这项工作得到 NIH 赠款 R01 GM113602 (WFM) 和 NSF 1144247 (AL) 的支持。我们承认马克 Slabodnick 和娜塔莉柯克兰开发的一些协议的初始版本和信息讨论。我们感谢卡丽娜 Perlaza 和格雷松刘易斯对手稿的批判性阅读。

1. 从单细胞或细胞片段培养Stentor和建立Stentor文化
<ol><li>准备衣藻衣藻文化作为食物用于Stentor。<ol>
<li>从商业供应商 (材料表) 获得衣藻衣藻细胞。</li>
		<li>采用无菌技术13, 在商用衣藻中建立500毫升液体培养衣藻。</li>
		<li>将衣藻文化保持在一盏灯下, 浓度接近饱和度 (大约 1), 每周两次稀释。 注:衣藻文化可以在振动筛上生长。</li>
		<li>定期检查衣藻的文化是否健康, 通过在幻灯片上放置一滴水, 覆盖它与盖玻片, 并检查它在显微镜下40X 放大。 注: 如果被细菌污染或衣藻细胞聚集成簇, 请勿使用Stentor喂养的培养基。如果出现上述任一问题, 请启动新的衣藻区域性。</li>
	</ol></li>
	<li>从商业供应商 (材料表) 获得Stentor 斑细胞。</li>
	<li>如果需要Stentor细胞从他们的自然栖息地, 收集他们从池塘, 湖泊, 或河流11。<ol>
<li>从池塘、湖泊或河流中收集Stentor , 找到一些植被相对平静、阴凉、清澈的地方。 注意: 在浮萍生长的地方发现Stentor的几率较高。</li>
		<li>在一个容易倾倒的容器中收集至少2升的水。</li>
		<li>在碎屑和微粒物质沉淀到容器的底部后, 轻轻地填充几个较小的容器, 检查Stentor, 等待几秒钟后, 每个收集的碎屑, 以解决在大容器。</li>
		<li>在某个位置完成样本集合后, 移动到至少10米以外的新位置, 并在那里重复取样的集合。 注: 并非每个池塘都有Stentor的人口, 因此可能需要对多个池塘进行取样。</li>
		<li>将样品返回实验室, 将水从容器中转移到单独的培养皿中。</li>
		<li>在每个培养皿, 搜索Stentor下的显微镜与斜光在5X 放大倍数。使用1毫升吸管将单个Stentor细胞转移到一个玻璃斑块的井中, 其中至少有100µL 的可商用的巴氏杀菌泉水 (PSW)。 注意: Stentor单元格在连接到基板时具有小号状形状 (图 1)。游泳Stentor细胞比附着在基板上的细胞的延伸程度小 (视频 1)。</li>
		<li>在 PSW 洗Stentor至少3x。通过去除井中约90% 的水, 同时保持井中的Stentor细胞, 然后在井中加入500µL 的 PSW 来进行洗涤。 注意: 在处理Stentor使用吸管与吸管提示10µL 或更小, 削减约0.5 毫米关闭的吸管尖端与剪刀, 以避免受伤的细胞由于剪切力, 是产生的大细胞流通过太小的尖端 op发生.</li>
	</ol></li>
	<li>在每次喂食Stentor前准备衣藻。<ol>
<li>转移1毫升的衣藻培养, 如在步骤1.1 成1.5 毫升离心管。离心机在 2095 x g 3 分钟。</li>
		<li>取出上清液, 悬浮1毫升 PSW 中的颗粒。离心机在 2095 x g 的3分钟内取出上清液, 并在500µL 的 PSW 中并用重悬颗粒。 注: 因此, 洗涤和集中衣藻将被称为 "准备衣藻" 在以下步骤。轻触介质对Stentor有害, 因此在喂养Stentor前洗涤衣藻是很重要的。</li>
	</ol></li>
	<li>如果需要克隆Stentor区域性, 请从单个Stentor单元格或单元格片段启动区域性。<ol>
<li>由于单Stentor细胞在 PSW 中生长不好, 通过过滤器对现有的、健康的Stentor文化中的500µL 培养基进行筛选, 制备条件介质。</li>
		<li>将条件介质的500µL 转移到玻璃点板的一个井中。</li>
		<li>将一Stentor转移到含有条件介质的玻璃斑片井中。尽量用尽可能少的培养基进行转移。</li>
		<li>每个Stentor 5 µL 每48小时的准备衣藻。当Stentor划分时, 计算井中单元格的个数, 并在每个单元格中添加5µL 的准备衣藻。</li>
		<li>保持Stentor在阴凉的地方, 因为细胞是敏感的光 (例如, 在透明塑料盒覆盖纸巾)。</li>
		<li>交换Stentor介质在井与新鲜被适应的媒介每 96 h。<ol>
<li>在步骤1.5.1 中准备新的条件介质。</li>
			<li>在井中轻轻吹打液体, 使所有Stentor细胞从井底分离出来。</li>
			<li>用1毫升的吸管小心地从油井中吸取液体, 确保所有的细胞都保持在井中。在井中添加500µL 的新鲜条件介质。</li>
		</ol>
</li>
		<li>当井中的单元数超过20时, 将单元格移动到较大的容器中, 例如, 一个宽口的玻璃罐子。<ol>
<li>在蒸压宽口玻璃罐中加入20毫升的 PSW。 注: 热处理玻璃器皿的Stentor可以用仔细的洗涤和冲洗代替。</li>
			<li>小心地将介质从井中向上和向下移除, 从井底分离出所有的细胞。收集所有Stentor细胞使用1毫升吸管尖端, 并轻轻地把它们转移到玻璃罐子。</li>
		</ol>
</li>
