Name: long-term live-cell imaging to assess cell fate in response to paclitaxel
Uploaded: 2018-05-14T13:00:00.000+00:00
Duration: 8 min 29 s
Description: Watch this Scientific Journal Video about Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel at JoVE.com

AFB、微型飞行器和 NJG 希望感谢瑞安昆顿对《 TDRFP-NLS 》的手稿和艾利安 Salic 的评论。AFB 和微型飞行器由研究院生物分子药理训练补助金5T32GM008541 资助。NJG 是沙米姆·卡齐和阿什拉夫 Dahod 乳腺癌研究实验室的成员, 并得到 NIH 赠款 GM117150 和 CA-154531、凯琳 Grunebaum 基金会、史密斯家族奖计划、塞尔学者计划和黑色素瘤研究的支持。联盟。

本协议的重点是使用非转化和染色体稳定的视网膜色素上皮 (RPE-1) 细胞线的活细胞成像实验。然而, 只要细胞培养条件根据需要进行调整, 这个协议就可以适应任何黏附细胞线。所有程序必须遵守机构生物安全和道德准则和规章。
1. 为活细胞成像准备细胞
<ol><li>
使用新鲜解冻和早期通道 RPE-1 细胞表达人组蛋白 H2B 融合到荧光蛋白 (例如H2B-GFP) 或 FUCCI 系统 (在引用45中可以找到有关如何生成 FUCCI 表达式单元的详细协议)评估有丝分裂细胞的命运。
	<ol>
<li>为了测量核破裂的频率, 使用 RPE-1 细胞表达 H2B-GFP 和一串二聚体的红色荧光蛋白融合到一个单一的核定位信号 (TDRFP-NLS)。 注: 三种绿色荧光蛋白的构造与单个核定位信号 (GFP3-NLS) 相融合, 也被用来演示破裂 (如参考42)。</li>
		<li>在适当的生长培养基上维持10厘米组织培养板上的细胞。RPE-1 细胞维持在苯酚无红, Dulbecco 的改良鹰培养基/营养混合物 F-12 (DMEM:F12) 补充10% 胎牛血清 (血清), 100 毫升/ml 青霉素, 100 微克/毫升链霉素。</li>
	</ol>
</li>
	<li>
从细胞中吸取培养基, 用10毫升无菌、室温磷酸盐缓冲盐水 (pbs) 冲洗组织培养皿, 去除残留培养基, 然后吸入 PBS。
	<ol>
<li>加入2毫升0.25% 胰蛋白酶与乙二胺四乙酸 (EDTA) 的细胞和孵化37°c 3 分钟或直到大多数细胞已脱离板块。 注意: 不要在胰蛋白酶中保持细胞比需要的时间长。</li>
	</ol>
</li>
	<li>
添加10毫升的完整培养基, 收集 trypsinized 细胞与10毫升血清 stripette 和免除15毫升锥管。
	<ol>
<li>在室温下离心 180 x g以3分钟颗粒状细胞。</li>
	</ol>
</li>
	<li>吸上清液, 小心不要扰乱颗粒, 然后通过轻轻吹打血清 stripette 中上下的培养基, 彻底并用重悬10毫升新鲜培养基中的颗粒。</li>
	<li>
使用 hemocytometer 或自动单元计数机计算单元格46。
	<ol>
<li>将新锥管中的细胞稀释成3万细胞/全培养基的 mL。</li>
		<li>板 3万 RPE-1 细胞 (1 毫升容量) 每井的12井玻璃底 (#1 5 厚度) 成像盘, 以达到所需的细胞密度第二天 (30–50% 汇合)。 注意: 始终用手套处理盘子, 小心不要碰玻璃底部。</li>
	</ol>
</li>
	<li>允许细胞生长在37°c 的组织培养孵化器, 直到他们完全附着和扁平的玻璃底成像板。虽然细胞可以在4小时内附着, 但建议细胞不使用药物治疗, 直到第二天才能成像。</li>
	<li>
添加紫杉醇 (溶解在二甲基亚砜 (亚砜)) 到理想的最终浓度在完全培养基和彻底混合。
	<ol>
<li>用同等体积的亚砜制备完全介质, 单独用作控制。</li>
		<li>将含有药物或亚砜的培养基加热至37摄氏度, 然后再加入细胞。这将防止由于突然温度变化引起的焦漂移。</li>
		<li>添加1毫升的培养基, 其中含有紫杉醇或亚砜单独的12井成像皿的个别水井。</li>
