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&amp;rsquo;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 10 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 4.5 mM glucose, 2 mM Glutamax. Adjust solution to 290 mOsm with mannitol and filter solution through a 0.2 &amp;micro;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 1 M solution to increase pH of dPBS and culture medium to 7.75.</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma (Merck, Damstadt, Germany)</td><td>S5761</td><td>10 mM NaHCO&lt;sub&gt;3&lt;/sub&gt; 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&amp;nbsp; 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 &amp;micro;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 &amp;micro;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 &amp;micro;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>Straight tip</td></tr><tr><td>Dumont #5/45 fine forceps</td><td>Fine Science Tools (CA)</td><td>11253-25</td><td>Angled 45&amp;deg; 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)。
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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

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

斑马鱼原代细胞的培养与转染

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

科研

发育生物学

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 ...

生物学

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

医学

Healthy Brain-pituitary Slices for Electrophysiological Investigations of Pituitary Cells in Teleost Fish

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the clear majority have been performed on dissociated primary cells. To improve our understanding of how teleost pituitary cells, behave in a more biologically relevant environment, this protocol shows how to prepare viable brain-pituitary slices using the small freshwater fish medaka (Oryzias latipes). Making the brain-pituitary slices, pH and osmolality of all solutions were adjusted to values found in body fluids of freshwater fish living at 25 to 28 &#176;C. Following slice preparation, the protocol demonstrates how to conduct electrophysiological recordings using the perforated whole-cell patch-clamp technique. The patch-clamp technique is a powerful tool with unprecedented temporal resolution and sensitivity, allowing investigation of electrical properties from intact whole cells down to single ion channels. Perforated patch is unique in that it keeps the intracellular environment intact preventing regulatory elements in the cytosol from being diluted by the patch pipette electrode solution. In contrast, when performing traditional whole-cell recordings, it was observed that medaka pituitary cells quickly lose their ability to fire action potentials. Among the various perforation techniques available, this protocol demonstrates how to achieve perforation of the patched membrane using the fungicide Amphotericin B.

The article describes an optimized protocol for making viable brain-pituitary tissue slices, using the teleost fish medaka (Oryzias latipes), followed by electrophysiological recordings of pituitary cells using the patch-clamp technique with the perforated patch configuration.

健康脑-垂体切片对硬骨鱼类鱼垂体细胞电生理的研究

Electrophysiological investigations of pituitary cells have been conducted in numerous vertebrate species, but very few in teleost fish. Among these, the ...

神经科学

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 ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

原发性生肌前体细胞/成肌细胞从基底哺乳动物支系文化准备

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

Here, we present a reproducible in vitro electroporation protocol for genetic manipulation of primary cerebellar granule cell precursors (GCPs) that is cost-effective, efficient, and viable. Moreover, this protocol also demonstrates a straightforward method for the molecular study of primary cilium-dependent Hedgehog signaling pathways in primary GCP cells.

高效且具有成本效益的电穿孔方法研究颗粒细胞前体中的原代纤毛依赖性信号通路

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

猪视网膜色素上皮细胞原代培养

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

尼罗罗非鱼肠单细胞悬液制备用于单细胞测序

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

Source: Terzi, A., et al., <a href="https://app.jove.com/v/62165">ROS Live Cell Imaging During Neuronal Development.</a> J. Vis. Exp. (2021)
The video demonstrates a method for culturing primary retinal ganglion cells from zebrafish embryos. Early-stage embryos undergo dissection to isolate the eyes, which are subsequently mechanically dissociated into a suspension of retinal ganglion cells. This cell suspension is cultured on coverslips coated with adhesive proteins to facilitate axon development.

斑马鱼胚胎原代视网膜神经节细胞培养物的产生

1. Preparation of solutions

	E2 media (1x)
	
		Prepare 100x E2A (500 mL), 500x E2B (100 mL) and 500x E2C (100 mL) solutions by combining all components ...