		<li>不要用Stentor的文化把罐子上的盖子拧紧, 让空气有足够的通道。</li>
		<li>每48小时添加 PSW 到罐中, 以保持Stentor密度在大约20细胞/毫升。用眼睛估计细胞的密度。 注: 或者, 用1毫升吸管将空气泡进罐子, 使细胞从墙壁中分离出来, 吸干1毫升的培养基, 并计算出吸管尖端的细胞数。</li>
		<li>每48小时, 饲料准备衣藻 Stentor文化在罐子 (参见步骤 1.4)。开始与喂养的文化与200µL 的准备衣藻。随着养殖量的增加, 逐渐增加用于喂养多达1毫升的衣藻的准备量。</li>
		<li>当文化卷达到罐子容量的90% 左右时, 将文化转移到更大的容器中。<ol>
<li>用1毫升的吸管将罐子内的文化Stentor , 从玻璃中分离出来。</li>
			<li>将罐子的全部内容倒入2杯玻璃容器中。</li>
			<li>用大约25毫升的 PSW 将罐子冲洗到2杯玻璃容器中, 收集剩下的Stentor。</li>
		</ol>
</li>
	</ol></li>
	<li>在2杯玻璃容器中保持健康的文化。<ol>
<li>饲料Stentor培养2毫升的衣藻每100毫升的培养每 4-5 天。每 4-5 天将 PSW 添加到玻璃容器中, 以保持Stentor密度约20细胞/毫升。 注: 450 毫升是2杯容器持有的最大容积。</li>
		<li>每周一次, 检查5X 解剖显微镜下的培养轮虫, 真菌和其他生长。为了延长养殖的健康水平, 使用1毫升的吸管去除污染微生物以及异常形状和无色的Stentor细胞。</li>
		<li>当玻璃容器满90% 时, 分裂文化。<ol>
<li>在玻璃容器中加入25毫升的 PSW。用25毫升的吸管把Stentor从玻璃上分离出来。将大约50% 的文化移动到一个新的2杯玻璃容器中。</li>
			<li>在两种文化中添加25毫升的 PSW, 并继续保持这两种文化, 如议定书本节所述。 注意: 由于Stentor细胞对高温敏感, 所以保持在保存培养基的房间温度在25摄氏度或更低。或者, 文化可以保存在孵化器中。有关Stentor培养的疑难解答, 请参阅表 1 。</li>
		</ol>
</li>
	</ol></li>
</ol>2. 通过切割Stentor细胞诱导再生
<ol><li>使用针拉器, 使几个针从毛细管使用程序如下: 热-735, 拉动-100, 速度-110, 时间-150, 压力-400。</li>
	<li>在50毫升的 PSW 中准备4% 纤维素溶液。<ol>
<li>添加1克的纤维素 (粘度: 1500 cP) 到50毫升的 PSW。</li>
		<li>在4摄氏度孵育至少8小时, 以促进纤维素的溶解。</li>
		<li>在室温下保持4% 纤维素溶液。</li>
	</ol>
</li>
	<li>在执行切口后, 准备一个用于储存细胞碎片的玻璃斑片。<ol>
<li>过滤消毒培养基从一个健康的文化, 500 毫升每片玻璃现货需要很好。</li>
		<li>在所需的每一个水井上转移500毫升的灭菌介质。</li>
	</ol>
</li>
	<li>收集一个健康的Stentor (有一个定义小号形状, 充满活力的蓝绿色, 并没有大的空泡) 在2µL 液滴, 并把它放在盖玻片或滑动。添加2µL 4% 纤维素 (图 2)。让Stentor在切割前减速 (视频 2)。</li>
	<li>将玻璃针与切割表面平行, 以防折断针头。</li>
	<li>使用立体解剖显微镜, 找到玻璃针的尖端, 并将其靠近细胞 (图 3A)。与针接触时观察Stentor合同 (图3B 和 C)。 注: 如果单元格的方向是使前端与后端分离困难, 则用针侧轻轻旋转。</li>
	<li>轻轻按收缩的Stentor与玻璃针的一侧切割的细胞在两个 (图 3D 和 E,视频 3)。将这两个碎片分开, 确保它们之间没有细胞质连接, 以避免碎片融合 (图 3F)。</li>
	<li>检查两个片断是否有至少一个 macronuclear 节点每个通过检查片段在解剖显微镜下与斜光照。 注意: 至少有一个 macronuclear 节点对细胞存活至关重要。在切割过程中或之后, 细胞可能会脱落其薄膜 (透明外壳) 和蓝绿色颜料。在大多数情况下, 这不会影响细胞的长期生存能力。</li>
	<li>将碎片移到步骤2.3 中准备好的玻璃点板的井中。</li>
	<li>剪切多个单元格, 因为切细胞的一小部分不会再生。</li>
	<li>如果在一个会话中执行多个切口, 则当玻璃针严重涂上Stentor残渣或其尖端破裂时, 将其更换。另外, 在硅垫片上轻轻擦拭针即可清洁针头。 注: 玻璃针可用于多个细胞切割疗程。</li>
	<li>将玻璃斑片与湿度室中的细胞碎片保持在一起。</li>
	<li>进入《议定书》4节, 以了解Stentor再生结果的成像和解释的细节。</li>
</ol>3. 蔗糖或尿素处理诱导 Membranellar 带和口腔器械再生
<ol><li>蔗糖 Membranellar 带和口服器械的去除<ol>
<li>在 PSW 中准备25% 的蔗糖溶液。</li>
		<li>添加500µL 25% 蔗糖在 PSW 到一个离心管与一顶盖。</li>
		<li>在每管中准备3离心管, 1 毫升 PSW, 用于洗涤细胞后的蔗糖处理。</li>