	</ol>
</li>
</ol>2. 建立活体细胞成像显微镜
<ol><li>用光学清洗器清洁成像盘的玻璃底部, 清除可能干扰成像的任何指纹或灰尘。使用足够的光清洁剂来润湿整个玻璃表面。</li>
	<li>将玻璃底部的盘子放在成像盘适配器的显微镜阶段, 从盘子中取下塑料盖, 然后用玻璃顶的房间盖上盘子。确保房间有一个阀门, 允许稳定的空气流动由 5% CO2组成。 注: 要 humidify 5% CO2, 气体通过插入到无菌水浴住房的油管流动。这使得气体变得平衡到95% 的湿度。</li>
	<li>使用控制显微镜的软件程序启动/校准编码阶段。这将确保精确的 X Y 坐标和防止焦距漂移。</li>
	<li>使用相衬光学和执行科勒照明 (在参考47中详细描述) 来关注单元格, 以聚焦光线并提供最佳对比度。</li>
	<li>
使用采集软件来确定白光和所有使用的荧光通道的最佳曝光时间 (在相机上给予75% 像素饱和度的曝光是理想的, 前提是这一量的光线对细胞没有毒性)。
	<ol>
<li>使用采集软件从每个井中选择几个不重叠的视图, 以便进行成像。选择成像区域, 其中细胞已经很好地粘在玻璃底部, 并在50% 和70% 之间汇合。避免成群细胞的区域, 因为这将使以后分析困难。 注意: 必须从每个井中都有非重叠的视图字段, 以避免两次跟踪相同的单元格。如果细胞具有高度的运动性, 那么可能需要从一个中心点获得几张图像, 并将它们缝合在一起, 使一个大的视野。</li>
	</ol>
</li>
	<li>激活显微镜的调焦功能, 以确保所有点在实验期间保持在焦点上。</li>
	<li>为了评估有丝分裂细胞的命运, 设置成像软件, 从每个选定的视野收集图像每10分钟, 高达 96 h. 未受干扰的有丝分裂持续20–40分钟, 因此10分钟间隔将提供足够的取样, 以确定细胞分裂的时间。发现核破裂, 这是一个瞬态事件, 获取图像每5分钟。 注意: 确保计算机驱动的图像采集软件有自动更新, 屏保和节能模式被禁用, 因为这些往往会干扰图像采集的长期实验。</li>
	<li>启动成像实验。定期确认图像采集在视频过程中运行顺畅。</li>
</ol>3. 对紫杉醇反应的细胞命运的视频分析
<ol><li>确认影像细胞在整个成像实验过程中都是健康的。用亚砜治疗的控制细胞应该是可行的, 并积极增殖。 注意: 如果细胞表现出压力的迹象, 不要定量视频, 而是集中精力优化成像条件48。成像压力的迹象包括起泡/死亡细胞、无法附着在盘子上的细胞, 以及显示低有丝分裂指数和/或长时间有丝分裂的细胞。可能的压力来源包括在成像室或毒性48内的温度波动或 CO2级别。</li>
	<li>当用10X 或20X 的目标成像整个视野时, 可能存在数以百计的单个细胞。因此, 为了帮助定量, 使用可用的软件工具将视图域划分为较小的象限。分别在每个象限内评分单元格 (如步骤3.3 中所述). 1–3.6. 2)。</li>
	<li>
分析视频时, 使用相衬和/或荧光图像跟踪合并视图中的每个单元格。使用软件分析工具创建合并视图。
	<ol>
<li>从视频开始, 确定一个单一的界面细胞, 并跟踪其进展, 通过细胞周期使用相光学。要跟踪单个单元格, 请观察从框架到框架的单元格。由于其扁平形态学 (相对比评估) 和缺乏 DNA 凝结 (如 H2B-GFP 评估) (图 1A), 确定间期细胞。</li>
	</ol>
</li>
	<li>
通过观察细胞舍入 (使用相对比光学) 和/或染色体凝结 (使用 H2B-GFP) 来识别进入有丝分裂的细胞 (图 1A-c)。细胞四舍五入和染色体凝结都容易被眼睛视觉化。
	<ol>
<li>注释细胞进入有丝分裂的时间。继续跟踪细胞, 直到它到达它的命运 (后期如步骤 3.5, 细胞死亡的有丝分裂, 在步骤 3.6.1, 或有丝分裂的滑移, 如步骤 3.6.2)。</li>
	</ol>
</li>
	<li>控制单元应有效地对齐其染色体, 并在1小时内输入后期 (图 1D)。通过相位对比光学可视化后期, 当细胞开始捏成两个或通过可视化的极地移动染色体标记为 H2B-GFP (图 1A)。注释细胞经历后期的时间。</li>
	<li>
相比之下, 紫杉醇治疗的细胞将保持与凝聚染色体的圆形数小时 (3-40 小时) (图 1BD)。
