作者感谢乳腺癌研究基金会和先进医学研究基金会的支持。

这里描述的所有方法都已被波士顿儿童医院机构生物安全委员会批准。
1. 细胞制剂
<ol><li>
解冻 KHOS 细胞
	<ol>
<li>预热培养基,即, Dulbecco 的改性鹰培养基辅以 10% (卷/卷) 胎小牛血清 (血清) 和 1% (卷/卷) 青霉素/链霉素。</li>
		<li>从液氮罐中取去低温小瓶, 将瓶子的底部浸入37摄氏度水浴中的温水中, 轻轻摇动低温瓶以加速解冻过程。保持低温小瓶的盖子不接触水, 以避免污染。一旦细胞解冻, 通过喷洒70% 乙醇溶液和擦拭瓶子来消毒低温小瓶。</li>
		<li>在层流罩中, 立即从低温小瓶中吸出细胞悬浮液, 并在10毫升加热培养基中轻轻悬浮 (1.1.1 中描述)。离心机细胞悬浮在大约 200 x g 为 5-7 分钟。</li>
		<li>并用重悬细胞颗粒与10毫升加热培养基, 并转移到一个 T75 瓶。用10毫升的吸管轻轻地用吸管多次悬浮细胞。</li>
		<li>将烧瓶放入一个37°c 孵化器与 5% (卷/卷) CO2和湿润气氛。允许单元格附加至少12小时。</li>
		<li>孵育细胞大约 3-7 天直到 80-100% 汇合。每 2-3 天改变培养基。</li>
	</ol>
</li>
	<li>
分裂 KHOS 和 N 细胞
	<ol>
<li>从烧瓶中移除培养基介质。</li>
		<li>用5毫升不含钙和镁的无菌 PBS (1x) 冲洗细胞, 去除不附着的细胞或分数。</li>
		<li>在烧瓶中加入1毫升0.05% 胰蛋白酶-edta 溶液, 并简单地旋转烧瓶, 以确保胰蛋白酶-edta 均匀分散。</li>
		<li>在37摄氏度孵化器或 3-5 分钟的室温下孵育烧瓶 2-3 分钟。检查显微镜下的细胞在大约400x 倍放大, 以确保细胞被四舍五入和分离。</li>
		<li>一旦细胞分离, 添加10毫升培养基, 并轻轻地将培养基上下移几次, 用10毫升的吸管将细胞团聚体分解成单个细胞悬浮。</li>
		<li>使用单元格计数器计数单元格。将细胞悬浮到新的 T75 烧瓶中, 密度为 2 x 106细胞或比例为1:4。</li>
		<li>允许细胞生长到 80-100% 汇合之前, 播种的 QPI 实验。</li>
	</ol>
</li>
	<li>
QPI 种子 KHOS 细胞
	<ol>
<li>种子 KHOS-6 井板中的 A 和 N 细胞密度为 5万-30万细胞/以及5毫升培养基后计数的细胞计数器。 注: 在图像采集过程中, 播种密度依赖于细胞增殖率和成像时间周期, 以 < 100% 汇合。</li>
		<li>孵育细胞至少12小时, 以允许附着。</li>
		<li>在进行 QPI 前, 用新鲜的媒介改变培养基。</li>
	</ol>
</li>
</ol>2. QPI 收购
<ol><li>
盖板滑动设置
	<ol>
<li>用75% 乙醇溶液浸泡15分钟, 清洗好几次, 用运行去离子水冲洗, 并消毒. 把盖子滑入一个不育的层流罩中晾干。</li>
		<li>小心地把盖子滑到一个6井板上, 以避免明显的气泡。确保盖板底部浸入培养基中 (最小5毫升/井)。</li>
		<li>将盘子放入孵化器 (37 °c, 5% CO2和湿润气氛) 到平衡至少15分钟。如果在盖子上有雾, 用无菌棉签擦拭干净。</li>
	</ol>
</li>
	<li>
用显微镜成像细胞
	<ol>
<li>打开软件初始化系统, 包括曝光时间的自标定、模式对比度和全息图噪声。请确保这些值是可接受的 (以绿色或黄色范围显示)。</li>
		<li>把6井板放到 QPI 显微镜的舞台上。</li>
		<li>单击 "实时捕获", 然后在 "软件焦点" 中选择 "手动"。通过调整 "显微镜设置" 中的工作距离, 粗略地聚焦细胞的相位图像, 获得相图中细胞的轮廓。在 "软件焦点" 中更改为 "自动"。</li>
		<li>点击 "新实验" 来创建一个新的实验。首先使用鼠标或数字键盘上的箭头键选择图像采集位置。在每次选择后单击 "记住"。通常 3-5 个位置为每个样品被选择。</li>
		<li>设置 "Timelapse" 部分的成像间隔和时间段。 注意: 要清楚地跟踪癌细胞, 间隔应短于5分钟, 48 小时设置为组合 QPI 分析的时间段。</li>
		<li>通过单击 "捕获" 来启动实验, 它将自动聚焦并在设置时间点获取图像。</li>
	</ol>
</li>
</ol>3. 数据分析
<ol><li>
细胞形态学分析
	<ol>
<li>单击 "识别单元格"。调整 "背景阈值" 的数值设置, 使单元格区域与背景噪音很好地分离。调整 "对象大小" 的设置编号, 以确保每个单元格都有一个核。为需要比较的样本保持一致的参数, 因为这可能会影响最终的面积和体积测量。如果需要, 请使用 "手动更改" 手动修改单元格段。</li>
		<li>使用软件中的 "分析数据" 功能对单元格进行分析。通过单击 "新分析" 开始新的数据分析。将所选图像拖动到 "源框架" 选项卡和单元格形态学参数, 包括单元格区域 (µm2)、光学厚度 (µm) 和每个单元格的卷 (µm3)。选择散点图或直方图模式, 并将数据导出为表或数字。</li>
	</ol>
</li>
	<li>
细胞增殖分析
	<ol>
<li>为不同的时间点选择至少5个图像 (例如、初始、12 h、24 h、36 h 和 48 h)。记录由软件提供的单元格编号。</li>
		<li>为了估计加倍时间, 在与初始数正常化后绘制细胞数, 并拟合指数生长曲线。</li>
	</ol>
</li>
	<li>
细胞运动分析
	<ol>
<li>在软件中使用 "跟踪单元" 功能。单击 "新分析" 开始新的数据分析。在 "源框架" 选项卡中, 将选定时间段 (例如、4小时后的 24 h 孵化) 中的图像拖动一系列. 通过单击 "添加单元格" 选择模式下的单元格随机选择 10-30 单元格。排除边缘的单元格和移出视图的区域。</li>
		<li>仔细检查图像序列中的跟踪。如果系统失去对单元格的跟踪或跟踪错误的单元格, 请通过单击 "确定" 以识别单元格, 或单击 "选择" 模式下的 "修改位置" 来修改单元格位置, 手动调整跟踪单元格。</li>
		<li>选择 "剧情运动" 中的细胞轨迹的玫瑰图, 或在 "剧情特征" (如运动速度 (µm)、运动 (µm)、迁移 (µm) 和迁移直接性等与运动相关的参数上绘制图。将数据导出为表或数字。</li>
	</ol>
</li>
</ol>