		<li>使用1毫升吸管 (图 4, 箭头 a) 收集 30-60 Stentor细胞在他们的培养基1毫升到一个单独的离心管中。</li>
		<li>收集所有的细胞从管在125µL 最终体积使用200µL 吸管在一个单一的平局。将它们转移到管中, 500 µL 25% 蔗糖 (在步骤3.1.2 中制备), 以获得625µL 的20% 蔗糖溶液 (图 4, 箭头 B)。开始秒表。</li>
		<li>孵化Stentor在这20% 蔗糖溶液和轻摇旋转离心管在机架1分钟。</li>
		<li>在一个绘图中收集所有单元格 (调整吸管到200µL 最大容量以便于收集)。</li>
		<li>保持细胞在吸管尖端, 直到秒表显示2分钟的蔗糖治疗。</li>
		<li>将Stentor弹出到步骤3.1.3 中的一个离心管中 (图 4, 箭头 C)。在机架上轻轻旋转管子。</li>
		<li>将细胞再洗两次, 在 PSW 的两个剩余的离心管中, 每一次都在步骤3.1.3 中制备 (图 4、箭头 D 和 E)。 注: 单拉细胞采集技术在洗涤之间不重要。</li>
		<li>进入《议定书》4节, 了解Stentor再生动力学的成像和解释细节 (图 4, 箭头 F 和 G)。</li>
	</ol>
</li>
	<li>尿素 Membranellar 带及口服器械的去除方法<ol>
<li>在 PSW 中制备4% 尿素溶液。</li>
		<li>添加300µL 4% 尿素在 PSW 到一个离心管与一顶盖。</li>
		<li>在尿素处理后, 在每管中准备3离心管, 含1毫升 PSW, 用于洗涤细胞。</li>
		<li>使用1毫升吸管 (图 4, 箭头 a) 收集 30-60 Stentor细胞在他们的培养基1毫升到一个单独的离心管中。</li>
		<li>收集所有的细胞从管在300µL 最终体积使用1毫升吸管在一个单一的平局。将它们转移到管中, 300 µL 4% 尿素 (在步骤3.2.2 中制备), 以获得600µL 的2% 尿素溶液 (图 4, 箭 B)。开始秒表。</li>
		<li>孵化Stentor在这2% 尿素溶液和轻摇旋转的离心管在机架1分钟。</li>
		<li>在单个绘图中收集所有单元格。</li>
		<li>保持细胞在吸管尖端, 直到秒表显示2分钟的尿素治疗。</li>
		<li>将Stentor弹出到步骤3.2.3 中的一个离心管中 (图 4, 箭头 C)。在机架上轻轻旋转管子。</li>
		<li>将细胞再洗两次, 在 PSW 的两个剩余的离心管中, 每一次都在步骤3.2.3 中制备 (图 4、箭头 D 和 E)。 注: 单拉细胞采集技术在洗涤之间不重要。</li>
		<li>进入《议定书》4节, 了解Stentor再生动力学的成像和解释细节 (图 4, 箭头 F 和 G)。</li>
	</ol>
</li>
</ol>4. 细胞再生的成像和分析
<ol><li>如果使用直立显微镜, 则使用吊滴法对单个细胞进行图像再生。<ol>
<li>将100µL 的 PSW 放在玻璃片的井中。</li>
		<li>使用10或20µL 吸管, 在4µL 培养基中隔离 1 Stentor细胞 (它们所在的介质)。将液滴存入 22 x 22 毫米2盖玻片的中间 (图 5)。 注: 如果细胞 1) 不健康 (异常形状或严重空泡) 或 2) 仍然有 membranellar 带或口服器具 (为化学治疗实验), 不要使用成像。</li>
		<li>在之前的水滴周围的培养基中再排列4滴Stentor , 同时留出足够的空隙。</li>
		<li>反转盖玻片与水滴, 并轻轻地把它放在玻璃点板的井, 在 PSW 添加到步骤4.1。</li>
		<li>注: 井中的 PSW 将减少水滴的蒸发 (图 5)。</li>
		<li>通过以下步骤 4.1.1 4.1.4 准备剩余的细胞进行成像。</li>
		<li>用所需的时间分辨率在显微镜下成像再生细胞。或者, 用解剖立体显微镜观察再生。 注: 再生将在细胞切割或蔗糖或尿素治疗后立即开始。然而, 在再生开始后, 可见的新口腔结构将形成约3小时。</li>
	</ol>
</li>
	<li>对于每个时间点, 为每个单元格分配三个再生阶段之一。为此, 请将每个单元格与演示阶段的代表性图像进行比较 (图 4和图 6)。<ol>
<li>将阶段1分配给尚未 membranellar 带的单元格 (图 6A)。</li>
		<li>用 membranellar 带将阶段2分配给单元格。 注: 治疗后 membranellar 带出现 3-6 小时 (图 4,图 6B)。在这个阶段的最后, membranellar 带后端的附加曲率会出现在口腔普利摩顿出现之前。</li>
		<li>用口服普利摩顿将阶段3分配给细胞。 注: 口服普利摩顿是一种内陷在 membranellar 带后端治疗后出现 6-8 小时 (图 4,图 6C 和 6D)。再生是完成时, 细胞有一个 membranellar 带在其前端和特点小号细胞形状 (图 4,图 6E)。大多数Stentor细胞在8-9 小时内完全再生, 自再生开始。</li>
	</ol>
</li>
	<li>在每个时间点以堆积框图的形式绘制每个再生阶段的单元格百分比 (图 7)。 注意: 这种类型的情节允许在细胞的种群中的再生动力学的可视化。</li>
</ol>

