这一项目由挪威生命科学大学和挪威研究理事会资助, 拨款243811和 244461 (水产养殖项目)。我们感谢挪威生命科学大学的卢尔德卡伦谭维持鱼类设施。

在这项研究中进行的动物实验得到了挪威生命科学大学的批准, 遵循了研究动物护理和福利的指导方针。
1. 解决方案的准备工作
<ol><li>根据制造商的指示, 校准 osmometer 和 pH 计仪器, 以确保正确的测量。</li>
	<li>
准备500毫升的 Ca2 +和毫克2 +免费 Dulbecco 的磷酸盐缓冲盐水 (dPBS), 调整 pH 值 7.75 (步骤 1.2.1) 和渗透到 290 mOsm/千克 (步骤 1.2.2) 之前, 无菌过滤 (步骤 1.2.3)。
	<ol>
<li>在仔细搅拌溶液的同时, 仔细添加1米氢氧化钠溶液的水滴, 将 ph 值调整为7.75。</li>
		<li>使用校准的 osmometer 测量溶液的渗透性, 并计算增加渗透压至 290 mOsm/千克所需的甘露醇量。添加所需量的甘露醇, 然后再次测量溶液的渗透, 以确保正确的调整。 注: 所需的甘露醇量取决于渗透压测量, 这通常是不同批次之间的差异。甘露醇的一个分子等于1渗透粒子。</li>
		<li>无菌-用0.2 µm 过滤器过滤 dPBS 溶液。 注: 该溶液可储存在4摄氏度, 至少3月。</li>
	</ol>
</li>
	<li>准备500毫升的莱博维茨 (L-15) 培养基没有 l-谷氨酰胺和碳酸氢盐, 并补充它与10毫米 NaHCO3 (步 1.3.1), 4.5 毫米葡萄糖 (步 1.3.2) 和2毫米商业上可利用的谷氨酰胺 (步 1.3.3)。调整解决方案, 以 290 mOsm/千克 (步骤 1.3.4), 无菌过滤 (步骤 1.3.5), 并添加2.5 毫升的青霉素-链霉素溶液 (步骤 1.3.6)。 注: 该溶液可储存在4摄氏度, 至少4周。<ol>
<li>添加420毫克的 NaHCO3每500毫升培养基, 以达到10毫米溶液。</li>
		<li>每500毫升培养基中添加405毫克葡萄糖以达到4.5 毫米溶液。</li>
		<li>在500毫升培养基中加入5毫升的谷氨酰胺溶液 100x, 达到2毫米溶液。搅拌直到所有溶质溶解。</li>
		<li>使用 osmometer (如在步骤1.2.2 中执行), 用甘露醇将解决方案调整为 290 mOsm/千克。</li>
		<li>使用0.2 µm 过滤器对 L-15 介质进行无菌过滤。</li>
		<li>在无菌过滤后, 加入2.5 毫升青霉素-链霉素溶液, 对应于50毫升的青霉素和50µg/毫升链霉素。</li>
	</ol>
</li>
	<li>50毫升的胰蛋白酶溶液, 使用1毫克/毫升 (0.1%) 胰蛋白酶 ii 型 S 溶解在修改后的 dPBS (在步骤1.2 中制备)。使整除数2毫升的无菌塑料管, 并储存在-20 °c, 直到使用。 注: 2 毫升的整除数可以储存在-20 摄氏度, 至少3月。</li>
	<li>用1毫克/毫升 (0.1%) 胰蛋白酶抑制剂类型的 i 型, 制备50毫升胰蛋白酶抑制剂溶液, 并辅以2µg/毫升 DNase i 溶解在修改后的 dPBS 中 (在步骤1.2 中制备)。使整除数2毫升的无菌塑料管, 并储存在-20 °c, 直到使用。 注: 2 毫升的整除数可以储存在-20 摄氏度, 至少3月。</li>
</ol>2. 设备的准备
<ol><li>用火抛光玻璃吸管细胞离解的尖端。使用2种不同尺寸的小贴士 (0.6–0.8 µm 的中间开口和0.4–0.6 µm 的小开口), 在同时转动吸管的同时, 与火焰的时间和距离进行播放。将玻璃吸管放在一个干净的玻璃容器中, 然后用高压釜 (用105摄氏度的固态模式45分钟)。</li>
