我们感谢日本东北大学的 Dr. 三, 他们分享了稳定表达 mCherry 组蛋白 H2B 和 GFP-α-蛋白的 HeLa 细胞。这项工作得到了一笔对 DV (R00CA178188) 和西北大学启动资金的支持。

1. 双胸苷阻滞和释放: 试剂制剂
<ol><li>使500毫升 Dulbecco 的改良鹰培养基 (DMEM) 培养基补充 10% FBS, 青霉素, 和链霉素。</li>
	<li>使100毫米的胸苷在无菌水和储存在等分在-80 ° c。</li>
</ol>2. 有丝分裂进展的固定细胞成像协议 (图 1A)
<ol><li>在1天, 种子〜 2 x 105 HeLa 细胞到一个6井板的井盖滑动 (用70% 乙醇和紫外线辐射灭菌) 和2毫升的 DMEM 培养基。在37° c 和 5% CO2的湿润培养箱中生长细胞24小时。</li>
	<li>1st胸苷块: 在2天, 彻底混合的要求体积的胸苷在新鲜的 DMEM 媒体 (100 毫米的股票, 2 毫米最终浓度)。</li>
	<li>在6井板的每一个井中加入2毫升的胸苷含培养基, 并孵育18小时。</li>
	<li>3天, 吸入培养基, 用2毫升 1x PBS 冲洗两次细胞, 再用新鲜 prewarmed DMEM;在新鲜的 prewarmed DMEM 培养基中培养细胞, 9 h 释放细胞。</li>
	<li>2nd胸苷块: 再次添加2毫升的胸苷含培养基 (2 mM 最终浓度) 的细胞在每个井的 6-井板。</li>
	<li>再孵育18小时</li>
	<li>4天, 吸入培养基, 用2毫升 1x PBS 冲洗细胞两次, 再用新鲜 prewarmed DMEM。</li>
	<li>将 siRNAs (控制和 Cdt1) 和转染试剂分别用于血清自由生长培养基10分钟, 将它们混合在一起, 在 RT 中孵育20分钟。在每一个转染 siRNA 的最终浓度为 100 nM。将反应混合物添加到已被胸苷洗掉的细胞中。</li>
	<li>孵育细胞在37° c 为 9-10 h 释放从堵塞和固定在盖子滑动的细胞与4% 煤灰为 20 min 在 RT。 注意: 粉煤灰是有毒的, 穿戴适当的保护。</li>
	<li>
免疫细胞后, permeabilizing 与 0.5% (v/v) 洗涤剂为10分钟在 RT 和洗涤细胞两次与 1x PBS 5 分钟每。
	<ol>
<li>在 RT 1x PBS 中用 1% BSA (w/v) 在1小时内阻断细胞, 然后用50µL 的原代抗体治疗细胞 (小鼠抗α-蛋白抗体稀释 1:1, 000, 兔 anti-Zwint1 抗体稀释 1:400, 和小鼠抗磷ɣ H2AX 稀释 1:300) 在 1% (w/v)BSA 在 1x PBS 中为1小时, 在 37 oC。</li>
		<li>冲洗后的细胞与 1x pbs 三次, 治疗细胞50µL 的第二抗体为每盖玻璃 (Alexa 488 和罗丹明红色在1:250 稀释 1% (瓦特/v) BSA 在 1x PBS) 为 1 h 在 RT。</li>
		<li>在用 1x pbs 清洗两次细胞后, 每次5分钟, 用 DAPI (0.1µg/毫升在 1x pbs) 治疗细胞, 在 RT 5 分钟。</li>
		<li>在用 1x PBS 清洗两次电池5分钟后, 将盖板滑向适当的安装介质, 在清晰的显微镜下滑动。</li>
	</ol>
</li>
	<li>图像的细胞为 immunostained 蛋白60X 或 100X 1.4 NA 计划-物镜 DIC 油浸没目标安装在一个倒置的高分辨率共焦显微镜配备了适当的相机, 必要的图像质量所需的。</li>
	<li>使用适用于显微镜的软件, 在室温下获得0.2 µm 厚度的 z 栈的图像。</li>
</ol>3. 有丝分裂进展的活细胞成像协议 (图 3A)
<ol><li>在1天, 种子大约 0.5-1 x 105 HeLa 细胞稳定表达 mCherry-H2B 和 GFP-α-蛋白 (根据细胞类型和他们表达的蛋白质略有变化) 到35毫米玻璃底菜与1.5 毫升的 DMEM 培养基和增长他们在湿化孵化器为24小时, 在 37 oC 和 5% CO2。</li>
	<li>1st胸苷块: 在2天, 在盘中的细胞中加入1.5 毫升的胸苷含有的培养基 (100 毫米胸苷, 2 毫米最终浓度,)。</li>
	<li>孵育18小时。</li>
	<li>3天, 吸入含有胸苷的培养基, 三次冲洗细胞, 用2毫升 1x PBS, 一次用新鲜 prewarmed DMEM。</li>
	<li>siRNAs (控制和 Hec1) 和血清自由生长培养基的转染试剂分别为10分钟, 将它们混合在一起, 孵育20分钟。在每一个转染 siRNA 的最终浓度为 100 nM。将反应混合物添加到已被胸苷洗掉的细胞中。</li>
	<li>在1.5 毫升的新鲜 prewarmed DMEM 培养基中培养8小时的细胞, 从块中释放细胞。</li>
	<li>2nd胸苷块: 在盘中的细胞中加入1.5 毫升的胸苷含培养基 (2 mM 最终浓度)。</li>
	<li>再孵育18小时</li>
	<li>4天, 吸入培养基, 三次冲洗细胞, 2 毫升 1x PBS, 一次与新鲜 prewarmed DMEM。然后在新鲜的 prewarmed 莱博维茨的 (L-15) 培养基中生长细胞, 并辅以 10% FBS 和20毫米 HEPES, 在 pH 7.0 为 8 h 释放细胞从块。</li>
	<li>将碟放在高分辨率共聚焦显微镜的温度控制箱中, 在开始成像前已经开启了至少30分钟, 以稳定舞台和实验温度。</li>
	<li>聚焦在明亮的领域与60x 目标, 直到细胞是可见的, 然后手动筛选的舞台上的选择区域。</li>
	<li>建立了光和激光功率/曝光、图像采集参数, 以及实验时间的使用显微镜图像采集软件的选择。使用荧光蛋白 (488 激发; 385 nm 发射) 和 mCherry (561 nm 激发和 385 nm 发射) 的过滤器来获取图像。</li>
	<li>通过分别获取脉冲透射光和荧光图像, 进行时间间隔试验, 每10分钟可达到16小时。</li>
	<li>获取图像作为十二1.0 µm 分离的 z 平面在9小时后, 从双胸苷块使用适当的显微镜的软件释放。</li>
	<li>分析图像跟踪个别细胞有丝分裂进展, 最后组装相应的电影与源软件斐济/ImageJ。</li>
</ol>

