我们感谢 t. Fritsch, a. Asseburg, i. 林德和 S. m。Tokarski 为优秀的动物护理和技术支持。我们感谢 Köster 实验室的所有成员进行了激烈而有益的讨论。我们感激地承认德意志 Forschungsgemeinschaft (高 1949/5-1) 和联邦下萨克森, Niedersächsisches Vorab (VWZN2889) 提供的资金。

这里描述的所有动物工作符合法律规定 (欧盟指令 2010/63)。养护和处理鱼类已得到地方当局和不伦瑞克技术大学动物福利代表和下萨克森国家消费者保护和食品安全办公室 (LAVES, 德国奥登堡) 的批准;亚利桑那州§4 (02.05) TSchB。
1. 斑马鱼胚胎主要细胞的制备
<ol><li>2天后受精 (dpf) 斑马鱼胚胎的制备
	<ol>
<li>1天: 根据您的斑马鱼设施经理8的规格, 设置几条斑马鱼菌株的通道。</li>
		<li>2天: 交配鱼和收集鸡蛋8直接产卵后, 在10厘米培养皿 (塑料)。用巴斯德吸管 (塑料) 去除死亡或被污染的鸡蛋。用 Danieau 30% (5.8 毫米氯化钠) 洗鸡蛋 1x, 0.07 毫米氯化钾, 0.04 毫米硫酸镁, 0.06 毫米硝酸钙, 5 毫米 2-[4-(2-羟乙基) piperazin-1 基] 磺酸, pH 7.2)8与 0.0001% (w/v) 亚甲基蓝色。交换培养基到 Danieau 30% 没有亚甲基蓝, 因为亚甲蓝可能导致自体荧光, 并在28摄氏度的夜间孵化卵。 注: 以每条鱼线的最低100个卵开始, 以获得足够数量的细胞。不要在一个培养皿中饲养超过150个胚胎。</li>
		<li>3天: 去除死亡或被污染的胚胎, 并交换培养基到 Danieau 30%。通过计数确定胚胎数量。在28摄氏度的晚上孵化胚胎。 注: 当使用转基因线表达荧光记者, 筛选 1 dpf 或 2 dpf 可能是必要的。 注意: 对于更大数量的胚胎, 建议采取一个黑白图像的各自培养皿 (图 1A) 和量化的数量的胚胎与成像软件。</li>
		<li>4天: 胚胎现在是 2 dpf。要去除 chorions, 添加1毫升链霉蛋白酶, 浓度为1毫克/毫升至10毫升 Danieau 30%8 , 并在室温下的振动筛上孵化胚胎, 直到所有 chorions 分离 (根据环境温度20–40分钟)。用 Danieau 30% 洗净, 去除链霉蛋白酶和 chorions, 在室温下保持胚胎, 直到进一步使用。</li>
	</ol></li>
	<li>可复用聚 l-赖氨酸镀膜玻璃底菜的研制 注: 商用玻璃底菜仅供一次性使用, 价格昂贵。下面的过程描述了如何从标准实验室材料中准备可再用的自制玻璃底菜。<ol>
<li>钻一个直径为10毫米的孔到标准细胞培养皿 (直径6厘米, 塑料) 的底部 (图 1B)。用自来水彻底冲洗盘子底, 除去钻孔粉尘。</li>
		<li>在盘子底部的孔周围涂上硅脂, 然后用油脂作为胶水附着盖玻片。确保油脂密封的差距, 盘子底部和盖玻片。 注意: 确保使用的盖玻片的厚度适合于以后的成像应用程序。</li>
		<li>彻底清洗自制玻璃底菜, 但小心, 用冷水和肥皂。用去离子水冲洗玻璃底菜三次, 去除肥皂。空气干燥盘盖子和盘子底部并且存放他们在一个干净的箱子, 直到进一步用途。</li>
		<li>在文化准备的天 (天 4, 看见 1.1.4): 用 70% (v/v) 乙醇滋润盘子盖子和盘子底部的里面。将碟子盖和盘子底部朝向内侧, 在一个无菌的工作台, 层流和紫外线光。空气干燥, 直到乙醇蒸发, 然后应用 UV 光20分钟。经过这种处理, 菜肴被组装和认为是无菌的。</li>
		<li>对于涂层, 吸管200µL 的聚 l-赖氨酸 (0.1 毫克/毫升) 在每一个玻璃底部碟, 并通过打破表面张力与吸管尖端的盖玻片传播液体。让干燥60分钟, 然后洗1x 与无菌1x 磷酸盐缓冲盐水 (PBS)。取出液体。把盘子放在长凳下, 直到进一步使用。 注: 其他涂层可根据实验目的进行测试。我们发现聚 l-赖氨酸足以支持神经元的生长, 而未经任何附加涂层处理的塑料似乎有利于成纤维细胞的生长 (图 1E, F)。 注: 自制玻璃底碟可多次使用。要交换盖玻片, 用温水冲洗, 小心地分离盖玻片和去除剩余油脂与 70% (v/v) 乙醇和肥皂。</li>
	</ol></li>
	<li>原代细胞的制备与电镀
	<ol>