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<li>Tartar, V. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/jez.1401310105/full">Grafting experiments concerning primordium formation in Stentor coeruleus.</a> Journal of Experimental Zoology Part A. 131 (1), 75-121 (1956).</li>
<li>Lillie, F. R. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/jmor.1050120105/abstract">On the smallest parts of Stentor capable of regeneration; a contribution on the limits of divisibility of living matter.</a> Journal of Morphology. 12 (1), 239-249 (1896).</li>
<li>Morgan, T. H. <a target="_blank" href="https://www.jstor.org/stable/1535709">Regeneration of proportionate structures in Stentor.</a> The Biological Bulletin. 2 (6), 311-328 (1901).</li>
<li>Slabodnick, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+macronuclear+genome+of+Stentor+coeruleus+reveals+tiny+introns+in+a+giant+cell%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">The macronuclear genome of Stentor coeruleus reveals tiny introns in a giant cell.</a> Current Biology. 27 (4), 569-575 (2017).</li>
<li>Tang, S. K. Y., Marshall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-repairing+cells%3A+How+single+cells+heal+membrane+ruptures+and+restore+lost+structures%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Self-repairing cells: How single cells heal membrane ruptures and restore lost structures.</a> Science. 356 (6342), 1022-1025 (2017).</li>
<li>Slabodnick, M., Prevo, B., Gross, P., Sheung, J., Marshall, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+cytoplasmic+flow+during+single-cell+wound+healing+in+Stentor+coeruleus%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Visualizing cytoplasmic flow during single-cell wound healing in Stentor coeruleus.</a> Journal of Visualized Experiments. 82 (82), 50848(2013).</li>
<li>Tartar, V. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aTartar%2C+V+%22Biology+of+Stentor%22">Biology of Stentor</a>. , Pergammon Press. Oxford. (1961).</li>
<li>Slabodnick, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+kinase+regulator+mob1+acts+as+a+patterning+protein+for+stentor+morphogenesis%5BTitle%5D)+AND+%22PLOS+Biology%22%5BJournal%5D)">The kinase regulator mob1 acts as a patterning protein for stentor morphogenesis.</a> PLOS Biology. 12 (5), e1001861(2014).</li>
<li>Harris, E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHarris%2C+E+%22The+Chlamydomonas+sourcebook%3A+Introduction+to+Chlamydomonas+and+its+laboratory+use%22">The Chlamydomonas sourcebook: Introduction to Chlamydomonas and its laboratory use</a>. 1, Academic Press. Cambridge. (2009).</li>
</ol>