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作者声明他们没有竞争的财政利益。

双胸苷同步的最关键的优点是它在短时间内提供了一个增加的有丝分裂细胞的样本大小, 其中许多细胞进入有丝分裂一致, 从而也使分析染色体对齐, 两极纺锤体形成, 染色体分离, 效率更高。
许多调控蛋白复合物和信号通路致力于确保正常进展通过有丝分裂和解除管制这一过程可能导致肿瘤。动态细胞成像的 HeLa 细胞稳定表达 mCherry-H2B 和 GFP-蛋白和释放从双胸苷块显示, 大多数控制细胞分离他们的染色体在后期在大约60分钟后, 纳布 (图 3B)。另一方面, 与 Hec1 功能扰动的 HeLa 细胞的活细胞成像显示, 大多数细胞在有丝分裂阶段停留在长时间内, 染色体失调, 其次是后期发作或凋亡 (图 3C)。
过量的胸苷抑制了 S 期的 DNA 合成, 从而阻断了 G1/S 期11的细胞。虽然双胸苷块同步是研究细胞周期和有丝分裂过程中最常用的方法, 但需要对该方法的一些局限性进行批判。重要的是, 需要确保化学物质被彻底冲洗 (7-8 小时), 这样效果是完全可逆的, 细胞可以正常地通过细胞周期的其余部分进行。细胞可能不会进入第二个循环, 导致有丝分裂不正确的进展。同步细胞的比例可能不够高, 单胸苷块和双胸苷块可能是必要的, 以增加同步细胞的数量, 特别是如果你有兴趣研究一个特定阶段的有丝分裂。胸苷治疗的期间必须优选由于不同的敏感性在细胞类型之间根据世代时间。例如, 仓鼠细胞有16小时的世代时间, 并与胸苷治疗 11 h 最佳同步17。在这项研究中, 我们治疗 16-18 h 与胸苷的 HeLa 细胞。此外, 过量的胸苷不仅能防止 DNA 合成, 而且还可以杀死细胞的重要部分11,12,18 , 需要密切注意以监视此损失。本研究中使用的胸苷储存液应在无菌蒸馏水中制备, 并应在细胞培养基中彻底混合, 以避免其沉淀后加入 siRNA 转染细胞培养基, 并确保其均匀在培养细胞周围分布。
为了研究靶有丝分裂蛋白在有丝分裂过程中的作用, 理想的细胞将被 siRNA 击倒在 1st胸苷块期间或在 1 st 的发行期间的兴趣蛋白质水平.胸苷块, 取决于需要的时间获得有效的蛋白击倒19,20,21。另一方面, 为了研究在有丝分裂过程中 Cdt1 等多功能蛋白的作用, 在从 2nd胸苷块的释放过程中, 必须对 siCdt1 细胞进行治疗, 以获得 G2/M 的特定损耗。Cdt1 是 DNA 复制起源许可的关键角色15。为了了解其在有丝分裂过程中的特殊作用而不影响其在许可中的作用, Cdt1 需要特别在 G2/M 阶段被耗尽, 这可以通过双胸苷同步来有效地实现, 从而避免了需要诉诸其他难以执行的有丝分裂特定的摄动方法。由于 dna 复制起源的许可中断, 导致 dna 损伤反应的 Cdt1 在异步培养中的损耗, 这反过来又混淆了对这种蛋白质在有丝分裂过程中可能的功能的分析。因此, 双胸苷同步技术提供了一种独特的实验方法, 以产生细胞, 经历了正常的 G1 和 S 相, 但缺乏 Cdt1 功能在 G2 和 M 阶段。我们的同步协议的特殊意义在于它抑制了特定阶段的多功能蛋白 (G2/M), 以研究它们在有丝分裂过程中的新功能。因此, 这种方法不仅有助于研究在不同细胞周期阶段具有多种功能的蛋白质的作用, 而且还能用于染色体排列、国民党附着体和染色体分离过程中的任何有丝分裂蛋白。
对于活细胞成像, 从双胸苷块释放后, 细胞应在预热莱博维茨的 (L-15) 培养基中进行孵育, 并辅以 10-20% FBS 直到影像开始。细胞进入有丝分裂在可变时间点后, 从胸苷块的释放取决于细胞类型。图像捕捉或固定的开始时间需要根据细胞类型进行优化。对于活细胞成像, 利用传统的共焦显微镜可能导致漂白在长期成像。另一方面, 高分辨率共焦显微镜给出了一个清晰的高分辨率图像, 其漂白效应的程度最小。
该协议的关键步骤是胸苷治疗的持续时间, 胸苷的正确冲洗, siRNA 转染的时间, 以及成像的开始时间。

