盖mini-magnetic/hot板。 </li><li>水位以上的烧杯放置在一个泡沫塑料盘。 </li><li>将在泡沫塑料盘的小孔中的温度计。热拨号需求将略高于2（温度为34℃）和搅拌转盘应〜5-6。 </li></ol></li><li> PBS： <ol><li>准备无菌PBS，填补4-6毫升15无菌15毫升管（一定要保持无菌 技术）。 </li><li>管和股票瓶放在旁边的引擎盖上冰。 </li></ol></li><li> Carbogen： <ol><li>打破了一半5毫升stripette，推动通过一个橡胶塞，所以stripette的一角伸出广角端的塞子。 </li><li> 400 - 500ML的DH 2 O（stripette断头应在DH 2 O），放置在一个500ml烧瓶的瓶塞。 </li><li>将烧瓶的侧开管。 </li><li>切过一个黄色的枪头的提示和连接管端（切端朝外）。 连接carbogen坦克5毫升stripette的一角。 </li></ol></li><li>准备木瓜sol&#39;n： <ol><li> *使用注意无菌技术。 </li><li>混合在无菌的50毫升管。 </li></ol></li><li>过滤木瓜sol&#39;n到解离小瓶： <ol><li> 25毫升stripette（或地方新上一个20毫升柱塞注射器的尖端0.2微米的注射器过滤器通过使用一个Steriflip过滤单元和转移解离小瓶， 取出柱塞和使用stripette（25毫升）的注射器转移到所有的木瓜蛋白酶sol&#39;n。 </li><li>解离瓶过滤器）。 </li><li>第小瓶，在第一个通过孔插入无菌注射器和附加0.2μm的无菌注射器过滤器，注射器的基础。 </li><li>连接的carbogen过滤器和封口膜这个地方。放置在发泡胶盘/持有人的小瓶。 *必须移动的微型磁力搅拌棒。 *气体必须到小瓶。 *温度必须稳定在34 ° C。 </li></ol></li><li>夹层的准备： <ol><li>将发动机罩下的解剖镜下。 </li><li>莱铝箔所有清扫工具，喷以70％乙醇，倒出多余的和干引擎盖下离开。 </li></ol></li><li>准备神经介质： <ol><li>将新鲜的神经介质的孵化器30毫升。 </li></ol></li><li>莱1铝箔的大型广场和12个小方格。保留铝箔斩首剪刀。用冰水填写一个小的发泡胶盒，并保留所有罩外。 </li><li>准备麻醉（氯胺酮和0.075毫升0.075毫升xzylazine）。 </li><li> 100板，至少有12个幼崽（P0 - P2的）。对小鼠，确保整个垃圾的基因型有一个匹配。如果基因型是未知的，从每一个单独的盘上的小狗的板块细胞，保持准确的记录鼠标号码和与之相配的细胞培养皿，确保胶质基质的遗传背景是在细胞培养的统一。 </li></ol> 2）解剖<ol><li>腹腔注射麻醉的第一个动物。动物表演时的镇静和不响应甩尾测试;在冰为30秒（直到低温）。冲洗一个小铝箔平方米，开盖itation剪刀，并用70％乙醇的负责人。 </li><li>杀头，让头落在铝箔广场和引擎盖下移动。轻轻地取出成一个冰冷的PBS15毫升管的大脑。广场上冰管，同时消除未来的大脑。 </li><li>重复这些步骤，直到有3个冰大脑。在显微镜下的第一个夹层菜倒到所有3个大脑和PBS。删除大脑中的相应部分（VTA）和使用转移吸管放入木瓜sol&#39;n段。第一分部进去时，启动一个计时器</li><li>重复以前的3个步骤，直到所有的大脑正在木瓜蛋白酶消化。平均消化时间应在2小时。第一部分已在最后进去的时间约1小时 尝试分裂的区别。 </li><li> *在消化过程中： <ol><li>确保在洗澡的温度是34 ° C。 </li><li>段可能会粘在第一个搅拌棒，但随着时间的推移应该传播出去。如果他们不这样做，挖掘瓶底部。 </li></ol></li></ol> 3）Trituration： <ol><li>使用移液管，转移大脑段无菌15毫升管（尽量避免多余的木瓜蛋白酶sol&#39;n），并把它们洗干净回暖胶质介质从孵化器2毫升3倍。将在管内的胶质介质后，允许部分解决和不扰民的细分，然后小心地取下尽可能sol&#39;n。更改移液器，以避免污染！ **不要让被删除sol&#39;n。 </li><li>开始使用一个新的移液管中的胶质媒体2毫升triturations。磨碎的25倍（避免气泡出租），并让管坐3分钟，直到未解离段解决。使用移液管，以消除和尽可能保持尽可能多的sol&#39;n互不干扰段底部。保存在一个新的（无菌）15米管上清液。 </li><li>重复上一步一个1000μl移液器和200μL的移液器使用，直到完全分离。 </li><li>磨碎用移液管的10倍，或待细胞完全悬浮在sol&#39;n。 </li></ol> 4）电镀细胞<ol><li>使用无菌黄色枪头，覆盖每个镊子尖，仔细下降为中心的玻璃内的每一道菜，以及可能的幻灯片环。 </li><li>放在hemacytometer和计数的细胞液中加入10μl。 （不包括垃圾群众）。 </li><li>乘以10的细胞数量。这是细胞数/μL。这个数字除以1,000,000-1,500,000（或想要的密度）。这是μL每道菜添加的数量。 </li><li>使用枪头滑过每道菜的中心，以及戒指，当你添加适当μL/碟。钢板他们轻轻地切换为每道菜的提示。 </li><li>加入100μL（稀释）GDNF的每道菜的幻灯片环内sol&#39;n。 </li><li>此时，放置在孵化器的托盘，并带来了在寒冷的房间里的神经介质。 </li><li>使细胞一夜之间解决。 </li><li>第二天：删除环（使用新的无菌枪头，覆盖每道菜的镊子提示） </li></ol> 5）有丝分裂的抑制作用：第二天<ol><li>加入200μLFDU股票1.8毫升无菌的MEM稀释1000X股票1:10 FDU等分。 </li><li>加入20μL稀释FDU sol&#39;n每道菜的外部环。经常更改枪头。封面和返回的孵化器。 *打扰尽可能少地为未来7-10天的菜肴。检查感染的菜肴和删除任何受感染的菜肴，在感染的最初迹象。文化3个星期内测试的准备。 </li></ol>媒体/解决方案 神经元的中等 （200ML） 对神经胶质细胞**最佳，如果空调使用前洗/ trituration一夜之间从烧瓶<table style="width: 435px; height: 231px;" border="0"><tbody><tr><td> 成分 </td><td> 金额 </td><td> 注释 </td></tr><tr><td> BSA的5％ </td><td> 0.5摹</td><td>分数V </td></tr><tr><td>纪念液体</td><td> 94.0毫升</td><td>西格玛</td></tr><tr><td> DMEM培养液</td><td> 80.0毫升</td><td>西格玛</td></tr><tr><td> F - 12液体</td><td> 20.0毫升</td><td>西格玛</td></tr><tr><td>葡萄糖45％液体</td><td> 1.50毫升</td><td>六西格玛解决方案</td></tr><tr><td>谷氨酰胺200MM </td><td> 0.5毫升</td><td> Aliquotted六西格玛解决方案</td></tr><tr><td> Diporzio浓。 </td><td> 2.0毫升</td><td>六西格玛解决方案</td></tr><tr><td>液体过氧化氢酶</td><td> 0.1毫升</td><td></td></tr><tr><td> Kynurenic酸0.5M </td><td> 200μL </td><td>在1N的NaOH </td></tr><tr><td>盐酸5N </td><td> 50μL </td><td></td></tr></tbody></table><ol><li>结合在250米成分（牛血清白蛋白进入最后）升塑料瓶。 </li><li>过滤器，标签和冷藏。 </li></ol> Kynurenic酸 （FW = 189.2） 0.5M = 94.6mg/ml 设为8毫升股票：无菌分装到200μL756.8mgKA/8ml 1N氢氧化钠和吸管 DiPorzio媒体 一）DiPorzio浓。股票： <table style="width: 620px; height: 251px;" border="0"><tbody><tr><td colspan="3"> 必要性 </td><td colspan="3"> 结合 </td><td colspan="3"> Alliquot </td></tr><tr><td> 添加剂 </td><td> 溶剂 </td><td> 管 </td><td> 金额 </td><td> 毫升 </td><td> 毫升/管 </td><td> 浓。 </td><td> 金额 </td><td> ＃Aliq。 </td></tr><tr><td>胰岛素</td><td> 20mm的盐酸（1） </td><td>塑料</td><td> 250毫克瓶</td><td> 10 </td><td> 1 </td><td> 25mg/ml </td><td> 25毫克</td><td> 10 </td></tr><tr><td>转</td><td>汉克的</td><td></td><td> 500毫克瓶</td><td> 5 </td><td> 1 </td><td> 100mg/ml </td><td> 100毫克</td><td> 5 </td></tr><tr><td>超氧化物歧化酶</td><td>汉克的</td><td></td><td> 70mg瓶</td><td> 14 </td><td> 1 </td><td> 5mg/ml </td><td> 5毫克</td><td> 14 </td></tr><tr><td>腐胺</td><td>汉克的</td><td></td><td> 50毫克</td><td> 3 </td><td> 0.12 </td><td> 20mg/ml </td><td> 2.4mg </td><td> 21 </td></tr><tr><td> NA 2 SEO 3 </td><td>汉克的</td><td></td><td> 0.104mg </td><td> 10 </td><td> 0.5 </td><td> 10μg/ml </td><td> 5.2μg </td><td> 20 </td></tr><tr><td> T3 </td><td> 10MM的NaOH </td><td></td><td> 2毫克</td><td> 10 </td><td> 0.1 </td><td> 0.20mg/ml </td><td> 0mg </td><td> 100 </td></tr><tr><td>孕酮</td><td> 100％乙醇</td><td>玻璃</td><td> 12.5mg </td><td> 10 </td><td> 0.05 </td><td> 1.25mg/ml </td><td>使用吸管</td><td></td></tr><tr><td>皮质醇</td><td> 100％乙醇</td><td>玻璃</td><td> 20毫克</td><td> 10 </td><td> 0.02 </td><td> 2.00mg /毫升</td><td>使用吸管</td><td></td></tr></tbody></table><ol><li> 20MM盐酸=41.5μl浓度。 