	<li>准备一个带冰块的聚苯乙烯盒子。</li>
	<li>用1.5 毫升的改性 dPBS (在步骤1.2 中准备) 将垂体转移到解剖过程中, 并将其放在冰上。</li>
	<li>准备一个15毫升管与10毫升的修改 dPBS (准备在步骤 1.2), 并保持在冰, 直到细胞离解。</li>
	<li>准备新鲜或解冻整除数的胰蛋白酶和胰蛋白酶抑制剂溶液 (准备在步骤1.4 和 1.5), 并保持在冰上, 直到使用。</li>
	<li>在开始解剖前, 将水浴设置为26摄氏度, 使水在垂体化学离解之前达到所需的温度。</li>
	<li>在使用前、期间和之后, 用70% 乙醇清洁所有的解剖工具, 包括用于解剖和细针的细钳和用于钉住鱼的蜡板。在整个过程中使用干净的手套 (经常更换和清洗), 以避免细胞的潜在污染。</li>
</ol>3. 垂体解剖和细胞离解
<ol><li>弄死将鱼浸泡在冰层中 (0 摄氏度)。将鱼放在冰层中至少1分钟, 以确保不可逆的低温休克。 注意: 固定化和平衡损失将在几秒钟后发生。确保鱼完全淹没在冰水中, 不躺在冰上, 因为后者会导致窒息和潜在的皮肤烧伤, 而不是快速和人性化的低温休克。</li>
	<li>在解剖显微镜下快速将鱼放在网中的蜡板上, 通过颈部的针刺来破坏脊柱。</li>
	<li>用细针将鱼头钉在蜡板上, 通过在前面 (嘴上) 插入一根针, 在头部的两侧 (脑后) 将头部固定在解剖前。</li>
	<li>查看解剖显微镜下的鱼, 并轻轻地刮掉头上的鳞片使用细钳与角度提示。</li>
	<li>小心地插入从头部一侧皮肤下的细钳的角度尖端, 并删除头骨屋顶, 慢慢地移动钳在嘴的方向, 同时牢牢抓住包围头骨屋顶的镊子。</li>
	<li>用有角尖的镊子完全切断脊髓。 注意: 时间是一个重要的因素, 所以工作有效和准确, 以限制从解剖垂体的时间, 直到细胞被镀在盘子里。经过一定的实践, 垂体解剖应该采取周围2–3分钟每条鱼, 其中只有大约十年代是需要采摘垂体时, 大脑已经暴露。</li>
	<li>小心翻转的大脑朝口, 以暴露垂体和解剖它与清洁钳与一个直接的提示。将脑垂体转移到含有1.5 毫升改性 dPBS 的塑料管上 (详情见步骤 2.3) 放在冰上。检查钳在显微镜下的尖端, 以确保垂体成功地添加到管, 并没有留在镊子。 注: 根据需要, 解剖尽可能多的垂体, 这取决于要同时准备的培养皿的数量。作为一个经验法则, 计算至少10垂体从成年鱼每碟有一个适当的密度细胞 (这取决于下游的应用)。</li>
	<li>在室温下将垂体在台式离心机中旋转。小心地删除 dPBS 使用玻璃吸管, 并注意避免删除任何垂体。</li>
	<li>加入1毫升的胰蛋白酶溶液 (在步骤1.4 中准备), 以洗涤垂体, 将其在台式离心机中旋转, 用玻璃吸管除去大部分液体, 然后再加入1毫升的胰蛋白酶溶液。</li>
	<li>在26摄氏度的水浴中孵化出30分钟的管子, 最好是轻轻摇动, 或者在潜伏期的时候轻拍一下管子。</li>
	<li>在室温下将垂体在台式离心机上旋转, 确保所有垂体都位于管的底部。用玻璃吸管除去大多数胰蛋白酶溶液, 加入1毫升的胰蛋白酶抑制剂溶液 (在步骤1.5 中制备), 以使胰蛋白酶失效并洗涤垂体。</li>
	<li>在室温下用台式离心机将垂体在管子底部收集。用玻璃吸管除去大部分液体, 加入1毫升的胰蛋白酶抑制剂溶液 (在步骤1.5 中制备)。将管子放在26摄氏度的水浴中20分钟, 最好是轻轻摇动, 或者交替地在潜伏期内轻轻地轻摇管子以使胰蛋白酶失效。</li>