<li>在文化准备的天 (天 4, 看见 1.1.4): 通过使用新鲜的塑料巴斯德吸管转移胚胎入无菌细胞培养盘 (直径 6 cm)。去除液体的步骤-明智的, 直到所有的胚胎聚集在一个大的下降直径约2厘米或更少。</li>
		<li>将该菜与胚胎放在无菌工作台中, 添加 CO2独立培养基 (辅以 10% (v/v) 过滤牛血清, 1 x 谷氨酰胺和 1.2% (v/v) 1万 U 青霉素-链霉素; 培养基与所有补充剂是在以下称为 "细胞培养培养基"), 直到盘子半填充。 注: 可根据实验目的对 CO2独立介质的替代品进行测试, 如 neurobasal 培养基、DMEM 培养基或莱博维茨 L-15 培养基。co2-独立媒介和莱博维茨的 L-15 媒介有好处不需要 CO2孵化器。</li>
		<li>为了去除蛋黄, 用200µL 吸管尖把胚胎上下移。成功的 deyolking 可以被介质的混浊所识别。</li>
		<li>用 70% (v/v) 乙醇和另一种细胞培养皿填充细胞培养皿, 新鲜细胞培养培养基。使用1000µL 吸管尖端将胚胎移植到无菌细胞过滤器 (40 µm;图 1C)。把过滤器放在手柄上, 用乙醇蘸到盘子里, 这样所有的胚胎都能被淹没5秒. 紧接着, 用新鲜细胞培养培养基将过滤器与胚胎浸泡在盘子里。 注: 细胞过滤可多次重复使用。在自来水下用软刷子清洁, 将其贮存在70% 乙醇中, 干燥和 UV, 在使用前直接处理在无菌工作台上 (参见 1.2.4)。</li>
		<li>将胚胎移植到无菌的1.5 毫升反应管中 (大约100胚胎在一管中)。添加胶原酶 (2 型) 在细胞培养基中稀释, 最终浓度为4毫克/毫升, 总体积为1毫升。在30转每分钟45分钟的室温下, 在垂直管转子上孵化出具有胚胎的试管。</li>
		<li>分离剩余的细胞团块通过吹打胚胎胶原酶混合物上下与1000µL 吸管尖端。然后用排气槽 (40 µm) 过滤细胞悬浮液, 通过无菌细胞过滤器;图 1D)成50毫升锥形管。用大约10毫升的新鲜细胞培养基冲洗过滤器。</li>
		<li>颗粒细胞通过离心3分钟在 180 x g。小球可能几乎看不见。小心地清除上清和并用重悬细胞在200µL 新鲜细胞培养培养基每30最初使用的胚胎。 注: 要获得可见颗粒, 建议至少从100个胚胎开始。</li>
		<li>在自制的聚 l-赖氨酸包覆玻璃底菜 (见 1.2) 的玻璃区域直接在步骤1.3.7 中获得的200µL 的细胞悬浮液吸管。在无菌工作台下, 室温下孵育60分钟。添加6毫升新鲜细胞培养培养基和孵化的主要细胞在28摄氏度。</li>
		<li>使用28摄氏度的倒置显微镜执行所需的成像应用。每天交换细胞培养基。在电镀 (磷酸二铵) 后的几天内, 培养物可以用于成像。</li>
	</ol></li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57872/57872fig1.jpg"> 图 1: 斑马鱼胚胎的原代细胞培养.(a) 1 磷酸二铵胚胎的黑白图像, 可由软件工具处理, 以分析胚胎数量。(B) 用钻孔 (直径10毫米) 制作的细胞培养皿 (直径6厘米) 用于制备可重复使用的自制玻璃底菜。(C) 使用简单手柄的细胞过滤 (40 µm) 作为 "着陆网", 将 deyolked 胚胎浸入乙醇中, 并迅速将其转移到新鲜细胞培养基中。(D) 用排气槽的细胞过滤器 (40 µm) 用于在胶原酶介导的离解后过滤细胞。(E) 在5磷酸二铵后, 在涂有聚 l-赖氨酸的玻璃上播种的初级细胞主要形成有明显扩展的神经元。缩放条 = 100 µm. (F) 5 磷酸二铵在未涂敷的处理过的塑料后, 成纤维细胞样的 overgrow 培养。刻度条 = 100 µm (E) 和 (F) 是通过 epifluorescent 显微镜获得的。(G) 在1磷酸二铵中从野生型斑马鱼中提取的初级细胞的光图像。纹状肌细胞和神经元簇可以很容易地观察到。刻度条 = 50 µm (H) 转基因线 Tg (ptf1a: eGFP) jh1 的培养细胞, 表达 eGFP 在 GABAergic 神经细胞的神经元祖体和视网膜细胞种群的子集29,30,31. 刻度条 = 50 µm (G) 和 (H) 是通过共聚焦激光扫描显微镜获得的, 其使用的是 (B) 所示的玻璃底碟。<a href="https://www.jove.com/files/ftp_upload/57872/57872fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
2. 原代细胞与质粒 DNA 的转染