HCl/25ml。 </li><li> 1mg/ml的股票，并添加104μl至10ml。 </li></ol> 二）DiPorzio媒体股票： <table style="width: 618px; height: 211px;" border="0"><tbody><tr><td> 添加剂 </td><td> 数量（毫升） </td><td> 金额（毫升）X2 </td><td> 终浓度。微克/毫升 </td><td> 终浓度。克分子浓度 </td></tr><tr><td>孕酮</td><td> 0.05 </td><td> 0.1 </td><td> 0.06 </td><td> 200nm的</td></tr><tr><td>皮质醇</td><td> 0.02 </td><td> 0.04 </td><td> 0.04 </td><td> 125nM </td></tr><tr><td>汉克的的BSS </td><td> 6.21 </td><td> 12.42 </td><td></td><td></td></tr><tr><td>胰岛素</td><td> 1 </td><td> 2 </td><td> 25 </td><td></td></tr><tr><td> NA 2 SEO 3 </td><td> 0.5 </td><td> 1 </td><td> 0.01 </td><td> 30NM </td></tr><tr><td> T3 </td><td> 0.1 </td><td> 0.2 </td><td> 0.02 </td><td> 30NM </td></tr><tr><td>超氧化物歧化酶</td><td> 1 </td><td> 2 </td><td> 5 </td><td></td></tr><tr><td>腐胺</td><td> 0.12 </td><td> 0.24 </td><td> 2.4 </td><td> 15NM </td></tr><tr><td>转</td><td> 1 </td><td> 2 </td><td> 100 </td><td></td></tr></tbody></table><ol><li>使用无菌15毫升聚丙烯管添加孕激素和皮质醇。使用吸管抽吸和真空中速度的乙醇蒸发：（使用5毫升stripette一半，中途破入管非常小心，不要吸乙醇液体安全保持Kimwipes地方吸管，然后将小费。 500μL商标管一侧。 </li><li>他们在上面的图表中出现的顺序添加其后等分。 </li><li>除了胰岛素，这使得sol&#39;n阴天后，加1N氢氧化钠20μL中的pH值。 Sol&#39;n应该从黄色变为粉红色，立即清除。此外，除了后转，立即加入20μL1N氢氧化钠中的pH值和防止沉淀物的形成。 </li><li>绘制成血清吸管和划分成5等份（一批）的毫升。 </li><li>商店@ -20 ° C。 </li><li>五月2批一次。 </li></ol> 解离媒体 A.木瓜蛋白酶（1瓶）： **电镀准备清扫前一天。 <table style="width: 536px; height: 156px;" border="0"><tbody><tr><td> 成分 </td><td> 金额&lt;/ STRONG&gt; </td><td> （注）终浓度。 </td></tr><tr><td>半胱氨酸水</td><td> 7.8mL </td><td> 1MM半胱氨酸</td></tr><tr><td>木瓜蛋白酶</td><td>变化（瓶*190μl 44.0mg/ml） </td><td> 20单位/毫升（共400个单位 20毫升值得sol&#39;n） </td></tr><tr><td> H＆B浓度。 </td><td> 2ML </td><td></td></tr><tr><td> Carbogen </td><td> 95％O2，5％CO2 </td><td></td></tr><tr><td>盐酸5N </td><td>加入10μl </td><td></td></tr><tr><td> 0.5％酚红</td><td> 20μL </td><td> 0.001％ </td></tr><tr><td> Kynurenate 0.5M </td><td>加入10μl </td><td> （1N氢氧化钠）0.5MM </td></tr></tbody></table><ol><li>添加木瓜蛋白酶半胱氨酸水。 </li><li> H＆B浓度，kynurenate，盐酸，酚红。 </li><li>过滤器分解小瓶（设置方向），并连接到carbogen。 </li></ol> B. H＆B浓缩液（100毫升的5倍） ： <table style="width: 583px; height: 243px;" border="0"><tbody><tr><td> 配料 </td><td> 兆瓦 </td><td> Powder/50ml H 2 O </td><td> 浓。 （M） </td><td> 联合收割机（毫升） </td><td> 终浓度。 （MM） </td></tr><tr><td>氯化钠</td><td> 58.44 </td><td> 11.699克</td><td> 4 </td><td> 14.5 </td><td> 116 </td></tr><tr><td>氯化钾</td><td> 74.56 </td><td> 3.728克</td><td> 1 </td><td> 2.7 </td><td> 5.4 </td></tr><tr><td>碳酸氢钠3 </td><td> 84.01 </td><td> 4.2摹</td><td> 1 </td><td> 13 </td><td> 26 </td></tr><tr><td>的NaH 2 PO 4 · H 2 O </td><td> 137.99 </td><td> 6.90摹</td><td> 1 </td><td> 1 </td><td> 2 </td></tr><tr><td> 硫酸镁 </td><td> 120.38 </td><td> 6.019克</td><td> 1 </td><td> 0.5 </td><td> 1 </td></tr><tr><td> EDTA（ED2 - SS） </td><td> 292 </td><td> Sigma公司5％（请 5g/100ml股票） </td><td> 0.134 </td><td> 0.3722 </td><td> 0.5 </td></tr><tr><td>血糖</td><td> 180 </td><td>适马45％ </td><td> 2.5 </td><td> 5 </td><td> 25 </td></tr><tr><td> TC H 2 O </td><td></td><td></td><td></td><td> 62.93 </td><td></td></tr></tbody></table><ol><li>结合股票的解决方案的金额。 </li><li>分成4毫升等分。 </li><li>新增分装后，加入木瓜蛋白酶半胱氨酸水。 </li></ol> C.半胱氨酸水（1X157.5毫升）： <table style="width: 644px; height: 137px;" border="0"><tbody><tr><td> 配料 </td><td> 兆瓦 </td><td> 组件 粉（毫克） </td><td> H 2 O（ML） </td><td> 股票浓度（MM） </td><td> 联合收割机（毫升） </td><td> 终浓度。 （MM） </td></tr><tr><td> 氯化钙 </td><td> 147.2 </td><td> 736 </td><td> 10 </td><td> 500 </td><td> 0.6 </td><td>为1.9mm </td></tr><tr><td>半胱氨酸</td><td> 121.7 </td><td> （1.5） </td><td> 24 </td><td> 20MM（0.02M） </td><td> 10 </td><td> 1.27MM （0.88） </td></tr><tr><td>训练班水</td><td></td><td></td><td></td><td></td><td> 146.9 </td><td></td></tr></tbody></table><ol><li>请和的0.5M氯化钙股票sol&#39;n保持在4 ° C </li><li>与其他成分的半胱氨酸使用sol&#39;n并结合</li><li> 15.75毫升和存储分为10等分，分频在4 ° C </li></ol> GDNF的制备 <ol><li>分装准备： <ol><li>灭菌去离子水溶解2.4mL无菌，冻干颗粒5μgGDNF的。本GDNF的sol&#39;n =2.08μg/mL浓度。 </li><li>分配到76.9μl在无菌离心管分装2.08μg/mLGDNF的sol&#39;n。这每小瓶= 160ng。 </li><li>储存在-20 ° C。 </li></ol></li><li>除了文化： <ol><li>解冻为每8菜1整除。 </li><li>加入723.1μl神经介质/等分稀释分装。这将使160ng/800μlGDNF的sol&#39;n。 </li><li>这sol&#39;n100μL加入终浓度为10ng GDNF的每毫升的媒介，在每道菜2ML。 </li></ol></li></ol> 复旦的制备 FDU - sol&#39;n 1000X股票： <table style="width: 564px; height: 80px;" border="0"><tbody><tr><td> 成分 </td><td> 金额 </td><td> （注）终浓度。 </td></tr><tr><td>尿苷</td><td> 247毫克</td><td> 16.5毫克/毫升</td></tr><tr><td> 5 FDU（5 fluorodeoxyuridine） </td><td> 100毫克（瓶） </td><td>6.7毫克/毫升</td></tr><tr><td>训练班水</td><td> 15毫升</td><td></td></tr></tbody></table><ol><li>超过15毫升16.5毫克/毫升尿苷一点点。 </li><li>添加15毫升尿苷sol&#39;n每瓶100毫克的FDU 6.7毫克/毫升sol&#39;n FDU。 </li><li>在-20 ° C到200μL分装和冻结的鸿沟</li></ol>稀释使用： <ol><li>抑制非神经元细胞的生长。加入200μL的股票，以1.8毫升的MEM稀释1000倍的股票1:10。 </li><li>加入20μL稀释FDU每道菜的外部环。经常更改吸管提示。封面和返回的孵化器。 </li></ol>