在细胞周期中, 细胞经历了一系列高度调控和世俗控制的事件, 以精确复制他们的基因组和增殖。在哺乳动物中, 细胞周期由相间和 M 相组成。在相间, 由三个阶段-G1, S 和 G2, 细胞复制它的基因组并且经历成长是必要的为正常细胞周期进展1,2。在 M 阶段, 由有丝分裂 (前期, 中期, 中期, 后期, 和末期) 和胞, 父母细胞产生两个基因上相同的子细胞。在有丝分裂中, 姊妹染色的复制基因组是凝聚 (前期) 和捕获在他们的 kinetochores 通过微管组装的有丝分裂纺锤 (中期), 驱动他们的对准在中期板块 (中期), 其次是他们的当姐妹染色被分裂对和被运输对相反纺锤杆 (后期) 时, 相等的离析。两个子细胞的物理分离由肌动蛋白为基础的收缩环 (末期和胞) 的活动。粒是一种专门的蛋白质结构, 在染色的丝区域装配, 作为主轴微管的附着点。它的主要功能是驱动染色体捕获, 对齐, 并帮助纠正不正确的主轴微管附着, 同时调解主轴组件检查点, 以维护染色体分离的保真度3,4。
细胞同步技术是了解细胞周期进展过程中的分子和结构事件的理想工具。这一方法已被用来丰富细胞群体在特定阶段的各种类型的分析, 包括基因表达谱, 细胞生化过程分析, 和检测蛋白质的亚单位定位。同步哺乳动物细胞不仅可以用于单个基因产品的研究, 也可用于涉及整个基因组分析的方法, 包括基因表达的微阵列分析5, miRNA 表达式模式6,平移调节7, 蛋白质组蛋白分析的修改8。同步也可以用来研究基因表达或蛋白质敲掉或敲出, 或化学物质对细胞周期进展的影响。
细胞可以在细胞周期的不同阶段同步。物理和化学方法都被广泛用于细胞同步。细胞同步的最重要的标准是同步应该是 noncytotoxic 和可逆的。由于药物对细胞同步的潜在不良影响, 化学依赖性方法对研究关键细胞周期事件是有利的。例如, 羟基、amphidicolin、含羞草和洛伐他汀可用于 G1/S 阶段的细胞同步, 但由于它们对它们抑制的生物化学通路的影响, 它们激活了细胞周期检查点机制, 并杀死了一个重要单元格的分数9,10。另一方面, 通过添加胸苷对生长介质的反馈抑制 DNA 复制, 称为 "胸苷阻滞", 可以在某些点11,12,13中逮捕细胞周期。在 G2/M 阶段也可以通过处理 nocodazole 和 RO-3306914来同步单元格。Nocodazole, 防止微管组装, 具有较高的细胞毒性。此外, nocodazole 细胞可以通过有丝分裂的滑移回到相早熟。双胸苷阻滞细胞在 G1/S 阶段和释放后的块, 细胞被发现进行同步通过 G2 和有丝分裂。在高分辨率的共聚焦显微镜下, 通过细胞固定或活体成像, 可以观察胸苷块释放的细胞周期的正常进展。当细胞进入并通过双胸苷块释放后有丝分裂时, 可以特别研究有丝分裂蛋白的摄动效应。Cdt1 是一种多功能的蛋白质, 在 G1 阶段参与 DNA 复制起源许可, 并且在有丝分裂过程中也需要粒微管附着体 (15。为了研究 Cdt1 在有丝分裂过程中的作用, 我们需要采取一种方法, 在 G1 阶段避免其损耗对复制许可的影响, 同时在 G2/M 阶段只会影响其损耗。在这里, 我们提出了详细的协议的基础上的双胸苷块, 以研究的有丝分裂作用的蛋白质执行多个功能在不同阶段的细胞周期的固定和活细胞成像。

<table>
	<tbody>
		<tr>
			<td>DMEM (1X)</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>DPBS (1X)</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Leibovitz&#8217;s (1X) L-15 medium</td>
			<td>Life Technologies</td>
			<td>21083-027</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Serum reduced medium (Opti-MEM)</td>
			<td>Life Technologies</td>
			<td>319-85-070</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin</td>
			<td>Life Technologies</td>
			<td>15070-063 (Pen Strep)</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Dharmafect2</td>
			<td>GE Dharmacon</td>
			<td>T-2002-02</td>
			<td>Store at 4 degree C</td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>MP Biomedicals LLC</td>
			<td>103056</td>
			<td>Dissolved in sterile distiled water</td>
		</tr>
		<tr>
			<td>Cdt1 siRNA</td>
			<td>Life Technologies</td>
			<td></td>
			<td>Ref 10</td>
		</tr>
		<tr>
			<td>Hec1 siRNA</td>
			<td>Life Technologies</td>
			<td></td>
			<td>Ref 13</td>
		</tr>
		<tr>
			<td>HeLa cells expressing GFP-H2B</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells expressing GFP-&#945;-tubulin and mCherry H2B</td>
			<td>Generous gift from Dr. Kozo Tanaka of Tohoku university, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>F8775</td>
			<td>Toxic, needs caution</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>D9542</td>
			<td>Toxic, needs caution</td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-tubulin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Sc32293</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Rabbit ant-Zwint1</td>
			<td>Bethyl</td>
			<td>A300-781A</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Mouse anti-phospho-&#947;H2AX (Ser139)</td>
			<td>Upstate Biotechnology</td>
			<td>05-626, clone JBW301</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Alexa 488</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>Rodamine Red-X</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>BioLite 6 well multidish</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td></td>
		</tr>
		<tr>
			<td>35MM Glass bottom dish</td>
			<td>MatTek Corporation</td>
			<td>P35GCOL-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse TiE inverted microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disc for confocal</td>
			<td>Yokagawa</td>
			<td>CSU-X1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra 888 EM-CCD Camera</td>
			<td>Andor</td>
			<td>iXon Ultra EMCCD</td>
			<td></td>
		</tr>
		<tr>
			<td>4 wave length laser</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation System for Microscopes</td>
			<td>Tokai Hit</td>
			<td>TIZB</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-elements software</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

combining mitotic cell synchronization and high resolution confocal microscopy to study the role of multifunctional cell cycle proteins during mitosis