<ol><li>并用重悬过滤和颗粒细胞的步骤1.3.7 中获得的 1x PBS 而不是细胞培养培养基。用台盼蓝染色整除细胞悬浮液, 以确定计数室9中的细胞数。</li>
	<li>将50万个细胞与10µg 超纯质粒 DNA 混合在1.5 毫升反应管中, 并用 1x PBS 将总容积调整为100µL。 注: 在实验中, 我们主要采用了基于质粒 pCS2+10的表达结构。pCS2+ 克隆的开放阅读框架的表达是由人巨细胞病毒 (CMV 启动子) 无处不在的启动子驱动的。其他表达式构造和启动子可以测试 (请参见代表结果和图 2 和图 3)。</li>
	<li>将细胞 DNA 组合立即转移到电穿孔试管 (0.4 厘米), 将试管放在电穿孔装置中, 并 electroporate 以下设置: 一次性脉冲、指数衰减、280伏、950µF。</li>
	<li>直接电穿孔后, 将细胞 DNA 组合转化为1.5 毫升反应管, 300 µL 新鲜细胞培养基。</li>
	<li>板200µL 的细胞悬浮, 如1.3.8 所述, 并按照1.3.9 所述进行, 根据使用的表达结构, 荧光蛋白的表达可能会在几个小时或第二天检测出来。</li>
</ol>3. 固定原细胞的染色
注意: 亚细胞结构也可以由经典的染色, 而不是使用荧光融合蛋白记者。对于斑马鱼的主要细胞, 我们使用以下标准协议, 以示范染色核, F 肌动蛋白和乙酰蛋白荧光标记。
<ol><li>在细胞培养皿或多井板上放置的聚 l-赖氨酸涂覆的板细胞, 如上文所述 (见 1.3.8)。</li>
	<li>为修复, 去除介质和覆盖的细胞与4% 多聚甲醛在 1x PBS。在4摄氏度的振动筛上孵化细胞10分钟。用 1x PBS 在室温下洗涤细胞 3x 5 分钟。确保 1x PBS 完全覆盖单元格, 并在振动筛上执行洗涤步骤。</li>
	<li>为了阻止和 permeabilize 固定细胞, 覆盖细胞与 1x PBS 含有5% 脱脂牛奶和0.3% 海卫 X-100。在振动筛的室温下放置单元格10分钟。洗涤细胞如3.2 所述。</li>
	<li>要标记乙酰蛋白, 一个标志为轴突11, 稀释主要抗体 1:2, 000 在 1x PBS 包含1% 脱脂牛奶。用这个溶液覆盖细胞, 在晚上4摄氏度的时候将它们孵化在一个振动筛上。第二天, 洗涤细胞如3.2 所述。</li>
	<li>稀释二次抗体共轭与绿色 fluorochrom 荧光素异硫氰酸酯 (FITC) 1:100 在 1x PBS 含有1% 脱脂牛奶和孵化的细胞与此解决方案1小时在室温下在黑暗中 (盖碟,如一个盒子或铝箔) 在一个振动筛。洗涤细胞如3.2 所述。</li>
	<li>为了同时染色肌动蛋白细胞骨架和细胞核, 孵化细胞在 1x PBS 补充与罗丹明12共轭与红 fluorochrome (1:50) 和 4 ', 6-diamidino 2 苯基吲哚 (DAPI)13 (100 ng/毫升) 为10分钟在房间里温度在黑暗中的振动筛。洗涤细胞如3.2 所述。</li>
	<li>要准备用于成像的单元格, 请在干净的表面放置一个玻璃物体载体 (显微镜滑动) 并放置在它的安装介质上。用镊子把一个固定的和染色的细胞从盘子里拿出来, 并把它放在与目标载体面对的细胞的下落上。请确保安装介质在盖板的整个区域上扩散。让我们在黑暗中干涸吧。</li>
	<li>将固定和装入的单元格存储在4摄氏度的黑暗中, 直到执行所需的成像应用程序。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57872/57872fig2.jpg"> 图 2: 电穿孔表达结构的转染.(A) 假定的神经元转染 eGFP 1 磷酸二铵。(B) 表达内质网靶蛋白 KDEL 的横纹肌细胞 (2 磷酸二铵)。(C) 在2磷酸二铵 MitoTag YFP 转染的神经元簇内的两个神经元。(D) 用 DCX tdTomato、MitoTag YFP 和 pCS-H2B-mseCFP 三转染的细胞 (2 磷酸二铵)。(E) pSK-无人参与: mCherry 转进入初级细胞 (1 磷酸二铵), 由双转基因胚胎携带的转基因 tg (atoh1a: Gal4TA4) hzm222和 Tg (4 xuas: KGFPGI) hzm332导致 GFP 表达后脑的神经祖细胞。刻度条 = 10 µm (E) 是通过共聚焦激光扫描显微镜获得的, 使用玻璃底部的菜肴, 如图 1B所示。(F) 5 磷酸二铵固定斑马鱼原发神经元的荧光染色。蓝色: DAPI (核);红色: 罗丹明 (肌动蛋白);绿色: 乙酰蛋白 (神经元)。