作者没有什么可透露的。

培养Stentor提出了许多挑战。首先, 要进行需要大量细胞的实验, 你需要维持大量的Stentor文化, 因为当Stentor浓度超过20细胞/毫升时, 它们的培养会变得不健康。第二, 可以污染Stentor文化的有机体往往比Stentor的分裂更快, 压倒了文化 (一种常见的污染物是轮虫)。因此, 有必要定期检查显微镜下的培养物, 去除污染物。有时, 需要从被污染的文化中手动解救出来的少量细胞开始新的文化。这增加了维持健康Stentor文化所需的时间。第三, 将单个细胞扩展到400毫升培养中需要至少一个月, 因为Stentor细胞周期为 3-5 天。第四, 不像其他模型纤毛虫, Stentor很少进入囊肿形式, 他们不能被冻结。
培养Stentor的一个重要方面是选择合适的食物有机体。以前描述了各种培养Stentor的方法。其中一个建议使用脱脂牛奶培养细菌, 然后喂养Stentor。这种技术在培养Stentor中是有效的;然而, 基因组技术的应用需要纯样本来避免从未定义的食物有机体中的基因组读数引起的混淆。使用目前的协议, Stentor可以生长在质量和, 因为他们的食物,衣藻, 已经排序, 存在的基因组污染的食物有机体可以检测和控制。由于未知的原因, 鞑靼人不得不补充他的股票, 回到他发现Stentor之前。通过目前的协议, 我们已经能够保持Stentor多年。
Stentor的再生实验一般都很简单, 但是有一些关键的细节要记住。关于2节, 切割Stentor细胞的一半可以掌握在几分钟内。掌握更先进的显微外科手术Stentor可能需要一周的练习。当执行蔗糖或尿素治疗 (第3节), 如果在成像开始大部分细胞仍然有他们的 membranellar 带, 增加孵化时间 10-三十年代, 当蔗糖治疗下一步执行。不要增加蔗糖治疗的时间超过3分钟孵化, 因为它会导致细胞死亡。
Stentor再生的影像学需要在长时间内对大细胞进行成像。4节详述的成像方法只能与直立显微镜一起使用。如果相反的显微镜是可用的, 那么细胞可以放在一个幻灯片或盖玻片在小室。一种方法是用石油果冻制造出一个腔室, 并盖上另一盖玻片以防止蒸发。另外, 单个单元格的腔室可以通过在 1 x 1 厘米2平方的硅间隔 (材料表)上制造一个孔, 并在一个盖玻片上放置间隔, 将单元格置于腔内, 并覆盖了另一个盖玻片室。当成像时, 如果Stentor不正确的方向来观察每一个再生阶段的特征特征, 则要牢牢地敲击板材使细胞收缩。观察细胞扩展以确定再生阶段 (完全延长需要大约四十五年代)。如果单元格仍处于错误的方向, 请重复敲击。图 1和图 6所示的图像由立体声变焦显微镜拍摄;然而, 所有的实验细节都可以使用5X 解剖立体显微镜进行。
除了培养和成像Stentor的另一个挑战是更新的跟踪, 特别是确定阶段所需的时间。如果再生细胞是完全地面向与口头普利摩顿的区域清楚地看见, 阶段的证明需要几秒钟。然而, 有时, Stentor细胞是在一个方向, 以防止明确分配的再生阶段, 从而花更多的时间来识别。培养个体再生细胞所需的大量时间可能会延缓所有再生细胞的量化, 从而降低分期的时间精度, 并要求观察者在移动到一个新的方向。因此, 这个实验既是时间又是劳动密集型的。基于这些原因, 很有必要开发自动检测Stentor细胞的方法, 并在视频显微数据中指定再生阶段。这些也将允许更多的重现性实验, 增加样本量, 并消除人类的偏见。
高度敏感的基因组和蛋白质学方法的出现已经开始将单个细胞的研究达到目的。对于这种单细胞分析, Stentor细胞的巨大大小使得它成为概念验证实验的理想测试对象。为了使这些实验成为可能, 培养Stentor是根本的, 因此这里所描述的方法应该在进一步发展更先进的单细胞技术方面发挥作用。

细胞不是简单的酶袋, 而是高度复杂的机器, 其组件被仔细地缩放到正确的尺寸, 并安排在定义良好的位置。单个细胞的形态发生是细胞和发育生物学的关键过程, 但其分子机制未知1、2。虽然一些培养的细胞类似斑点, 单细胞有机体可以有极其复杂的结构, 以纤毛虫3,4所见的复杂皮层模式为例。
也许一个高度结构化细胞的最极端的例子是Stentor 斑, 一个巨大 heterotrichous 纤毛远亲与嗜和草履虫相关。Stentor是1毫米长, 覆盖着超过100条纵向条纹的蓝色颜料交替与行纤毛组织由平行栈的微管丝带, 运行长度的整个细胞。细胞是喇叭状 (图 1), 与 membranellar 带和口腔设备 (OA) 在其前端, 和一个霍尔德法斯特, 将细胞附着在基底上的后端。除了明显的前后极性, 细胞也显示了一个独特的手性模式, 这样, 睫状行之间的间距逐渐增加顺时针方向。这就导致了一个不连续的地方, 最窄的行满足最宽的行, 而这个区域的细胞表面, 称为条纹对比的轨迹, 可以诱导形成第二套前端结构时, 移植到另一个细胞5,使它正式等同于 Spemann 的组织者。因此, 发育生物学的所有关键过程都有类似的Stentor: axiation、模式形成和诱导。在胚胎中, 这些过程是由不同细胞之间的命运差异驱动的, 但在Stentor中, 它们必须由单个细胞内不同区域之间的命运差异驱动。定义Stentor内各区域之间的差异是一个谜。
如果Stentor的任何一部分被切断, 细胞中缺失的部分就可以再生, 在几个小时内产生正常细胞。如果一个细胞被切成两半, 甚至更小的片断, 每个片断重组成一个正常的, 但更小的细胞, 并恢复适当比例之间的细胞部分6,7。即使是微小的碎片 , 1/64 大小的原始细胞, 能够再生成一个小的, 但正常比例的细胞, 然后增长到全尺寸6。Stentor因此提供了一个独特的机会, 研究的机制, 细胞的大小和生长调节使用的外科方法, 通常适用于组织或整个有机体的水平。
Stentor的特性之一, 允许它从广泛的外科手术中再生是它包含一个单一的结节样线粒体 (图 1) 与大约5万拷贝整个基因组8。只要单元格片段包含至少一个 macronuclear 节点, 它就有能力完全再生。Stentor再生能力的另一个属性是它巨大的创伤愈合能力。虽然许多细胞类型能够愈合他们的伤口9, Stentor能够恢复从一个特殊的范围内的物理扰动。Stentor从剧烈扰动中恢复的一个例子, 以及Stentor10中细胞质流动的可视化方法。这些方法允许研究创伤和随后的再生如何影响细胞质的物理状态。
Stentor的巨大体积, 非凡的再生能力, 以及它体现了许多多细胞胚胎 (如组织者, axiation 和模式) 的发展现象, 吸引了许多发展生物学家在上个世纪的转折, 包括托马斯狩猎摩根7年。在50和60的过程中, 显微外科方法显示了一个惊人的阵列的再生和形态发生过程在这个单细胞生物11。然而, Stentor已经发展成为一个分子生物学模型系统, 只是最近。在过去的几年中, Stentor基因组的序列和组装8, 并提出了扰乱基因表达的方法, 利用 rna 干扰的喂养被开发12。
Stentor被发展成一个现代分子生物学模型有机体的原因之一是, 由于其长的细胞周期 (3 到5天), 生长大的文化是困难的。然而, 现代基因组和蛋白质蛋白方法要求的材料比以前少, 而且单个Stentor细胞的体积足够用于这些方法, 即使不诉诸超灵敏的方法来分析单个比Stentor小得多的细胞。协议1节详细介绍了从单个Stentor单元格中建立大型区域性的过程。同样的方法可以用来建立一个大的文化从细胞片段获得通过切割细胞。1节还提供了长期保持健康Stentor文化的指导方针。《议定书》2节提供了通过用玻璃针手工切割细胞来诱导细胞再生的方法。《议定书》3节专门讨论两种诱导特定细胞结构再生的方法 (membranellar 带和口服器械): 用蔗糖或尿素治疗细胞导致这些结构脱落, 其次是其再生。《议定书》4节详细介绍了在很长一段时间内对个体再生细胞进行成像的方法。第4节结束与再生阶段的描述和提示的再生动力学分析。