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没有宣布利益冲突。

在本研究中, 我们描述了一个体外, 无创, 无标签的方法, 使用 QPI 定量表征人骨肉瘤细胞的血管生成和非血管生成表型。采用综合、高通量的方法对多细胞参数进行了分析, 包括细胞面积、细胞厚度、细胞体积、增殖率、加倍时间、迁移直接、运动速度、迁移和运动。
与常规的细胞形态学和细胞行为评估方法相比, 该法不仅节省了时间和人工, 而且提供了更准确、更详细?...

在固体肿瘤的发展和进展中, 最早的检查点之一是获得血管生成表型, 这是癌症的标志。这一进展涉及各种生物化学和分子过程1,2,3。研究肿瘤进展这一关键步骤的技术挑战是缺乏连续和定量地对活癌细胞的血管生成和非血管生成性表型进行无偏定性和鉴别的工具。传统的检测方法用于研究血管生成和非血管生成细胞的细胞行为, 通常需要昂贵的试剂和仪器, 例如细胞增殖/迁移分析4,5, 6,7,8,9,10,11,12,13,14或互补的在体内评估4,5,6,8,15,16, 以及需要大量的专业知识和密集的时间和劳动消费。
最近, 定量相位成像 (QPI) 已经成为一种新的技术, 它能够对各种细胞形态学和行为参数进行时间推移和无标记的评估17,18,19,20,21,22. 与常规光学显微术不同, QPI 在光通过光学物体后按像素量化相移像素的变化, 并重建具有转换的光学厚度和体积的全息图像, 从而使直接对活细胞的分析和以下特点: (1) 定量成像, (2) 无创和时间推移成像, (3) 无标签成像, (4) 同时多参数成像。这些特性使 QPI 成为评估和理解细胞级病理过程的有力工具。
在最近的一项研究中, 我们利用 QPI 定量地定性和区分人骨肉瘤细胞的血管生成 KHOS 和非血管生成 KHOS-N 表型, 结合细胞形态学分析,扩散和运动23。利用图像分析软件, 对血管生成体与非血管生成性人骨肉瘤细胞的细胞形态和行为参数进行了定量比较, 并在这两个方面确定了五种特征差异。型.这种新的方法提供了一个综合和定量的平台, 以评估各种生物相关的细胞特征。