刻度条 = 10 µm (G) 用做爱狂人转染的神经元样细胞。在2磷酸二铵, 没有延伸是可见的。在5磷酸二铵, 形成了突起状结构。刻度条 = 25 µm (H) 神经元, 与 (F) 中的细胞相同, 周围有成纤维细胞样的单元格。缩放条 = 10 µm (I) 从携带转基因 Tg (XITubb:D sred) zf14828转染的转基因胚胎中提取的神经元做爱狂人。在12至15磷酸二铵之间, 突起发生大规模变性。刻度条 = 100 µm (fI) 的细胞在聚 l-赖氨酸涂层玻璃 (f, H) 或塑料 (G, i) 上播种, 在 L-15 培养基中培养, 在10% 过滤牛血清和神经元补充 B-27 (稀释 1:50) 和成像 epifluorescent 显微镜。<a href="https://www.jove.com/files/ftp_upload/57872/57872fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
4. 成年斑马鱼脑原细胞的制备
<ol><li>
脑提取
	<ol>
<li>选择至少90天龄的成年鱼。如果需要使用特定的单元格类型, 请选择一个转基因线, 在该行中, 特定于细胞的荧光报告器表达式将允许可视化所需的单元格。</li>
		<li>将鱼放在装有200毫升麻醉剂 Tricaine8 (0.2%) 的烧杯中, Danieau 30%。等到鱼停止移动。将麻醉鱼浸泡在装有200毫升冰水的烧杯中, 以弄死15分钟。</li>
		<li>用 70% (v/v) 乙醇填充培养皿 (直径6厘米)。用一把镊子把鱼放在尾巴上, 然后蘸到乙醇里。确保鱼完全浸入乙醇中5秒。</li>
		<li>根据古普塔和马林斯14的协议提取大脑, 以下 adaptions: 只使用蒸压或无菌包装的工具, 在无菌 1x PBS 中解剖头部。</li>
		<li>直接提取后, 将大脑放置在培养皿中 (直径3厘米), 填充无菌 1x PBS, 并将菜盘置于无菌的工作台内进行细胞培养。</li>
	</ol>
</li>
	<li>
原代细胞的脑分离与电镀
	<ol>
<li>放置两套无菌镊子 (蒸压), 一种无菌培养皿 (直径6厘米), 填充 70% (v/v) 乙醇, 一个无菌培养皿 (直径10厘米) 填充莱博维茨的 L-15 培养基补充 10% (v/v) 过滤牛血清, B-27 (1:50) 和 1.2% (v/v) 1万 U 型青霉素-链霉素和一个带手柄的无菌细胞过滤器 (40 µm;图 1C)在干净的长凳下。</li>
		<li>将细胞过滤器放入充满乙醇的培养皿中, 确保液位至少比滤网底部高5毫米。</li>
		<li>使用第一套镊子, 将大脑转移到已经放置在乙醇中的过滤器中, 并确保它完全被液体覆盖。1秒后, 将过滤器与大脑转移到含有莱博维茨 L-15 培养基的培养皿中, 上面描述的补充剂。</li>
		<li>使用第二套镊子, 将大脑转移到一个无菌的1.5 毫升反应管填充500µL 莱博维茨的 L-15 培养基与上述补充。将胶原酶 (2 型) 添加到最终浓度为4毫克/毫升的总体积为1毫升。</li>
		<li>在30转每分钟35分钟室温下, 在垂直管转子上孵化管。用1000µL 吸管尖端机械地将剩余的组织团簇吹打, 以帮助离解过程。</li>
		<li>当没有可见粒子停留在溶液中时, 停止离解。通过带有排气槽的无菌滤池过滤细胞悬浮液 (40 µm;图 1D)成50毫升锥形管。用大约10毫升的新鲜细胞培养基冲洗过滤器。 注: 当单细胞悬浮液达到时, 过滤步骤就不像胚胎分离那样必要了。大脑是一种软组织, 因此它更容易被均匀地分离成单细胞悬浮。</li>
		<li>颗粒细胞通过离心5分钟在 180 x g和并用重悬细胞颗粒成1毫升新鲜莱博维茨的 L-15 培养基与上述补充。</li>
		<li>吸管500µL 细胞悬浮 (50% 获得的细胞) 在一个自制的聚 l-赖氨酸镀膜玻璃底菜 (见 1.2) 或在一个井的24井板。缩小在较小的表面 (即, 96 井板的井的125µL 溶液) 的情况下。在无菌工作台下, 室温下孵育60分钟。然后添加必要量的新鲜培养基填充特定的容器和孵化的主要细胞在28摄氏度。</li>
		<li>使用28摄氏度的倒置显微镜执行所需的成像应用。培养基可以在电镀后几天用于成像。每天更换50% 的培养基。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