<table><tbody><tr><td>&lt;em&gt;Stentor coeruleus&lt;/em&gt; live culture</td><td>Carolina Biological Supply</td><td>131598</td><td></td></tr><tr><td>&lt;em&gt;Chlamydomonas&lt;/em&gt; &lt;em&gt;reinhardtii&lt;/em&gt; on agar</td><td>Carolina Biological Supply</td><td>152040</td><td></td></tr><tr><td>Pasteurized springwater, 1-quart bottle</td><td>Carolina Biological Supply</td><td>132458</td><td></td></tr><tr><td>Methyl cellulose, 1500 cP</td><td>Millipore Sigma</td><td>M0387</td><td></td></tr><tr><td>Pyrex glass spot plate</td><td>Fisher Scientific</td><td>13748B</td><td></td></tr><tr><td>Wide-mouth glass jar with screw cap, 60 mL</td><td>Carolina Biological Supply</td><td>715740</td><td></td></tr><tr><td>2-cup glass container with glass lid</td><td>Pyrex</td><td>1095600</td><td></td></tr><tr><td>CultureWell silicone sheet material</td><td>Grace Bio Labs</td><td>664475</td><td></td></tr><tr><td>Kimwipes</td><td>Kimberly Clark</td><td>34155</td><td></td></tr><tr><td>Posi-Click 1.7 mL microcentrifuge tube</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>Cover Glass</td><td>Fisher finest</td><td>12-548-B</td><td></td></tr><tr><td>TAP Growth Media, optimized for &lt;em&gt;Chlamydomonas&lt;/em&gt; culture</td><td>ThermoFisher</td><td>A1379801</td><td></td></tr><tr><td>Silicone spacer, CultureWell Silicone Sheet, 0.25mm Thick/13 cm x 18 cm</td><td>Grace Bio Labs</td><td>664475</td><td></td></tr><tr><td>Petrolatum, Petroleum Jelly, White</td><td>Spectrum</td><td>P1037</td><td></td></tr><tr><td>Borosilicate glass capillary tubes.&lt;br/&gt; OD: 1.2 mm, ID: 0.9 mm, length: 10 cm.</td><td>Sutter Instrument</td><td>B120-90-10</td><td></td></tr><tr><td>Needle puller P-87</td><td>Sutter Instrument</td><td></td><td></td></tr><tr><td>Stemi 2000 stereo microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Axiozoom V16, stereo zoom microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

methods for the study of regeneration in stentor

细胞需要能够再生他们的部分, 以恢复从外部扰动。单细胞纤毛Stentor 斑是研究创面愈合和后续细胞再生的优良模型生物。Stentor基因组最近获得了, 以及现代分子生物学方法, 如 rna 干扰。这些工具使得在分子水平上研究单细胞再生成为可能。该协议的第一部分包括从单个细胞或细胞片段中建立Stentor细胞文化, 以及维护Stentor文化的一般准则。大量培养Stentor允许使用有价值的工具, 如生物化学、测序和质谱。该协议的后续部分涵盖了诱导Stentor再生的不同方法。用玻璃针手工切割细胞可以研究大细胞部分的再生, 而用蔗糖或尿素处理细胞则允许研究位于细胞前端的特定结构的再生。给出了一种成像个体再生细胞的方法, 以及分期和分析再生动力学的一个专栏。再生的整个过程在三阶段被划分。通过对细胞的发育过程的动态可视化, 证明了再生时间的异质性。