	<ol>
<li>识别 mitotically 细胞死亡。 注意: 在有丝分裂过程中死亡的细胞通过相对比显微镜可视化, 因为细胞会疱疹、收缩和/或破裂 (图 1B)。如果成像 H2B-GFP, 染色体也会片段在细胞死亡期间。</li>
		<li>识别 mitotically 细胞, 并进行细胞滑移。 注意: 通过相对比显微镜观察细胞的有丝分裂滑移, 因为它们在不经过后期 (图 1C) 的情况下平退成相间和 decondense 染色体。发生有丝分裂的细胞会产生大的二、四倍体杂交细胞, 经常多核 (图 1C)。</li>
	</ol>
</li>
	<li>继续从原始视图字段跟踪单元格。跟踪整个视图字段后, 移动到从相同井获取的单独的视图字段并继续跟踪单元格。</li>
</ol>4. 影像分析识别有丝分裂后细胞的命运
<ol><li>
使用表达 FUCCI 系统的 RPE-1 细胞, 评估有丝分裂后的细胞命运 (如步骤3.6.2 中所述)。除了相对比成像, 有必要获得红色荧光 (指示 G1相) 和绿色荧光 (指示的 S/克2/M) 图像)。
	<ol>
<li>跟踪 FUCCI RPE-1 单元格, 如步骤3.3 至3.7 所述。<ol>
<li>为了确认 FUCCI 系统工作正常, 验证了控制单元对红绿荧光蛋白的替代表达, 并对活细胞成像数据进行了适当的分析。控制细胞应该从显示完全核红荧光到显示完全核绿色荧光在期间作为细胞进展从 G1到 S 阶段。细胞在有丝分裂完成过程中应继续呈现绿色荧光。在有丝分裂之后, 细胞在界面中应该再次表现出完全的红色荧光。 注意: 虽然有方法使用荧光迹线49来量化 FUCCI 系统中的 RFP 和 GFP 荧光强度, 但由于荧光颜色的变化是健壮的, 眼睛是可见的, 这通常是不必要的。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
用相衬显微镜识别有丝分裂中被逮捕的细胞, 并跟踪它们直到它们经历有丝分裂滑移, 如3.4 和3.6.2 所述。在 G1阶段 (图 2AC) 中, 细胞在有丝分裂过程中脱离分裂, 回到相间, 将会从在有丝分裂期间呈现绿色荧光转变为红色荧光。
	<ol>
<li>跟踪这些滑细胞使用相对比和 epifluorescent 成像的眼睛, 以评估他们的细胞命运 (图 2D)。<ol>
<li>标识重新输入单元周期的单元格。通过使用指示 G1/S 转换 (图 2A) 的 FUCCI 系统, 荧光表达式中的红-绿变化标识这些单元格。</li>
			<li>识别经过 G1单元周期逮捕的单元格。这些单元格由持续用于 > 24 h (图 2B) 的红色荧光表达式标识。</li>
			<li>识别在界面中死亡的细胞。这些细胞由细胞破裂/起泡/收缩使用相对比成像 (图 2C) 来识别。</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. 识别核包络破裂频率的视频分析
<ol><li>使用 RPE-1 细胞表达 H2B-GFP 和 TDRFP, 评估核包膜破裂。除了相对比成像, 获得红色荧光 (TRITC) 和绿色荧光 (FITC) 图像。每5分钟获取图像以可视化破裂。 注意: 要生成细胞系, 在其中, TDRFP-NLS 能有效地导入到细胞核中, 具有极小的细胞质荧光是非常重要的。</li>
	<li>如步骤3.3 至3.4 所述的图像单元格。TDRFP 荧光信号和 H2B-GFP 应在界面间进行协同定位。当有丝分裂 (由细胞四舍五入使用相光学和/或 H2B-GFP 的染色体凝结), 核信封将崩溃, TDRFP 的信号将成为细胞质 (图 3A)。有丝分裂后, 核包膜将在子细胞中进行改造, TDRFP 的信号将成为核。</li>
	<li>通过活细胞成像跟踪整个后续间期的子细胞。通过观察核局部 TDRFP 到周围细胞质的瞬态爆裂, 确定核破裂事件。在几分钟内, 核包将被修复, TDRFP 将被 relocalized 到原子核 (图 3A)。 注: 通常, 核 DNA, 如 H2B-GFP 的可视化, 将被视为在破裂部位外突出作为一个小的疱疹。</li>
	<li>在相间的过程中获得破裂事件的核的分数。要获得此分数, 请计算在所跟踪的单元格总数中发生破裂事件的单元格数。</li>
</ol>