在这里, 我们提出了两种不同的协议, 培养主要细胞从 2 dpf 斑马鱼胚胎或成年斑马鱼的大脑。
从 2 dpf 斑马鱼中制备原代细胞培养物, 对于任何有基本细胞培养技术经验的人来说, 都是相对容易执行的。然而, 为了获得良好和可重现的结果, 足够数量的胚胎作为起始材料是至关重要的 (100 是最低的)。在胚胎的饲养过程中, 必须避免所有可能的污染源, 以尽可能降低细菌和寄生虫的?...

斑马鱼 (斑马斑马,斑马) 是一个流行的模型脊椎动物为许多领域的基础和生物医学研究1。斑马鱼胚胎发育迅速,前子宫, 是透明的, 并适合显微镜下, 从而提供了良好的先决条件, 研究脊椎动物的发展, 在一个活生生的有机体。由于斑马鱼2的遗传驯良, 许多稳定的转基因报告线与细胞类型特异表达的各种荧光标记已建立允许观察特定的细胞群体。斑马鱼社区提供了各种各样的所谓的 Gal4-driver 线, 携带转基因表达合成 Kal4TA4 (或 KalTA3-equivalent GalFF) 基因与 Gal4-DNA-binding 领域的酵母融合到病毒转录激活域在细胞类型特异增强剂的控制下。这些驱动程序线被交叉到执行器线, 其中运载转基因由定义的上游激活序列 (无人驾驶) 融合到一个记者基因。Kal4TA4 蛋白结合到无人研究的元素, 从而激活细胞类型选择表达的报告基因3,4。这种方法允许对双转基因动物中几乎所有可用增强因子和报告元素进行高度多样的组合研究。
然而, 在一个整体和不断变化的胚胎中, 聚焦于单个细胞或其亚型含量的深度实时成像是有限的。为了解决特定细胞生物学问题的最高分辨率, 使用细胞培养往往是可取的。斑马鱼的一些细胞线存在, 但它们被认为是重选择5,6,7 , 他们的繁殖往往是耗时的。此外, 所有可用的细胞系都是成纤维细胞, 限制实验使用细胞培养到一种类型的单元。因此, 我们建立了一个有效和易于使用的协议, 直接从斑马鱼胚胎和斑马鱼的大脑中制备初级细胞, 以及提高文化的长寿和扩大栽培的多样性的方法。单元格类型。此外, 我们提出了一个程序, 染胚胎原细胞与表达结构的荧光细胞器标记。因此, 细胞形态和亚细胞结构可以用高的空间和时间分辨率来进行分析, 从而保持其关键特征。

