Name: a time-lapse, label-free, quantitative phase imaging study of dormant and active human cancer cells
Uploaded: 2018-02-16T13:00:00.000+00:00
Duration: 12 min 48 s
Description: 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

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 San Sebastian

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

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 ...

科研

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 ...

医学

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 ...

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification of lymphocyte subtypes is important for the study of immunology as well as diagnosis and treatment of various diseases. Currently, standard methods for classifying lymphocyte types rely on labeling specific membrane proteins via antigen-antibody reactions. However, these labeling techniques carry the potential risks of altering cellular functions. The protocol described here overcomes these challenges by exploiting intrinsic optical contrasts measured by 3D quantitative phase imaging and a machine learning algorithm. Measurement of 3D refractive index (RI) tomograms of lymphocytes provides quantitative information about 3D morphology and phenotypes of individual cells. The biophysical parameters extracted from the measured 3D RI tomograms are then quantitatively analyzed with a machine learning algorithm, enabling label-free identification of lymphocyte types at a single-cell level. We measure the 3D RI tomograms of B, CD4+ T, and CD8+ T lymphocytes and identified their cell types with over 80% accuracy. In this protocol, we describe the detailed steps for lymphocyte isolation, 3D quantitative phase imaging, and machine learning for identifying lymphocyte types.

We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types.

用三维定量相位成像和机器学习技术对淋巴细胞亚型进行无标签鉴定

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification ...

免疫与感染

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代谢标记和流式细胞仪测定细胞周期进程的动力学

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

生物学

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

3D球形模型中癌细胞入侵的时限解荧光成像和分析

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...

通过连续成像量化细胞死亡起始

Studying Cell Death Initiation Using a Digital Microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

作者聚焦:研究超敏反应中细胞死亡起始的简化方法 

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

使用数码显微镜研究细胞死亡起始

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as ...