作者没有什么可透露的。

长期活细胞成像的最关键的方面是确保成像细胞的健康。必须让细胞接触到极小的外部环境压力源, 例如温度、湿度和/或 CO2级的不合格条件。同样重要的是, 限制光损伤的荧光激发照明, 因为成像应力是已知的影响细胞行为的 50.限制光损伤可以以多种方式实现, 如在其他48中详细说明.一般而言, 最好使用最短的曝光时间来产生足够明亮的荧光信号, 从而?...

抗有丝分裂药物长期用于各种类型的实体肿瘤的化疗方案, 并经常显示出很大的功效1,2,3。机械化, 这些药物扰乱正常的有丝分裂进展, 并促进有丝分裂在迅速增殖的癌细胞的抑制。然而, 细胞的命运, 对有丝分裂的反应是高度可变的: 虽然细胞的一小部分经历细胞死亡直接从有丝分裂, 其他退出有丝分裂和返回到间期作为二、四倍体杂交细胞 (一个过程称为有丝分裂滑移)4, 5,6,7,8。这些界面细胞可以执行细胞凋亡, 接受永久性细胞周期骤停, 甚至重新进入细胞周期4,5,6,7,8,9 ,10,11。细胞通过滑入间期而逃避有丝分裂细胞死亡, 只会在药物去除后重新进入细胞周期, 因此可能有助于癌细胞的重新出现。此外, 从有丝分裂中滑出的细胞是二、四倍体杂交的, tetraploidy 被称为促进染色体不稳定, 驱动肿瘤复发12, 13, 14, 15.因此, 确定控制细胞命运的因素对抗有丝分裂药物治疗是至关重要的, 以优化目前的治疗方法。
在本议定书中, 我们描述的方法, 以直接观察和研究细胞的命运, 经过长期的有丝分裂, 以回应抗有丝分裂药物紫杉醇。紫杉醇是临床上行之有效的治疗, 在许多肿瘤类型, 包括乳房, 卵巢和肺部, 已证明是非常有效的16,17,18,19, 20. 紫杉醇是从红豆杉树皮中提取的植物生物碱, 稳定微管, 从而防止其动态性 21, 22.虽然紫杉醇抑制微管动力学不会影响从 g1到 g 2 的细胞周期进展,但该药物确实导致在有丝分裂期间主轴组件检查点的持续激活, 阻碍动粒微管附件 (此处深度查看 23, 24) 25.因此, 在紫杉醇治疗的细胞中防止后期发作, 并导致长期的有丝分裂停止。
本议定书将首先描述在活体细胞成像实验中识别有丝分裂细胞的方法。由于两种明显的细胞生物学变化, 有丝分裂可以在黏附组织培养细胞中可视化。首先, 染色体在核包络击穿前立即变得高度浓缩。虽然通常可以通过标准的相衬显微镜检测到染色体凝结, 但使用标记染色体的荧光标记 (例如荧光标记的组蛋白) 能更清楚地发现。其次, 有丝分裂细胞也可以通过细胞四舍五入所产生的戏剧性形态学变化来识别。
该协议将演示如何使用活细胞成像方法来跟踪发生长期有丝分裂骤停的细胞的命运。在有丝分裂中被捕的细胞经历了三种截然不同的命运。首先, 细胞在有丝分裂过程中会发生细胞死亡。这种现象很容易被光镜所形象化, 因为死亡细胞被观察到收缩、疱疹和/或破裂。第二, 细胞可以从有丝分裂中退出, 回到无染色体分离或胞质分裂的间期, 这一过程称为有丝分裂滑移。染色体的 decondensation 和/或有丝分裂细胞的扁平化容易识别这个过程。从有丝分裂中滑出的细胞也经常显示不规则的, 多裂的细胞核, 并且经常有几个微核的5。第三, 在有丝分裂中被拘捕的细胞可以在长时间延迟后进行有丝分裂, 并进行减数分裂。虽然不常见的较高药物浓度, 这种行为表明, 被逮捕的细胞可能已满足主轴装配检查点, 或主轴装配检查点部分减弱或缺陷。染色体分离和后续胞质分裂使用活细胞成像可以可视化后期发病。