	<li>通过加入2毫升的 L-15 培养基, 在26摄氏度和 1% CO2的情况下, 用35毫米的聚 d-赖氨酸包膜细胞培养皿, 在镀细胞之前, 将其孵化为至少10分钟。 注: 还可以使用塑料细胞培养皿。使用一个集中的玻璃底部的菜的主要好处是, 细胞被镀的区域是小的, 因此, 每道菜需要的垂体细胞较少。一些下游应用可能还需要一个玻璃底部 (即一些成像技术)。</li>
	<li>将15毫升管的冷却离心机设置为4摄氏度, 使其达到所需的温度, 然后开始机械分离。</li>
	<li>在室温下, 用胰蛋白酶抑制剂溶液 (从步骤 3.11) 在台式离心机上的垂体, 收集管子底部的管状物, 确保所有的垂体都位于管子的底部, 然后仔细地使用玻璃吸管去除大多数胰蛋白酶抑制剂溶液。 注: 在经核证的层流工作台 (黎巴嫩武装部队) 中, 执行协议的所有以下步骤, 以避免细胞污染。</li>
	<li>将1毫升的冰冷改良 dPBS (在步骤2.4 中制备) 到垂体组织片中, 并轻轻地吮吸在火焰抛光玻璃吸管 (在步骤2.1 中准备) 的组织件上下 (6–7x)。 注: 温度是一个基本的方面, 所以保持所有的解决方案在4摄氏度。从这一点开始, 尽可能在冰上执行所有步骤。</li>
	<li>在室温下在台式离心机上旋转组织件, 并仔细将含有分离细胞 (大约1毫升) 的 dPBS 的上部转移到放置在冰上的新的15毫升管上。避免将吸管移向管子底部, 使其在组织部分留下。</li>
	<li>重复步骤 3.16–3.17, 游离1毫升冰冷 dPBS 的细胞, 直到所有10毫升的 dPBS 使用。开始使用一个中型开口的玻璃吸管, 并切换到一个较小的开口朝向末端的玻璃吸管 (为最后的3–4毫升 dPBS 添加) 的离解。如果还有可见的组织片接近分离过程的末尾, 增加吹打到大约10x 的 2 dPBS 的最后步骤, 以分离, 最后留下残余的组织块后面。并用重悬的细胞, 并避免产生气泡, 因为它可能会导致细胞损伤时, 细胞附着在气泡表面。 注意: 这是该协议的关键步骤, 需要一些培训才能取得良好的效果。</li>
	<li>平衡管在离心机 (预冷到4°c) 和旋转向下的细胞在 200 x g 10 分钟在4摄氏度。一定要标记的管道的一侧, 细胞颗粒将。</li>
	<li>小心地从离心机上取出管子, 用玻璃吸管轻轻地将上清液转移到空的15毫升塑料管, 然后直接离心。一定要去除大部分上清, 但要注意避免丢失小 (通常是隐形的) 细胞小球。</li>
	<li>仔细并用重悬 L-15 培养基中50–100µL 细胞颗粒。 注意: 在这一步, 有可能计算细胞和/或执行生存能力测试 (如一个细胞计数的存在的台盼蓝使用 hemocytometer), 但请注意, 使用部分细胞悬浮为此目的将降低产量。避免使用大的悬浮体积, 因为它会增加细胞在盘子中心外扩散的风险。</li>
	<li>仔细滴下细胞培养皿中含有2毫升 L-15 培养基的离解细胞 (在步骤3.13 中制备)。允许细胞下沉到盘子的底部大约5分钟, 然后将它们移动到孵化器, 以避免细胞扩散到玻璃底部的凹陷部分外。</li>
	<li>孵化的菜在26摄氏度和 1% CO2 , 让所有的细胞彻底下沉, 并附着在盘子的底部。</li>
	<li>30分钟后, 看看显微镜中的细胞, 确保它们附着在培养皿上。</li>
	<li>继续孵化细胞在26°c 和 1% CO2。</li>
	<li>每3–4天小心地改变大部分 (但不是全部) 媒介。 注意: 避免更频繁地更改介质, 因为它可能会干扰细胞并使其与玻璃底部分离。在播种后一周内使用细胞进行实验。</li>
</ol>