研究细胞周期的各种调控事件, 以相依赖的方式提供了一个明确的理解细胞生长和分裂。在细胞周期的特定阶段, 细胞数量的同步被发现在这种实验中非常有用。与毒性相对较小的化学物质进行治疗时, 细胞的同步化可以在药物抑制剂的使用上获得好处, 用于研究相应的细胞周期事件, 并获取特定的有丝分裂阶段的丰富度。在这里, 我们描述了在细胞周期的不同阶段同步人类细胞的协议, 包括 S 相和 M 相的双胸苷块和释放程序, 以研究有丝分裂蛋白在染色体排列中的功能和隔离。本协议对于研究具有已建立的相间功能的多功能蛋白的有丝分裂作用非常有用。在我们的例子中, Cdt1 的有丝分裂作用, 一个关键的蛋白质复制原产地许可在 G1 阶段, 可以有效地研究, 只有当 G2/M-specific Cdt1 可以耗尽。我们用双胸苷同步描述了 G2/M-specific Cdt1 损耗的详细协议。我们还解释了细胞固定的协议, 和活细胞成像使用高分辨率共聚焦显微镜后, 胸苷释放。该方法也有助于分析有丝分裂蛋白在生理和扰动条件下的功能, 如 Hec1, Ndc80 复合物的一个组成部分, 因为它使一个获得大样本大小的有丝分裂细胞的固定和活细胞分析, 我们在这里展示。