Stentor的文化已可靠地建立和维护从个别细胞或细胞片段使用协议1节。视频 1显示了一个大型健康文化的典型例子。
在Stentor中用蔗糖处理方法在步骤3.1 中概述了再生再生的时间过程, 结合了4节中讨论的成像和分析方法 (图 7)。这个情节表明, 在细胞的人口所采取的时间内, 有一个小时长的传播到任何特定的阶段。这种类型的分析允许研究再生细胞的新生过程中的时间异质性。
以下是在许多蔗糖处理后迄今观察到的再生时间的摘要 (图 4和图 6)。阶段1是当Stentor细胞看起来像没有任何 membranellar 带的泪珠 (这个阶段开始后立即蔗糖冲洗)。这个阶段持续 3-6 h. 阶段2是当一个 membranellar 带出现并且增长 (3-6 h 在蔗糖处理以后)。这个阶段持续 3-4 h. 阶段3是当口头普利摩顿出现在 membranellar 带的后端 (6-8 h 在蔗糖治疗以后), 并且两个结构被移动往细胞的前端。这个阶段持续 1-2 小时。当 membranellar 带和口腔器械到达细胞的前端时, 这表明再生的完成。细胞采用了典型的Stentor喇叭状。在蔗糖治疗后, 细胞完全再生 8-9 小时。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57759/57759fig1.jpg"> 图1。Stentor的快照.membranellar 带和口腔器械显示在细胞的前端。霍尔德法斯特在细胞的后端。Stentor线粒体是结节样。一个营养良好的Stentor有绿色的食物液泡, 其中大部分是衣藻。刻度条为0.5 毫米.<a href="/files/ftp_upload/57759/57759fig1large.jpg" target="_blank">请单击此处查看此图的较大版本</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57759/57759fig2.jpg"> 图2。Stentor切割安装.细胞切割是通过手工操作玻璃针, 而看着细胞使用商用解剖立体显微镜。纸巾的目的是提供白色的背景, 以更好地看到细胞。<a href="/files/ftp_upload/57759/57759fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57759/57759fig3.jpg"> 图3。视频3的快照演示如何用玻璃针切割Stentor .(a) 在针头接触它之前立即Stentor 。(B) 在针和玻璃滑梯之间轻轻挤压的Stentor 。(C) 对针头的作用力作出反应的收缩Stentor 。(D) 用针边轻轻按压细胞的Stentor 。(E) Stentor现在切成两个, 但没有分开。(F) 两个Stentor碎片。刻度条为0.25 毫米.<a href="/files/ftp_upload/57759/57759fig3large.jpg" target="_blank">请单击此处查看此图的较大版本</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57759/57759fig4v2.jpg"> 图4。《议定书》3和4节的示意图可视化.这是一个说明性的协议, 以执行蔗糖或尿素处理和观察细胞再生。在底部面板中指示的时间从洗涤1的开始测量。<a href="/files/ftp_upload/57759/57759fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57759/57759fig5.jpg"> 图5。用直立显微镜对再生进行成像和/或直接观察的设置.在每4µL 液滴从盖玻片, 有一个细胞正在进行再生。水在水井的玻璃点板限制蒸发的水滴。此设置允许并行地重新生成多个单元格。<a href="/files/ftp_upload/57759/57759fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57759/57759fig6.jpg"> 图6。Stentor在 membranellar 带的各个阶段和口腔器官再生的快照.(A) 阶段1的特点是泪样细胞形状和没有 membranellar 带。(B) 阶段2的特点是 membranellar 带的外观, 一种纤毛的结构, 连续跳动。Membranellar 带在面板 B D 中标有白色虚线. (C 和 D) 阶段3的特点是口腔普利摩顿的外观 (用箭头标记)。口腔普利摩顿看起来像是 membranellar 带后端的内陷。口腔普利摩顿将向细胞前移动, 成为口腔器械。(E) 当细胞采用Stentor喇叭状形状的特征时, 再生就完成了, 而口腔器械 (标有箭头) 在细胞的前端扩大了。刻度条为0.25 毫米.<a href="/files/ftp_upload/57759/57759fig6large.jpg" target="_blank">请单击此处查看此图的较大版本</a>。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57759/57759fig7.jpg"> 图7。堆积盒图, 显示了蔗糖处理后所有再生阶段细胞的比例随着时间的推移而变化。该图显示了再生细胞的新生期时间内的异质性。"注册结束" 表示再生的完成。单元格数:28。<a href="/files/ftp_upload/57759/57759fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Movie 1" src="/files/ftp_upload/57759/57759video1.jpg"> 视频1。一个健康的Stentor文化的例子.通常, 如果文化是这集中的, 分裂文化被推荐。<a href="/files/ftp_upload/57759/Video1.mp4" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Movie 2" src="/files/ftp_upload/57759/57759video2.jpg"> 视频2。放慢Stentor与纤维素.<a href="/files/ftp_upload/57759/Video2.mov" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Movie 3" src="/files/ftp_upload/57759/57759video3.jpg"> 视频3。切割Stentor.在立体解剖显微镜下, 通过细胞按压玻璃针, 可以将单个细胞手动切割成多个片断。<a href="/files/ftp_upload/57759/Video3.mov" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>文化问题</td>
			<td>可能的补救办法</td>
			<td>可能的预防</td>
		</tr>
<tr>
<td>文化被轮虫或其他大有机体淹没。</td>
			<td>删除尽可能多的Stentor到一个新的文化菜。然后洗三次细胞。</td>
			<td>在收到Stentor时, 即使是从商业供应商那里购买, 也要彻底清洗。</td>
		</tr>
<tr>
<td>文化被真菌或其他小有机体淹没。</td>
			<td>找出一个角落里有最少的Stentor.在该区域, 尽可能多地删除媒体, 而不删除许多Stentor并添加新的 PSW。轻轻地混合, 但彻底 distrubute 入侵的有机体。跳过下一个喂食, Stentor会吃掉它们。</td>
			<td>定期观察文化, 通过交换新鲜 PSW 的培养基去除小杂质。</td>
		</tr>
<tr>
<td>
Stentor在彼此的顶端。</td>
			<td>分裂的文化, 使浓度小于20细胞/毫升。</td>
			<td>更经常地分裂文化。</td>
		</tr>
<tr>
<td>
Stentor细胞正在交配。</td>
			<td>每4天喂养一次, 而不是每5天进食一次。</td>
			<td></td>
		</tr>
<tr>
<td>文化有许多黑暗的废物材料。</td>
			<td>去除尽可能多的暗物质, Stentor废料, 尽可能不移除细胞。如果Stentor附着在暗物质上, 吸管向上和向下释放Stentor细胞从他们的废料, 然后删除介质与废料, 而不删除细胞。或者, 移除这些Stentor并相应地减少饲料。</td>
			<td>饲料Stentor 1 毫升的食物每100毫升的文化。</td>
		</tr>
<tr>
<td>不健康的前瞻 (空泡/臃肿或圆形) Stentor.
</td>
			<td>删除尽可能多的媒体, 而不删除许多Stentor单元格并添加新的 PSW。</td>
			<td>定期交换媒体。</td>
		</tr>
</tbody></table>表1。培养Stentor的疑难解答指南。