<ol>
	<li>Geiger, B. M., Behr, G. G., Frank, L., Caldera-Siu, A. D., Beinfeld, M. C., Kokkotou, E. G., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+defective+mesolimbic+dopamine+exocytosis+in+obesity-prone+rats.">Evidence for defective mesolimbic dopamine exocytosis in obesity-prone rats.</a> FASEB Journal. 8, 2740-2746 (2008).</li><li>Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+dopamine+quantal+size+in+midbrain+and+hippocampal+neurons.">Regulation of dopamine quantal size in midbrain and hippocampal neurons.</a> Behavioural Brain Research. 130, 203-207 (2002).</li><li>Pothos, E. N., Larsen, K. E., Setlik, W., Gershon, M. D., Krantz, D., Liu, Y. -. J., Edwards, R. H., Sulzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+transporter+expression+regulates+vesicle+phenotype+and+quantal+size.">Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.</a> The Journal of Neuroscience. 20, 7297-7306 (2000).</li><li>Pothos, E. N., Davila, V., Sulzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+recording+of+quanta+from+midbrain+dopamine+neurons+and+modulation+of+the+quantal+size.">Presynaptic recording of quanta from midbrain dopamine neurons and modulation of the quantal size.</a> The Journal of Neuroscience. 18, 4106-4118 (1998).</li><li>Sulzer, D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+mechanisms+that+regulate+quantal+size.">Presynaptic mechanisms that regulate quantal size.</a> Reviews in the Neurosciences. 11, 159-212 (2000).</li></ol>