还将描述活细胞成像方法, 以跟踪细胞的命运, 以避免有丝分裂细胞死亡的有丝分裂延迟。发生有丝分裂滑移的细胞在随后的界面中死亡, 触发持久性 G1细胞周期的停止, 或重新进入细胞周期以启动新一轮细胞分裂4。使用 FUCCI (荧光泛细胞周期指示器) 系统来确定细胞分裂后重新进入细胞周期的分数将被描述。FUCCI 允许直接可视化 G1/S 转换, 并且可以与长期活细胞成像同时使用, 这两个体外和在体内26,27。FUCCI 系统利用两个荧光标记的蛋白质, 截断形式的 hCdt1 (染色质许可和 DNA 复制因子 1) 和 hGeminin, 其水平振荡的基础上细胞周期的位置。在 G1阶段, hCdt1 (融合到红色荧光蛋白) 在高级别存在, 它的作用是许可 DNA 进行复制, 但泛素化 E3 泛素连接酶超临界 Skp2 并在 S/G 2/M 阶段降级, 以防止重新复制 DNA26。相比之下, hGeminin (融合到绿色荧光蛋白), 是 hCdt1 的抑制剂, 其水平峰值在 S/G2/M, 但泛素化由 E3 泛素连接酶APC Cdh1 和退化在有丝分裂结束和整个G 126. 因此, FUCCI 提供了细胞周期阶段的直接荧光读数, 因为细胞在 G1,和绿色荧光期间在 S/G2/m 期间呈现红色荧光。FUCCI 系统是一个重要的进步, 超过其他方法 (如 bromodeoxyuridine 染色), 以确定增生细胞, 因为它不需要细胞固定, 并允许单细胞成像不需要额外的药理同步细胞群的治疗方法。尽管在本协议中没有讨论, 还开发了额外的活细胞传感器来可视化细胞周期进展, 包括 G128的旋 B 传感器、DNA 连接酶-RFP 29 和 PCDNA-GFP 30 传感器 对于 s-相位和最近的 FUCCI-4 传感器, 它检测单元周期的所有阶段31。
最后介绍了一种检测核包络破裂的活细胞成像方法。最近的研究表明, 癌细胞的核包不稳定, 容易爆裂, 从而使 nucleoplasm 和细胞质的内容混合。这种现象, 称为核破裂, 可以促进 DNA 损伤和刺激先天免疫应答32,33,34,35,36,37,38,39,40,41,42,43,44. 虽然核破裂的根本原因仍然不完整, 但众所周知, 核结构的变形与核破裂的发生率42的增加有关联。紫杉醇治疗的一个众所周知的作用是在有丝分裂后产生惊人的异常核结构;因此, 用活细胞成像方法来量化核破裂将被描述, 同时也探讨紫杉醇治疗是否增加核破裂事件的频率。核破裂可以通过观察到的核靶荧光蛋白泄漏到细胞质中来检测 (例如与核定位信号融合的 RFP 串联二聚体重复 TDRFP)。这种渗漏是明显可见的眼睛, 这使得简单的定量的破裂事件。
该协议需要一个 widefield 荧光显微镜, 配备了编码阶段和调焦软件。编码阶段允许精确的自动移动到定义的 X Y 坐标, 而自动对焦软件在成像周期的持续时间内维护焦点的单元格。此外, 该协议要求设备保持37°c 的细胞与湿润 5% CO2大气。这可以通过在温度和空气控制的外壳内封闭整个显微镜, 或者使用在当地保持温度和环境的舞台顶部设备来实现。该协议中使用的相对比目标是一个0.30 的数值孔径的计划。然而, 20X 目标也足以在单个焦平面中识别圆形有丝分裂和扁平化的间相细胞。如果执行相对比成像 (如本方法所述), 盖子可以是玻璃或塑料。如果使用差动干涉造影 (DIC) 显微镜, 必须使用玻璃罩防止光的退极化。

