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, 从而有效地跟踪细胞, 从而限制毒性。然而, 如果细胞只被成像一次每 10–20 min, 更长的曝光 (那接近的像素饱和) 通常是好容忍的 (虽然这必须为每个荧光和细胞类型的经验主义地确定)。由于成像细胞对环境和成像应力都很敏感, 因此必须将控制细胞始终包括在活细胞成像实验中, 并进行监测以确认正常的增殖。
长期活细胞成像的一个限制是, 它需要装备一个现有的显微镜与环境室, 既保持温度和5% 加湿 CO2大气, 或一个舞台顶室, 也可以支持适当的温度和大气。虽然流动 CO2是最佳的, 但如果一个环境室与5% 加湿 co2不可用, 也可以使用 co2独立介质对单元格进行成像, 最多可达48小时。然而, 许多细胞类型是不能容忍这种媒介, 这必须通过测量细胞生长和生存能力的经验来确定。用矿物油覆盖细胞也可以防止介质蒸发。
对此方法的第二个主要限制是, 手动跟踪单元命运是非常费力和耗费时间的。但是, 单元跟踪程序是可用的, 可以优化以自动进行图像分析51,52,53。这种方法的一个进一步的限制是, 高度运动的细胞往往难以追踪超过一段时间。如果这是一个很大的问题, 整个井可以被成像和图像缝合在一起形成一个大的视野。许多软件程序支持此方法。
自体荧光是另一个必须与此协议解决的约束。酚红色游离培养基在活细胞成像期间减少背景自体荧光。同时也证明了在生长培养基、核黄素和吡哆醛中常见的两种维生素能降低荧光蛋白的钴。因此, 缺乏这些维生素的培养基也可用于荧光成像, 以增强对噪声口粮的信号。虽然塑料底菜成本较低, 可能提供足够的成像效果, 但它们也产生了大量的自体荧光, 对信噪比有负面影响。此外, 塑料底碟的厚度有较大的变化, 这会破坏许多显微镜的自动对焦功能。本协议中的玻璃底成像盘厚度为0.17 毫米, 是现代显微镜使用的理想玻璃。
最后, 包含多个视野和获取多个荧光通道的长期电影会产生大量的数据文件 (甚至每 tb 数兆字节), 这会给数据存储和备份带来问题。为减轻此问题, 建议使用 2 x 2 或 4 x 4 binning 获取所有图像, 前提是所产生的分辨率损失是可以接受的。Binning 不仅限制暴露时间 (即稳定表达 H2B-GFP 的单元格或 FUCCI 系统应具有小于500毫秒的曝光率), 而且还会大幅减小数据文件的大小 (作废 2 x 2 的图像是1/4 的文件大小。非作废图像)。
尽管有这些限制, 长期活细胞成像是跟踪个体细胞命运的最佳方法, 特别是当细胞命运高度异构时。随着更多的活细胞荧光标记和传感器变得可用, 活细胞成像方法将扩大到可视化和定量几个额外的细胞生物学方面。例如, 目前存在的活细胞传感器定量 DNA 损伤灶、p53 水平、细胞周期位置和凋亡26,54,55,56,57,58.因此, 成像实验有能力揭示的基础细胞属性, 决定了如何和为什么单细胞反应的化疗药物的可变性。