双胸苷块释放后细胞有丝分裂进展及微管稳定性研究 Cdt1 在 G1 阶段参与了 DNA 复制起源的许可。它在 S 相时被降解, 但在 G2/M 相中重新积累。为了研究其在有丝分裂中的作用, 内源性 Cdt1 需要用最合适的细胞同步技术, 即双胸苷块15, 在 G/M 相的特定阶段被耗尽。细胞在 2nd胸苷块释放后立即转染 siRNAs, 当细胞处于 S 阶段, 而在 G1 中复制源的许可也需要 Cdt1 功能的时候, 它早就完成了。用印迹 (图 1B) 验证了 9 h siCdt1 转染和双胸苷块释放后 Cdt1 的损耗。从双胸苷块释放后9小时的固定细胞显示, 大多数细胞处于中期阶段的控制和 Cdt1 siRNA 转染细胞。但从双胸苷块释放后10小时的固定细胞显示, 大多数 Cdt1 siRNA 治疗的细胞仍在晚期中期阶段被捕, 而控制细胞进入后期, 并正常分离他们的染色体 (图1C).为了了解 Cdt1 损耗对粒微管 (国民党) 稳定性的影响, 在从双胸苷块释放后的9小时内固定细胞, 然后进行冷处理, 只保留稳定的粒微管 (kMTs)。与对照组相比, Cdt1-depleted 细胞在冷处理后的 kMTs 相对较弱 (图 1D)。因此, 利用这种方法, 即使不使用活细胞成像, 也可以有效地研究有丝分裂蛋白 (s) 的功能扰动后正常有丝分裂细胞的进展。
图 2显示了在使用我们的协议 (在控制单元中也观察到) 细胞在 G2/M 阶段耗尽 Cdt1 时, DNA 复制源的许可不会受到扰动。在 G2/M 期内, Cdt1 细胞衰竭并没有导致磷酸化ɣH2AX 的积累, 这是 DNA 损伤的标志, 在随后的 G2 阶段;而 siRNA 转染的异步文化, 以耗尽 Cdt1 诱导积累的 phospho-ɣH2AX-positive 病灶, 可能是由于 dna 损伤不当导致 dna 复制许可。
活体细胞成像技术研究双胸苷块释放后细胞有丝分裂进展 在核包络破裂 (纳布) 后, 正常细胞 (在缺乏功能性有丝分裂蛋白扰动的情况下) 进入有丝分裂并进行后期起始, 在大约30-60 分钟内退出有丝分裂 (图 3B)。但 Hec1 的 rna 介导的击倒, 一个关键的粒蛋白, 是强大的国民党依恋形成所需的, 被发现延迟正常有丝分裂进程。在这种情况下, 大多数细胞进入有丝分裂后的纳布, 但不经历后期发作或退出有丝分裂, 即使在几个小时的延迟有丝分裂 (图 3C)。同样, 在有丝分裂过程中, rna 介导的本地化对 kinetochores 或有功能的任何其他蛋白质的影响可以通过使用双胸苷块释放后的活细胞成像来有效观察。除了研究有丝分裂的进展, 延迟和逮捕的性质, 其他一些关键的有丝分裂现象, 可以用这种方法来检测, 包括染色体对齐, 有丝分裂纺锤体的形成, 和定位的激活和沉默主轴总成检查点。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56513/56513fig1.jpg"> 图 1: 双胸苷同步后固定细胞有丝分裂进展及粒微管的稳定性分析.(a) 细胞同步、固定和免疫荧光的示意性表达。(B) 在从第二胸苷块释放出 9 h 后, Cdt1 被击倒的西方印迹。(C) 免疫荧光显微显示有丝分裂细胞固定9和10小时后释放从双胸苷块和治疗与控制 (顶) 或 Cdt1 siRNA (底部)。α-蛋白染色是在红色, DNA 是在绿色的细胞表达 GFP-组蛋白 H2B。缩放条 = 10 µm. 中期和中期细胞分别用黄色和白色箭头表示。(D) HeLa 细胞治疗后与控制 siRNA (顶部面板) 和后 Cdt1 耗尽 (底部面板) 9 小时后释放从双胸苷块后, 治疗冰冷莱博维茨的 (L-15) 培养基和固定。细胞是免疫染色的主轴 MT (绿色) 和粒标记, Zwint1 (红色) 和 DAPI 的 DNA 在黑白。缩放栏 = 5 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56513/56513fig1large.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/56513/56513fig2.jpg"> 图 2: G2/M 阶段期间的 Cdt1-depletion 不扰乱复制授权.