<table><tbody><tr><td>&lt;u&gt;Fish lines&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>AB (wild-type)</td><td></td><td></td><td>established by Streisinger and colleagues, available from the Zebrafish International Resource Center (ZIRC)</td></tr><tr><td>Tg(&lt;em&gt;ptf1a&lt;/em&gt;:eGFP)jh1</td><td></td><td></td><td>stable transgenic line in which the enhancer of the zebrafish gene&lt;em&gt; ptf1a &lt;/em&gt;drives expression of the fluorescent protein EGFP (Parsons &lt;em&gt;et al&lt;/em&gt;., 2007)</td></tr><tr><td>Tg(XITubb:DsRed)zf148</td><td></td><td></td><td>stable transgenic line in which the&lt;em&gt; Xenopus &lt;/em&gt;neural-specific beta tubulin promoter drives expression of the fluorescent protein DsRed&amp;nbsp; (Peri and N&amp;uuml;sslein-Volhard, 2008)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Equipment&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td></td><td>model 5804 R</td></tr><tr><td>ChemiDoc MP imaging system</td><td>BioRad</td><td></td><td>model XRS+, used to acquire black-and-white images of Petri dishes containing 1 da embryos</td></tr><tr><td>confocal laser scanning microscope</td><td>Leica microsystems</td><td></td><td>model SP8, equipped with 28 &amp;deg;C temperature box and a 63X objective</td></tr><tr><td>epifluorescent microscope</td><td>Leica microsystems</td><td></td><td>model DM5500B, equipped with 28 &amp;deg;C temperature box and a 40X objective</td></tr><tr><td>Gene Pulser Xcell with capacitance extender</td><td>BioRad</td><td>1652661</td><td>electroporation device</td></tr><tr><td>Horizontal shaker</td><td>GFL</td><td></td><td>model 3011</td></tr><tr><td>incubator for cell culture (28 &amp;deg;C)</td><td>Memmert</td><td></td><td>model incubator I</td></tr><tr><td>incubator for embryos (28 &amp;deg;C)</td><td>Heraeus</td><td></td><td>type B6120</td></tr><tr><td>light microscope</td><td>Zeiss</td><td></td><td>model TELAVAL 31</td></tr><tr><td>micro pipettes</td><td>Gilson</td><td></td><td></td></tr><tr><td>sterile work bench</td><td>Bio Base</td><td></td><td>with laminar flow and UV light</td></tr><tr><td>tweezers</td><td>Dumont</td><td></td><td>Style 5, Inox</td></tr><tr><td>vertical tube rotator</td><td>Labinco B.V.</td><td></td><td>model LD-79</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Software&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Image Lab Software</td><td>BioRad</td><td></td><td>for the ChemiDoc MP imaging system from BioRad</td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td></td><td>used for counting 1 dpf embryos by applying the Count particles-tool to the respective black-and-white images; Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/. (1997-2016).</td></tr><tr><td>LAS X</td><td>Leica Microsystems</td><td></td><td>for both confocal and epifluorescent microscopes from Leica Microsystems</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Plasmids&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>pCS-DCX-tdTomato</td><td>K&amp;ouml;ster Lab</td><td># 1599</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al.&lt;/em&gt;, 1994)</td></tr><tr><td>pCS-eGFP</td><td>K&amp;ouml;ster Lab</td><td># 7</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-H2B-mseCFP</td><td>K&amp;ouml;ster Lab</td><td># 2379</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-mClover</td><td>K&amp;ouml;ster Lab</td><td># 3865</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al.&lt;/em&gt;, 1994)</td></tr><tr><td>pCS-MitoTag-YFP</td><td>K&amp;ouml;ster Lab</td><td># 2199</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-ss-RFP-KDEL</td><td>K&amp;ouml;ster Lab</td><td># 4330</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-VAMP1-mCitrine</td><td>K&amp;ouml;ster Lab</td><td># 2291</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al.,&lt;/em&gt; 1994)</td></tr><tr><td>pSK-UAS:mCherry</td><td>K&amp;ouml;ster Lab</td><td># 1062</td><td>based on the pBluescript-backbone of Stratagene</td></tr><tr><td>Plasmid numbers refer to the database entries of the K&amp;ouml;ster lab. Plasmids are available upon request.