Watch this Scientific Journal Video about Methods for the Study of Regeneration in Stentor at JoVE.com

Methods for the Study of Regeneration in Stentor

Stentor再生的研究方法

巨大的纤毛, Stentor 斑, 是一个优秀的系统来研究再生和伤口愈合。我们提出了从单细胞或细胞片段中建立Stentor细胞培养的程序, 通过切割细胞诱导再生, 化学诱导 membranellar 带的再生和口腔器具, 影像学和细胞分析再生。

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

methods-for-the-study-of-regeneration-in-stentor

Research

JoVE Journal

Developmental Biology

Methods for the Study of Regeneration in Stentor

11.7K 次观看.  University of California San Francisco. 巨大的纤毛, Stentor 斑, 是一个优秀的系统来研究再生和伤口愈合。我们提出了从单细胞或细胞片段中建立Stentor细胞培养的程序, 通过切割细胞诱导再生, 化学诱导 membranellar 带的再生和口腔器具, 影像学和细胞分析再生。

视频: Methods for the Study of Regeneration in Stentor

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

使用提顿体系的研究斑马鱼再生的分子机制

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

科研

发育生物学

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

从斯塔利特海葵的反口Physa完全诱导再生息肉<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

颈动脉支架植入再狭窄的研究的小鼠模型

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

医学

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

调查器官脊髓联合培养物中生长多电极阵列功能再生

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

神经科学

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

涡固定，局部照射，组织移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

生物学

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

可视化细胞质流动过程中单细胞伤口愈合<em&gt;蓝斑宏声</em

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

对于侧线再生成年斑马鱼的含量

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

A Standardized Murine Model of Extracorporeal Shockwave Therapy Induced Soft Tissue Regeneration

Shockwave therapy (SWT) shows promising regenerative effects in several different tissues. However, the underlying molecular mechanisms are poorly understood. Angiogenesis, a process of new blood vessel formation is a leading driver of regeneration in softer tissues as well as a recently discovered effect of SWT. How the mechanical stimulus of SWT induces angiogenesis and regeneration and which pathways are involved is not fully understood. To further improve the clinical use of SWT and gain valuable information about how mechanical stimulation can affect tissue and tissue regeneration, a standardized model of SWT is needed. We, hereby, describe a standardized, easy to implement murine model of shockwave therapy induced regeneration, utilizing the hind-limb ischemia model.

<meta charset="utf8"></head><body>体外冲击波疗法诱导软组织再生的标准化小鼠模型

This article describes a standardized murine model of tissue regeneration via shockwave treatment.

体外冲击波疗法诱导软组织再生的标准化小鼠模型

Shockwave therapy (SWT) shows promising regenerative effects in several different tissues. However, the underlying molecular mechanisms are poorly ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

DiI灌注作为血管可视化的方法<em&gt;墨西哥恶性肿瘤</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Stentor再生的研究方法

摘要

探索更多视频

使用提顿体系的研究斑马鱼再生的分子机制

从斯塔利特海葵的反口Physa完全诱导再生息肉

颈动脉支架植入再狭窄的研究的小鼠模型

调查器官脊髓联合培养物中生长多电极阵列功能再生

涡固定，局部照射，组织移植

可视化细胞质流动过程中单细胞伤口愈合

对于侧线再生成年斑马鱼的含量

体外冲击波疗法诱导软组织再生的标准化小鼠模型

体外冲击波疗法诱导软组织再生的标准化小鼠模型

DiI灌注作为血管可视化的方法