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The importance of osmolality, pCO2, and pH.</a> General and Comparative Endocrinology. 178 (2), 206-215 (2012).</li><li>Wittbrodt, J., Shima, A., Schartl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medaka+-+a+model+organism+from+the+far+East.">Medaka - a model organism from the far East.</a> Nature Reviews Genetics. 3, 53-64 (2002).</li><li>Matsuda, M., 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=DMY+is+a+Y-specific+DM-domain+gene+required+for+male+development+in+the+medaka+fish.">DMY is a Y-specific DM-domain gene required for male development in the medaka fish.</a> Nature. 417 (6888), 559-563 (2002).</li><li>Kasahara, M., 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+medaka+draft+genome+and+insights+into+vertebrate+genome+evolution.">The medaka draft genome and insights into vertebrate genome evolution.</a> Nature. 447 (7145), 714-719 (2007).</li><li>Miyanishi, H., Inokuchi, M., Nobata, S., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Past+seawater+experience+enhances+seawater+adaptability+in+medaka,+Oryzias+latipes.">Past seawater experience enhances seawater adaptability in medaka, Oryzias latipes.</a> Zoological Letters. 2 (12), (2016).</li><li>Waymouth, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmolality+of+mammalian+blood+and+of+media+for+culture+of+mammalian+cells.">Osmolality of mammalian blood and of media for culture of mammalian cells.</a> In Vitro. 6 (2), 109-127 (1970).</li><li>Cameron, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid-base+homeostasis+-+past+and+present+perspectives.">Acid-base homeostasis - past and present perspectives.</a> Physiological Zoology. 62 (4), 845-865 (1989).</li><li>Claiborne, J. B., Edwards, S. L., Morrison-Shetlar, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid-base+regulation+in+fishes:+cellular+and+molecular+mechanisms.">Acid-base regulation in fishes: cellular and molecular mechanisms.</a> Journal of Experimental Zoology. 293 (3), 302-319 (2002).</li><li>Perry, S. F., Tufts, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish respiration. , (1998).</li><li>Hildahl, 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=Developmental+tracing+of+luteinizing+hormone+&#946;-subunit+gene+expression+using+green+fluorescent+protein+transgenic+medaka+(Oryzias+latipes)+reveals+a+putative+novel+developmental+function.">Developmental tracing of luteinizing hormone &#946;-subunit gene expression using green fluorescent protein transgenic medaka (Oryzias latipes) reveals a putative novel developmental function.</a> Developmental Dynamics. 241 (11), 1665-1677 (2012).</li><li>Strandab&#248;, R. A. U., 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=Signal+transduction+involved+in+GnRH2-stimulation+of+identified+LH-producing+gonadotropes+from+lhb-GFP+transgenic+medaka+(Oryzias+latipes).">Signal transduction involved in GnRH2-stimulation of identified LH-producing gonadotropes from lhb-GFP transgenic medaka (Oryzias latipes).</a> Molecular and Cellular Endocrinology. 372 (1-2), 128-139 (2013).</li><li>Verbalis, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+volume+regulation+in+response+to+changes+in+osmolality.">Brain volume regulation in response to changes in osmolality.</a> Neuroscience. 168 (4), 862-870 (2010).</li></ol>