<table><tbody><tr><td>T75 flask</td><td>Corning, NY, USA</td><td>353136</td><td></td></tr><tr><td>6-well plates&amp;nbsp;</td><td>Corning, NY, USA</td><td>3506</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium (DMEM)</td><td>Thermo Fisher Scientific, MA, USA</td><td>11965092</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Atlanta Biologicals, GA, USA</td><td>S11550</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Thermo Fisher Scientific, MA, USA</td><td>15140122</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Thermo Fisher Scientific, MA, USA</td><td>10010023</td><td></td></tr><tr><td>Beckman Z1 Coulter counter</td><td>Beckman Coulter, IN, USA</td><td>Z1&amp;nbsp;</td><td></td></tr><tr><td>HoloMonitor M4</td><td>Phase Holographic Imaging Phi AB, Lund, Sweden</td><td>M4</td><td>Microscope</td></tr><tr><td>Hololid</td><td>Phase Holographic Imaging Phi AB, Lund, Sweden</td><td>PHI 8020</td><td></td></tr><tr><td>HStudioM4</td><td>Phase Holographic Imaging Phi AB, Lund, Sweden</td><td>HStudioM4</td><td>Software</td></tr></tbody></table>

a time-lapse, label-free, quantitative phase imaging study of dormant and active human cancer cells

血管生成表型的获得是肿瘤休眠逃逸的重要组成部分。虽然一些经典的体外检测 (例如、增殖、迁移和其他) 和在体内模型已经开发, 以调查和表征血管生成和非血管生成细胞表型, 这些方法是时间和劳动密集型, 往往需要昂贵的试剂和仪器, 以及重要的专业知识。在最近的一项研究中, 我们用一种新的定量相成像 (QPI) 技术来进行血管生成和非血管生成性人骨肉瘤 KHOS 细胞的时间推移和无标记的特征。用 QPI 定量测量和分析了细胞参数的一个小组, 包括胞形态学、增殖和运动。这一新颖和定量的方法提供了机会, 以持续和非侵入性研究相关的细胞过程, 行为, 和肿瘤细胞的特点和其他细胞类型的简单和综合的方式。本报告描述了我们的实验性协议, 包括细胞制备、QPI 采集和数据分析。

图 1描述了典型的细胞形态学特征。图像显示为全息图(图1A-b) 和2D 图像 (图1C D)。光学细胞厚度 (从折射率和光学路径长度计算) 通过线剖面或整个细胞测量来量化。绘制了用于整个单元格的 KHOS 和 KHOS 单元的面积和厚度的散射图, 如图 1EG所示, 这...

Watch this Scientific Journal Video about A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells at JoVE.com

A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells

应用定量相成像技术对休眠和活性癌细胞表型进行了表征。用一种简单的方法对细胞增殖、迁移和形态学测定进行了综合分析。

休眠和活性人肿瘤细胞的时间推移、无标签、定量相成像研究

The acquisition of the angiogenic phenotype is an essential component of the escape from tumor dormancy. Although several classic in vitro assays (e.g., ...

a-time-lapse-label-free-quantitative-phase-imaging-study-dormant

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

7.4K 次观看.  Boston Children's Hospital. 应用定量相成像技术对休眠和活性癌细胞表型进行了表征。用一种简单的方法对细胞增殖、迁移和形态学测定进行了综合分析。

视频: A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

一个<em在体外系统来研究肿瘤休眠和转移性增长

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

科研

医学

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

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 the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

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 ...

癌症研究

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.

脑肿瘤细胞行为的纵向生命成像,以响应侵入性手术活检

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

M1巨噬细胞-4T1小鼠乳腺癌细胞在共培养中噬菌体活性的时移2D成像

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

生物学

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

透过玻璃看:时间推移显微镜和单细胞的纵向跟踪研究抗癌治疗

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

活细胞成像，以评估在斜接主轴扰动后元相定时和细胞命运的动态

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...