<table><tbody><tr><td>phenol red-free, DMEM:F12 media</td><td>Hyclone</td><td>SH3027202</td><td></td></tr><tr><td>10% fetal bovine serum (FBS)</td><td>Gibco</td><td>10438026</td><td></td></tr><tr><td>100 IU/ml penicillin, and 100 mg/ml streptomycin.</td><td>Gibco</td><td>15070063</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>0.25% Trypsin/EDTA</td><td>Gemini</td><td>50-753-3104</td><td></td></tr><tr><td>12-well glass bottomed imaging dish</td><td>Cellvis</td><td>P12-1.5H-N</td><td></td></tr><tr><td>Paclitaxel</td><td>TSZ Chem</td><td>RS036</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma</td><td>NC0215085</td><td></td></tr><tr><td>Environmental Chamber</td><td>In Vivo Scientific</td><td></td><td></td></tr><tr><td>5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas</td><td>Z03NI7432000201</td><td></td></tr><tr><td>15 mL Conical Tubes</td><td>Fisherbrand</td><td>05-539-12</td><td></td></tr><tr><td>10 cm polystyrene tissue culture plates</td><td>Falcon</td><td>08772E</td><td></td></tr><tr><td>10 mL Disposable Serological Pipette</td><td>Fisherbrand</td><td>4488</td><td></td></tr><tr><td>5804R 15 amp Centrifuge</td><td>Eppendorf</td><td>97007-370</td><td></td></tr><tr><td>1000 microliters Pipette</td><td>Gilson</td><td>Pipetman Classic</td><td></td></tr><tr><td>20 microliters Pipette</td><td>Gilson</td><td>Pipetman Classic</td><td></td></tr><tr><td>p1000 Pipette Tips</td><td>Sharp</td><td>p1126</td><td></td></tr><tr><td>p20 Pipette Tips</td><td>Sharp</td><td>P1121</td><td></td></tr><tr><td>RPE-1 Cells</td><td>ATCC</td><td>CRL-4000</td><td></td></tr><tr><td>Incubator Galaxy 170S</td><td>Eppendorf</td><td>CO17011005</td><td></td></tr><tr><td>Pipette Boy</td><td>Integra</td><td>156401</td><td></td></tr><tr><td>Hemacytometer</td><td>Fisher Scientific</td><td>0267110</td><td></td></tr><tr><td>Nikon Ti-E-PFS Inverted Microscope for Live Cell Imaging</td><td>Micro Video Instruments, Inc.</td><td>MEA53100</td><td></td></tr><tr><td>Prior X-Y Stage System and White Light LED Illuminator</td><td>Micro Video Instruments, Inc.</td><td>500-H117P1N4</td><td></td></tr><tr><td>Prior Proscan 3 Controller XYZ with Encoders</td><td>Micro Video Instruments, Inc.</td><td>500-H31XYZE</td><td></td></tr><tr><td>Andor Digital Camera</td><td>Micro Video Instruments, Inc.</td><td>D10NLC</td><td></td></tr><tr><td>Nikon NIS Elements AR Software</td><td>Micro Video Instruments, Inc.</td><td>MQS31000</td><td></td></tr><tr><td>SOLA LED Illuminator for Fluorescence</td><td>Micro Video Instruments, Inc.</td><td>SOLA-E</td><td></td></tr><tr><td>InVivo Environmental Chamber with CO2 Flow Control</td><td>Micro Video Instruments, Inc.</td><td>Ti-IVS-100</td><td></td></tr><tr><td>Ti-ND6-PFS Perfect Focus Motorized Nosepiece</td><td>Micro Video Instruments, Inc.</td><td>MEP59391</td><td></td></tr><tr><td>Ti-C System Condenser Turret</td><td>Micro Video Instruments, Inc.</td><td>MEL51000</td><td></td></tr><tr><td>Ti-C LWD LWD Lens Unit for System Condenser Turret</td><td>Micro Video Instruments, Inc.</td><td>MEL56200</td><td></td></tr><tr><td>Te-C LWD Ph1 Module</td><td>Micro Video Instruments, Inc.</td><td>MEH41100</td><td></td></tr><tr><td>CFI Plan Fluor DLL 10X Objectivena 0.3 wd 16mm</td><td>Micro Video Instruments, Inc.</td><td>MRH10101</td><td></td></tr><tr><td>CFI Super Plan Fluor ELWD 20xc ADM Objective</td><td>Micro Video Instruments, Inc.</td><td>MRH48230</td><td></td></tr></tbody></table>