作者没有什么要申报的。

体外细胞培养系统为研究人员提供了强有力的工具, 如果使用正确的方式1, 就可以回答过多的不同的生物学问题。重要的是要记住, 失去了与邻近细胞的连接的分离细胞可能已经获得了不同的功能性质比他们原来在体内。为了避免有误解从体外实验获得的结果的风险, 重要的是考虑调整原细胞培养过程的物种和细胞类型的兴趣。即使是对标准的文化程序和解决?...

细胞培养是分子生物学研究的主要工具之一, 它为回答从正常细胞生理学到药物筛选和致癌1等不同生物学问题提供了一个优秀的模型系统。从动物组织直接分离的主要细胞使用酶法和/或机械方法, 通常被认为比细胞系更具生物学意义, 因为生物反应可能更接近体内的情况。为了模拟细胞适应的特性, 获得生理上有意义的结果, 应针对每个物种和细胞类型对准备初级细胞培养的协议进行优化。
许多协议描述哺乳动物细胞系统的文化条件, 而类似的协议描述鱼类细胞的主要培养条件是相当稀少的比较。细胞容易受到温度、pH 值和渗透压的快速变化, 在离解过程中尤其脆弱。用于哺乳动物细胞培养的商业盐溶液和培养基不适合硬骨鱼类鱼, 特别是在 pH 缓冲系统和渗透压方面。因此, 重要的是要测量和调整这些参数的生理相关水平的解决方案的兴趣品种。
主要的垂体培养由几种硬骨鱼类鱼类组成, 包括鲤鱼 (鲤鱼)2,3, 草鱼 (草鱼艾达拉)4, 金鱼 (鲫鱼)5, 虹鳟 (虹鳟鱼虹鳟鱼)6, 欧洲鳗鲡 (安圭拉)7, 罗非鱼 (罗非鱼)8, 斑马鱼 (斑马斑马)9, 和大西洋鳕鱼 (Gadus morhua)10。除了将孵化温度调整到感兴趣的物种之外, 这些协议中的一些还在哺乳动物样的条件下孵化出细胞, 这种情况对感兴趣的物种可能是不理想的, pH 值从7.2 到7.5 在湿润的大气层中。包含 3-5% CO2。此外, 目前尚不清楚, 在不同的溶液中, 用于制备几种主要细胞培养液的渗透压是否调整和稳定。
目前的议定书是根据以前与大西洋鳕鱼10的初级文化有关的工作, 包括孵化温度、渗透力、ph 值和 ph 值缓冲系统的调整, 包括二氧化碳的分压 (2), 以鳉的生理学 (latipes)。鳉是一种小 (3–4厘米) 淡水鱼, 原产于东亚。这些天, 它被作为一个模型物种在世界各地的许多研究实验室, 因为它是相对容易繁殖和高度抵抗许多常见的鱼类疾病11。使用鳉作为模型有几个优点, 包括温度耐受度从4–40°c11, 短世代时间, 透明胚胎, 性别确定基因12, 和序列化基因组13, 以及许多其他可利用的遗传资源。
本协议中的主要养殖条件被优化, 以符合 medakas 在鱼设施中保存的26摄氏度的温度。此外, 渗透压从生活在盐水中的大西洋鳕鱼的 320 mOsm/千克减少到 290 mOsm/千克, 鳉生活在淡水中, 并符合鳉等离子14的正常渗透压。相比之下, 哺乳动物血浆的典型渗透压在 275–295 mOsm15的范围内。鱼类生活在各种温度下, 鳃与水直接接触, 使鱼的血液和胞外液的 pH 值和缓冲能力与哺乳动物不同。哺乳动物的文化媒介通常包括缓冲系统, 导致 pH 值约 7.4, 当媒体被平衡到标准大气 5% CO2在湿润空气在37摄氏度。ph 值是温度依赖性的, 中性酸碱度 (水中) 的价值随温度的降低而增加16。典型的硬骨鱼类鱼血浆 pH 值从7.7 到 7.917不等。该协议的优化包括减少从 ph 值为7.85 的 cod 保持在12°c, ph 为7.75 的鳉保持在26°c 通过增加 CO2从0.5% 到1%。
此外, 在鱼类和哺乳动物中, 碳酸氢盐的缓冲能力是相当不同的。CO2容易地被交换在鳃在鱼和2在水中是仅一小部分的2在肺18。改变温度或2会改变介质的 pH 值和缓冲度。因此, 对于孵化哺乳动物细胞的 pH 值和2的不均, 都不是最佳的鱼细胞, 因此, 培养基应优化与含有生理相关的鱼类价值的缓冲系统和特殊种类的兴趣。该协议描述了如何从鳉垂体中制备主细胞培养, 包括孵化温度、渗透、ph 和 ph 缓冲系统的调整, 以及在准备主细胞时需要考虑的其他重要参数。非哺乳动物物种的文化。