使用控制荧光 (顶部面板) 或cdt1 siRNAs (中面板) 或双胸苷同步 hela 细胞治疗的异步 hela 细胞, 在2和胸苷洗出过程中用cdt1 siRNA, 然后在10小时内固定.释放后 (底部面板) 染色的 DAPI (伪色绿色) 和 anti-phospho-ɣH2AX 抗体 (红色)。缩放栏 = 10 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56513/56513fig2large.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/56513/56513fig3.jpg"> 图 3: 在同步细胞中通过活细胞成像分析有丝分裂进展.(a) 单元同步和活细胞成像的示意图表示。mCherry-组蛋白 H2B 和 GFP-α-蛋白稳定表达的 HeLa 细胞从双胸苷块释放8小时, 让他们进入 G2/M 阶段从 S 阶段逮捕。活体成像是使用高分辨率共聚焦显微镜开始8小时后, 释放的细胞从胸苷块和持续16小时从该点。这里显示的静止图像从视频捕获每10分钟的控制细胞, 而不扰动任何有丝分裂蛋白 (B) 或细胞耗尽的 Hec1 亚基的 Ndc80 复合物 (C)。缩放栏 = 10 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56513/56513fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis at JoVE

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

我们提出一个协议的双胸苷同步 HeLa 细胞后, 分析使用高分辨率共聚焦显微镜。这种方法是获得大量的细胞, 同步进行从 S 期到有丝分裂的关键, 使研究的有丝分裂作用的功能蛋白, 也具有相间的作用。

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

combining-mitotic-cell-synchronization-high-resolution-confocal

Nanyang Technological University

Research

JoVE Journal

Biology

14.2K 次观看.  Northwestern University. 我们提出一个协议的双胸苷同步 HeLa 细胞后, 分析使用高分辨率共聚焦显微镜。这种方法是获得大量的细胞, 同步进行从 S 期到有丝分裂的关键, 使研究的有丝分裂作用的功能蛋白, 也具有相间的作用。

视频: Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

科研

生物学

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.

结合母细胞和波动测试磁分选，以分析基因组不稳定性的过程中有丝分裂细胞衰老<em&gt;酿酒酵母</em

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

同步<em&gt;新月柄杆菌</em&gt;对细菌细胞周期的研究

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

无细胞化验，研究染色质解聚在有丝分裂的结束

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

活体细胞荧光显微镜观察微生物细胞生长过程中的基本过程

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

基于显微镜的测定，用于使用SNARE运输研究和分析回收内体

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...