long-term live-cell imaging to assess cell fate in response to paclitaxel

活细胞成像是一种强大的技术, 可以用来直接可视化的生物现象在单个细胞的长期时间。在过去的十年中, 新的和创新的技术大大提高了活细胞成像的实用性。在 37 °C 和 5% CO2单元格培养条件下, 单元格现在可以保持焦点并连续数天进行映像。此外, 可以同时获取多个表示不同实验条件的视场, 从而提供高通量的实验数据。活细胞成像通过允许直接可视化和时间定量的动态细胞事件提供了一个显著的优势比固定细胞成像。活体细胞成像还可以识别在使用基于人口的化验方法时可能遗漏的单个细胞行为的变化。在这里, 我们描述活细胞成像协议, 以评估细胞命运决定后, 抗有丝分裂药物紫杉醇治疗。我们展示的方法, 以可视化的 mitotically 被逮捕的细胞直接死于有丝分裂或滑回之间的界面。我们还描述了如何使用荧光泛的细胞周期指示器 (FUCCI) 系统来评估产生于有丝分裂滑移的周期性细胞的分数, 能够重新进入细胞周期。最后, 我们描述了一种用于识别核包络破裂事件的活细胞成像方法。

使用上述协议, RPE-1 表达 H2B-GFP 的细胞以抗有丝分裂或车辆控制 (亚砜) 治疗, 并通过活细胞成像进行分析。分析显示, 100% 的控制有丝分裂 RPE-1 细胞开始后期平均22分钟后进入有丝分裂 (图 1A, 1D)。相比之下, 紫杉醇治疗的 RPE-1 细胞在长达数小时的时间内出现了严重的有丝分裂 (图 1D)。影像显示, 48% 的这些 mitotically 被...

Watch this Scientific Journal Video about Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel at JoVE.com

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

活细胞成像提供了丰富的信息, 单个细胞或整个人口, 是无法实现的固定细胞成像单独。这里描述了用抗有丝分裂药物紫杉醇治疗后细胞命运决定的活细胞成像协议。

长期活细胞成像对紫杉醇反应细胞命运的评估

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

long-term-live-cell-imaging-to-assess-cell-fate-response-to

Research

JoVE Journal

Cancer Research

9.9K 次观看.  Boston University School of Medicine. 活细胞成像提供了丰富的信息, 单个细胞或整个人口, 是无法实现的固定细胞成像单独。这里描述了用抗有丝分裂药物紫杉醇治疗后细胞命运决定的活细胞成像协议。

视频: Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

实时成像来研究抗紫杉烷乳腺癌微管动态失稳

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

科研

癌症研究

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

TIP60 短暂耗竭后乳腺上皮细胞迁移的活体影像学研究

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

建立过度表达 dr3 的细胞系, 评估抗有丝分裂治疗的凋亡反应

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

医学

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

共聚焦成像在肝癌细胞中观察到的异常有丝分裂细胞死亡相关

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

用于患者来源的肿瘤类器官药物评估的自动动力学成像

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

作者聚焦:为基于类器官的药物反应分析开发多重动力学分析

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

多重活细胞成像在患者来源的癌症类器官模型中用于药物反应

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

长期活细胞成像对紫杉醇反应细胞命运的评估

摘要

探索更多视频

此视频中的章节

实时成像来研究抗紫杉烷乳腺癌微管动态失稳

TIP60 短暂耗竭后乳腺上皮细胞迁移的活体影像学研究

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

建立过度表达 dr3 的细胞系, 评估抗有丝分裂治疗的凋亡反应

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

共聚焦成像在肝癌细胞中观察到的异常有丝分裂细胞死亡相关

多重活细胞成像在患者来源的癌症类器官模型中用于药物反应

多重活细胞成像在患者来源的癌症类器官模型中用于药物反应

多重活细胞成像在患者来源的癌症类器官模型中用于药物反应