</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Plastic and glass ware&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>BD Falcon Cell Strainer (40 &amp;micro;m)</td><td>FALCON</td><td>REF 352340</td><td>distributed by BD Bioscience, used as &amp;ldquo;landing net&amp;rdquo; to dip deyolked embryos into ethanol and to transfer them quickly to fresh cell culture medium</td></tr><tr><td>1.5 mL reaction tubes</td><td>Sarstedt</td><td>72690550</td><td></td></tr><tr><td>24-well plate</td><td>Sarstedt</td><td>83.3922</td><td></td></tr><tr><td>50 mL falconic tube</td><td>Sarstedt</td><td>62.547.004</td><td></td></tr><tr><td>96-well plate</td><td>Sarstedt</td><td>83.3924.005</td><td></td></tr><tr><td>EasyStrainer (40 &amp;micro;m)</td><td>Greiner Bio-One</td><td>542 040</td><td>with venting slots; used to filter cells after collagenase-mediated dissociation</td></tr><tr><td>electroporation cuvette (0.4 cm)</td><td>Kisker</td><td>4905022</td><td></td></tr><tr><td>glass coverslips</td><td>Heinz Herenz Medizinalbedarf GmbH</td><td>1051201</td><td></td></tr><tr><td>Microscope slides</td><td>Thermo Fisher Scientific (Menzel Gl&amp;auml;ser)</td><td>631-0845</td><td></td></tr><tr><td>Neubauer chamber</td><td>Henneberg-Sander GmbH</td><td>9020-01</td><td></td></tr><tr><td>Pasteur pipettes (plastic; 3 mL)</td><td>A. Hartenstein</td><td>PP05</td><td></td></tr><tr><td>Petri dishes (plastic; diameter 10 cm)</td><td>Sarstedt</td><td>821473</td><td>for zebrafish embryos</td></tr><tr><td>pipette tips</td><td>Sarstedt</td><td>Blue (1000 &amp;micro;l): 70762; Yellow (200 &amp;micro;l): 70760002; White (10 &amp;micro;L): 701116</td><td></td></tr><tr><td>sterile cell culture dishes (plastic; diameter 3 cm)</td><td>TPP Techno Plastic Products AG</td><td>93040</td><td></td></tr><tr><td>sterile cell culture dishes (plastic; diameter 6 cm)</td><td>Sarstedt</td><td>72690550</td><td></td></tr><tr><td>sterile Petri dishes (plastic; diameter 10 cm)</td><td>Sarstedt</td><td>83.3902</td><td>for brain dissection</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Chemicals and Reagents&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>sodium chloride</td><td>Roth</td><td>0601.1</td><td></td></tr><tr><td>4 % paraformaldehyde in 1x PBS</td><td>Sigma-Aldrich</td><td>16005</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>calcium nitrate tetrahydrate</td><td>Sigma-Aldrich</td><td>C1396</td><td></td></tr><tr><td>ethanol p.a. 100%</td><td>Sigma-Aldrich</td><td>46139</td><td></td></tr><tr><td>goat &amp;alpha;-mouse IgG (Fc specific) FITC conjugated</td><td>Thermo Fisher Scientific</td><td>31547</td><td></td></tr><tr><td>HEPES</td><td>Roth</td><td>9105.4</td><td></td></tr><tr><td>high vacuum grease</td><td>DOW CORNING</td><td>3826-50</td><td>silicon grease used for self-made glass bottom dishes</td></tr><tr><td>magnesium sulfate heptahydrate</td><td>Merck</td><td>105886</td><td></td></tr><tr><td>methylene blue</td><td>Serva</td><td>29198.01</td><td></td></tr><tr><td>Monoclonal Anti-Tubulin, Acetylated antibody</td><td>Sigma-Aldrich</td><td>T6793</td><td></td></tr><tr><td>Aqua-Poly/Mount (mounting medium)</td><td>Polyscience</td><td>18606</td><td></td></tr><tr><td>poly-L-lysine</td><td>Biochrom</td><td>L 7240</td><td></td></tr><tr><td>potasssion chloride</td><td>Merck</td><td>104938</td><td></td></tr><tr><td>Skim milk</td><td>Roth</td><td>68514-61-4</td><td></td></tr><tr><td>Texas Red-X Phalloidin</td><td>Thermo Fisher Scientific</td><td>T7471</td><td></td></tr><tr><td>Tricaine</td><td>Sigma-Aldrich</td><td>E10521</td><td>Synonym: Ethyl 3-aminobenzoate methanesulfonate</td></tr><tr><td>Triton X-100</td><td>BioRad</td><td>1610407</td><td></td></tr><tr><td>Trypan Blue</td><td>Gibco by Life Technologies</td><td>15250061</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Enzymes&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>collagenase (Type 2)</td><td>Thermo Fisher Scientific</td><td>17101015</td><td>dissolve powder in cell culture medium (8 mg/mL) and sterile-filter the solution, store aliquots at -20 &amp;deg;C</td></tr><tr><td>pronase (from Streptomyces griseus)</td><td>Roche</td><td>11459643001</td><td>distributed by Sigma-Aldrich, dissolve in 30% Danieau (10 mg/mL) and store aliquots at -20 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Medium and solutions for cell culture&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>1x PBS (Dulbecco&#039;s Phosphate Buffered Saline)</td><td>Gibco by Life Technologies</td><td>14190-169</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-independent medium</td><td>Gibco by Life Technologies</td><td>18045054</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>filtrated bovine serum (FBS)</td><td>PAN-Biotech</td><td></td><td>individual batch</td></tr><tr><td>glutamine 100x</td><td>Gibco by Life Technologies</td><td>25030081</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>Leibovitz&#039;s L-15 medium</td><td>Gibco by Life Technologies</td><td>11415049</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>PenStrep (10,000 U/mL)</td><td>Gibco by Life Technologies</td><td>15140148</td><td>distributed by Thermo Fisher Scientific</td></tr></tbody></table>