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% CO2</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 被拘捕的细胞经历了有丝分裂细胞死亡 (图 1B, 1E), 而其余52% 则经历了有丝分裂延迟 (图 1C, 1E)。
为评估 RPE-1 细胞有丝分裂后的结局, RPE-1 细胞表达 FUCCI 系统的方法分别采用低剂量 50 nM 紫杉醇、高剂量5µM 紫杉醇或车辆控制 (亚砜), 并通过长期活细胞成像分析。数据显示, RPE-1 细胞在 50 nM 紫杉醇治疗后出现有丝分裂滑移, 22% 死于随后的细胞周期 (图 2C, 2D), 72% 诱导细胞周期骤停 (图 2B, 2D), 和只有5% 重新输入 S 阶段 (图 2A, 2D)。相比之下, RPE-1 细胞的高剂量5µM 紫杉醇, 经历了有丝分裂的滑动, 35% 死于随后的细胞周期, 65% 诱导细胞周期拘捕, 并没有重新进入 S 阶段 (图 2D)。
有趣的是, 低剂量紫杉醇治疗促进核包膜破裂的 RPE-1 细胞有丝分裂后。在有丝分裂完成的女儿细胞被跟踪和得分的任何核破裂事件, 表明只有4% 的控制 (亚砜处理) RPE-1 子细胞破裂, 而23% 的女儿细胞治疗 10 nM 紫杉醇显示破裂 (图 3A, 3B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57383/57383fig1.jpg"> 图 1: 有丝分裂细胞的命运, 以应对抗有丝分裂治疗.用亚砜汽车控制 (A) 或5µM 紫杉醇 (B、C) 对 RPE-1 细胞进行稳定表达 H2B-GFP, 并采用时间推移相对比和 widefield 荧光显微镜对有丝分裂细胞的命运进行成像。当它们进入有丝分裂 (中间板), 直到它们开始后期 (A), 并经历有丝分裂细胞死亡 (B), 或者从有丝分裂滑回同一个间期 (C) 时, 都跟踪了界面细胞 (左面板)。白色箭头表示跟踪的单元格。(D) 控制细胞 (亚砜处理) 和抗有丝分裂处理细胞在有丝分裂中所用的时间量, 如量化的活细胞成像 (每个条件的 n = 100 个细胞)。(E) 以5µM 紫杉醇处理的细胞或细胞中, 经历后期、有丝分裂细胞死亡或有丝分裂滑移的细胞 (n = 100) 的分数。时间, h: 最小刻度条 = 50 µm.<a href="/files/ftp_upload/57383/57383fig1large.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/57383/57383fig2.jpg"> 图 2: 经历有丝分裂延迟的细胞的命运.用 50 nM 紫杉醇或5µM 紫杉醇对 FUCCI 系统进行稳定表达的 RPE-1 细胞, 用时间推移相对比法和 widefield 荧光显微镜对有丝分裂后定量细胞的命运进行了成像。细胞被评分为重新进入细胞周期, 由 FUCCI 系统中的红-绿荧光变化判断 (a);根据 > 24 h (B) 的持续红色荧光判断, 在 G1阶段中进行逮捕;或在随后的有丝分裂中死亡, 由细胞起泡/破裂 (C) 判断。白色箭头表示跟踪的单元格。(D) 所经历的每一个命运的细胞 (n = 100) 的分数。误差条表示从两个独立实验中的平均值的标准偏差。时间, h: 最小刻度条 = 50 µm.<a href="/files/ftp_upload/57383/57383fig2large.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/57383/57383fig3.jpg"> 图 3: 紫杉醇处理过的细胞核包膜破裂的频率.RPE-1 细胞稳定表达 TDRFP 和 H2B-GFP 的治疗与 10 nM 紫杉醇或车辆控制 (亚砜) 和成像使用时间推移相对比和 widefield 荧光显微术 (A)。在有丝分裂期间 (中期/后期) 核包裹被分解, TDRFP-NLS 成为细胞质。有丝分裂后, TDRFP relocalizes 到间期细胞的核 (破裂前)。核破裂事件是由 TDRFP 的服务业, 从细胞核到细胞质的间质细胞 (破裂), 其次是 TDRFP-nls 的孵育由核包络修复后 (破裂)。白色箭头表示跟踪的单元格。(B) 在紫杉醇或车辆控制的情况下, 在有丝分裂后出现核包膜破裂的细胞 (n > 100) 的分数。时间, h: 最小刻度条 = 50 µm.<a href="/files/ftp_upload/57383/57383fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

9.8K 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

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

长期高分辨率活体显微镜在肺与真空稳定成像窗口

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

科研

癌症研究

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

综合程序，以评估<em&gt;在体内</em&gt;癌症纳米医学的性能

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

将斑马鱼小儿脑肿瘤移植入免疫能力的宿主，用于长期研究肿瘤细胞行为和药物反应

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-&#946; is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-&#946;/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-&#946; displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-&#946; signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-&#946; signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Here, we present a protocol for live cell imaging of TGF-&#946;/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

腺病毒报告系统 TGF β/Smad3 信号通路的活体细胞成像

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

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 ...

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

利用微阵列研究微环境对癌症细胞表型的影响

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.