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (dPBS), without calcium chloride and magnesium chloride</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>D8537</td>
			<td>Adjust solution to pH 7.75 with 1M NaOH and 290 mOsm with mannitol.</td>
		</tr>
		<tr>
			<td>L-15 medium (Leibovitz), witout L-glutamine</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>L5520</td>
			<td>Supplement 500 ml culture medium with 10mM NaHCO3, 4.5 mM glucose, 2 mM Glutamax. Adjust solution to 290 mOsm with mannitol and filter solution through a 0.2 &#181;m PES sterile filter, before adding 2.5 ml Penicillin-Streptomycin solution (see below for details).</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>S5881</td>
			<td>Add drops of 1M solution to increase pH of dPBS and culture medium to 7.75.</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>S5761</td>
			<td>10 mM NaHCO3 equals 420 mg per 500 ml culture medium.</td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>63565</td>
			<td>Use to increase osmolality of dPBS and culture medium. Calculate correct amount needed to reach an osmolality of 290 mOsm.</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>G5400</td>
			<td>4.5 mM&#160; D-glucose equals 405 mg per 500 ml culture medium.</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco (Life Technologies, Paisley, UK)</td>
			<td>35050-061</td>
			<td>Alternative to L-glutamine, with increased stability. 2 mM Glutamax equals 5 ml of 100X stock in 500 ml culture medium.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>P0781</td>
			<td>Stock solution 10,000 units penicillin and 10 mg streptomycin per mL. Use 2.5 ml of stock solution in 500 ml L-15 medium (equivalent of 50 U/ml Penicillin and 50 &#181;g/ml Streptomycin).</td>
		</tr>
		<tr>
			<td>Trypsin type II-S</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>T7409</td>
			<td>Prepare 1 mg/ml in dPBS solution.</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor type I-S</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>T6522</td>
			<td>Prepare 1 mg/ml in dPBS solution, supplement with 2 &#181;g/ml Dnase I (see details below).</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>D5025</td>
			<td>Use in trypsin inhibitor solution (see above).</td>
		</tr>
		<tr>
			<td>0.2 &#181;m Polyethersulfone (PES) sterile filter system</td>
			<td>Corning Inc. (Corning, NY)</td>
			<td>431097</td>
			<td>Use for sterile filtration of dPBS and L-15 medium after adjustments.</td>
		</tr>
		<tr>
			<td>35 mm cell culture dish with glass bottom, poly d-lysine coated</td>
			<td>MatTek Corporation (Ashland, MA)</td>
			<td>P35GC-1.5-10-C</td>
			<td>Can also be replaced by plastic dish, depending on downstream application.</td>
		</tr>
		<tr>
			<td>Dumont #5 fine forceps</td>
			<td>Fine Science Tools (CA)</td>
			<td>11254-20</td>
			<td>Straigt tip</td>
		</tr>
		<tr>
			<td>Dumont #5/45 fine forceps</td>
			<td>Fine Science Tools (CA)</td>
			<td>11253-25</td>
			<td>Angled 45&#176; tip</td>
		</tr>
		<tr>
			<td>Stereo microscope SZ61</td>
			<td>Olympus Corp. (Tokyo, Japan)</td>
			<td></td>
			<td>Use for dissection of pituitaries.</td>
		</tr>
		<tr>
			<td>Alegra X-22R Centrufuge</td>
			<td>Beckman Coulter Inc. (Brea, CA)</td>
			<td></td>
			<td>With cooling option (similar to current model XR-30).</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Techne (Staffordshire, UK)</td>
			<td></td>
			<td>Any water bath with the possibility of adjusting temperature will do.</td>
		</tr>
		<tr>
			<td>Pasteur glass pipettes</td>
			<td>VWR (NY)</td>
			<td>612-1701</td>
			<td>Outer diameter 1.6 mm, fire polish and autoclave before use.</td>
		</tr>
		<tr>
			<td>Galaxy MiniStar table centrifuge</td>
			<td>VWR (NY)</td>
			<td>521-2844</td>
			<td>Any small table centrigue will do.</td>
		</tr>
		<tr>
			<td>Fine needles / insect pins</td>
			<td>Fine Science Tools (CA)</td>
			<td>26001-40</td>
			<td>Diameter 0.03 mm. Other fine needles can be used instead.</td>
		</tr>
		<tr>
			<td>Wax plate</td>
			<td></td>
			<td></td>
			<td>Custom made by adding melted paraffin wax in large petri dish.</td>
		</tr>
	</tbody>
</table>