culture and transfection of zebrafish primary cells

斑马鱼的胚胎是透明的, 在母体外迅速发育, 因此可以在完整和发育的脊椎动物体内进行动态生物过程的良好成像。然而, 在整个坐骑中, 不同细胞类型和亚胞结构的形貌的详细成像是有限的。因此, 我们建立了一个有效和易于使用的协议, 培养活原细胞从斑马鱼胚胎和成人组织。
简言之, 2 的 dpf 斑马鱼胚胎 dechorionated、deyolked、消毒, 并与胶原酶分离成单细胞。在过滤步骤后, 主要细胞被镀到玻璃底部的菜肴和培育了几天。新鲜的文化, 尽可能多的长期 differenciated, 可用于高分辨率共焦成像研究。培养物中含有不同的细胞类型, 在聚 l-赖氨酸涂层中, 有横纹肌细胞和神经元突出。为了专门用荧光标记蛋白对亚细胞结构进行标记, 我们还建立了一种电穿孔协议, 允许将质粒 DNA 转染到不同类型, 包括神经元。因此, 在操作者定义的刺激, 复杂细胞行为, 和细胞内动力学的主要斑马鱼细胞可以评估与高空间和时间分辨率。此外, 利用成年斑马鱼的大脑, 我们证明了所描述的离解技术, 以及基本的培养条件, 也为成年斑马鱼组织工作。

图 1G显示了一种典型的从野生型胚胎中提取的具有纹状细胞和神经元样细胞簇的透射光图像。为了更容易地识别某些细胞类型, 可以使用具有细胞类型特异表达的荧光蛋白的转基因线 (图 1H)。
转染 pCS2+-based 质粒11将增强的绿色荧光蛋白 (eGFP)15转化...

Watch this Scientific Journal Video about Culture and Transfection of Zebrafish Primary Cells at JoVE.com

Culture and Transfection of Zebrafish Primary Cells

我们提出了一个有效的和易于使用的协议, 以制备斑马鱼胚胎的原细胞培养的转染和活细胞成像, 以及一个协议, 以准备初级细胞从成年斑马鱼的大脑。

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 ...

culture-and-transfection-of-zebrafish-primary-cells

Research

JoVE Journal

Developmental Biology

12.4K 次观看.  Braunschweig University of Technology. 我们提出了一个有效的和易于使用的协议, 以制备斑马鱼胚胎的原细胞培养的转染和活细胞成像, 以及一个协议, 以准备初级细胞从成年斑马鱼的大脑。

视频: Culture and Transfection of Zebrafish Primary Cells

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

科研

发育生物学

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

在斑马鱼胚胎人乳腺癌细胞的侵袭行为

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

癌症研究

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

分离培养成年斑马鱼脑源性神经球

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Ortho- and Ectopic Zebrafish Xeno-Engraftment of Ocular Melanoma to Recapitulate Primary Tumor and Experimental Metastasis Development

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and development of novel strategies for the treatment of metastatic ocular melanoma. In this protocol we delineate a quick and efficient way to generate embryonic zebrafish models for both the primary and disseminated stage of ocular melanoma, using retro-orbital orthotopic and intravascular ectopic cell engraftment, respectively. Combining these two different engraftment strategies we can recapitulate the etiology of cancer in its totality, progressing from primary, localized tumor growth under the eye to a peri-vascular metastasis formation in the tail. These models allow us to quickly and easily modify the cancer cells prior to implantation with specific labeling, genetic or chemical interference; and to treat the engrafted hosts with (small molecular) inhibitors to attenuate tumor development. 
Here, we describe the generation and quantification of both orthotopic and ectopic engraftment of ocular melanomas (conjunctival and uveal melanoma) using fluorescently labelled stable cell lines. This protocol is also applicable for engraftment of primary cells derived from patient biopsy and patient/PDX derived material (manuscript in preparation). Within hours post engraftment cell migration and proliferation can be visualized and quantified. Both tumor foci are readily available for imaging with both epifluorescence microscopy and confocal microscopy. Using these models, we can confirm or refute the activity of either chemical or genetic inhibition strategies within as little as 8 days after the onset of the experiment, allowing not only highly efficient screening on stable cell lines, but also enables patient directed screening for precision medicine approaches.

Here, we present a protocol to establish versatile orthotopic and ectopic zebrafish xenograft models for ocular melanoma to assess the growth kinetics of the primary tumor, dissemination, extravasation and distant, peri-vascular metastasis formation and the effect of chemical inhibition thereon.

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

Here, we present an effective, easy, and fast 3D culture protocol for the formation of spheroids of two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte).

胚胎和肝脏斑马鱼细胞系球状体的3D培养方法

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known ...

环境科学

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

斑马鱼 Tol2 系统:一种模块化且灵活的基于网关的转基因方法

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...

<meta charset="utf8"></head><body>用于再生和发育研究的稳健斑马鱼细胞移植

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>作者聚焦:绘制发育和再生过程中斑马鱼神经系统中的细胞连接图

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

胚胎和幼体斑马鱼的高分辨率细胞移植

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...