这里所描述的方法，让精细的分辨率在全身或可在体内的方法，否则不中枢多巴胺神经元的形态和神经化学物质的功能。

primary dissociated midbrain dopamine cell cultures from rodent neonates

允许的孤立的多巴胺神经元突触前特点从大脑中的其它全身投入研究的多巴胺神经元的原代细胞培养创造能力。在我们的实验室，我们使用这些神经元，以评估使用的碳纤维安培，以及使用定量PCR和免疫细胞化学的多巴胺相关基因和蛋白质表达水平的多巴胺的释放动力学。在这个视频中，我们将向您展示我们如何产生这些文化灭鼠新生儿。 这一过程涉及几个步骤，包括电镀皮层胶质星形胶质细胞，神经细胞的培养基由胶质基质空调，中脑新生儿的解剖，消化，提取和多巴胺神经元的电镀和神经营养因子除了，确保细胞的存活。 应用适合这样的准备，包括电生理学，免疫细胞化学，定量PCR，视频显微镜（即实时与质膜融合水泡），细胞活力检测和其他毒理学屏幕。

Watch this Scientific Journal Video about Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates at JoVE.com

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

小学分离中脑多巴胺的细胞培养允许的多巴胺神经元突触前特性的研究。它们可用于监控实时多巴胺的释放动力学和多巴胺的胞外分​​泌的调节蛋白/ mRNA的水平。在这里，我们将向您展示如何从这些文化灭鼠新生儿。

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

primary-dissociated-midbrain-dopamine-cell-cultures-from-rodent

Research

JoVE Journal

Biology

14.8K 次观看.  Tufts University. 小学分离中脑多巴胺的细胞培养允许的多巴胺神经元突触前特性的研究。它们可用于监控实时多巴胺的释放动力学和多巴胺的胞外分​​泌的调节蛋白/ mRNA的水平。在这里，我们将向您展示如何从这些文化灭鼠新生儿。

视频: Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES细胞源性神经上皮细胞培养

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

科研

生物学

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

从晚期果蝇蛹的大脑原发性神经文化

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

游离的小鼠皮层神经元文化的制备

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

从海兔的神经细胞培养的高分辨率成像生长锥

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

建立从小学成人成纤维细胞培养鼠害

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

快速高密度的人类胚胎干细胞培养成纤维细胞去除

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...