preparation of a high-quality primary cell culture from fish pituitaries

主细胞培养是科学家常用的一种强有力的工具, 用于研究受控环境中孤立细胞的细胞性质和机制。尽管哺乳动物和鱼类之间的生理学存在巨大差异, 但鱼类的初级细胞培养协议往往是以哺乳动物的文化条件为基础的, 通常只进行少量的修饰。环境的差异不仅影响体温, 而且对血清中渗透、ph 值和 ph 值的缓冲能力等指标也有很显著的作用。由于细胞培养培养基和类似的工作解决方案旨在模仿细胞外液和/或血清的特性, 对其进行适应, 关键是这些参数是专门针对所讨论的动物进行调整的。
当前协议描述优化的鳉 (Oryzias latipes) 的主要区域性条件。该议定书提供了有关如何隔离和维持健康的分离垂体细胞超过一周的详细步骤, 包括以下步骤: 1. 渗透压对鳉血浆中发现的值的调节, 2. 调整孵化温度到正常的鳉温度 (这里在水族馆设施), 和3。将 pH 值和碳酸氢盐缓冲量调整到与生活在类似温度下的其他鱼类相比的价值。使用描述的协议的结果促进了生理上有意义的结果为鳉并且可以作为参考指南由科学家做主要细胞文化从其他非哺乳动物种类。

该协议描述了从鳉垂体的主细胞培养的制备, 并提供了在至少一个星期的文化中可以保持的健康细胞。该协议的基础上的生理相关值为鳉14 , 并进一步优化到垂体组织在成年鱼, 使用 pH 值7.75 和渗透 290 mOsm/千克在整个过程中从组织收获到电镀区域性中的单元格 (图 1和图 2)。
<p class="jove_content" fo:keep-together.with...

Watch this Scientific Journal Video about Preparation of a High-quality Primary Cell Culture from Fish Pituitaries at JoVE.com

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

在这里, 我们描述了一个协议, 以准备和维护主要的垂体细胞培养从鳉 (Oryzias latipes)。该协议中的优化条件是通过模拟鱼类的生理条件来考虑温度、渗透和 pH 等重要参数, 从而使生理上有更有意义的结果。

从鱼类垂体制备优质原代细胞培养

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled ...

preparation-high-quality-primary-cell-culture-from-fish

Research

JoVE Journal

Neuroscience

15.1K 次观看.  Norwegian University of Life Sciences. 在这里, 我们描述了一个协议, 以准备和维护主要的垂体细胞培养从鳉 (Oryzias latipes)。该协议中的优化条件是通过模拟鱼类的生理条件来考虑温度、渗透和 pH 等重要参数, 从而使生理上有更有意义的结果。

视频: Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

科研

神经科学

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of the nervous system often produce viable larvae and adults that have locomotion defective phenotypes that are difficult to adequately describe with text or completely represent with a single photographic image. Current modes of scientific publishing, however, support the submission of digital video media as supplemental material to accompany a manuscript. Here we describe a simple and widely accessible microscopy technique for acquiring high-quality digital video of both Drosophila larval and adult phenotypes from a lateral perspective. Video of larval and adult locomotion from a side-view is advantageous because it allows the observation and analysis of subtle distinctions and variations in aberrant locomotive behaviors. We have successfully used the technique to visualize and quantify aberrant crawling behaviors in third instar larvae, in addition to adult mutant phenotypes and behaviors including grooming.

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Here we describe a simple and widely accessible microscopy technique to acquire high-quality digital video of Drosophila adult and larval mutant phenotypes from a lateral perspective.

收购高品质的数字视频<em&gt;果蝇</em&gt;从横向角度看幼虫及成虫行为

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

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

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

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

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

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

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

一种从人类死后小脑中的激光捕获微解剖 purkinje 细胞中分离高质量 rna 的不锈钢协议

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

荧光活化细胞分选建立纯化的 Gabaep 或谷胱甘肽神经元的原代细胞培养

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...