这项工作是由美国国立卫生研究院和ACS，包括T32CA009135，CA133027，CA070723，和CA022443补助。比尔萨格登是美国癌症学会的研究教授。

1。可见质粒的细胞工程<ol><li>使用标准的限制性消化或同源重组技术引入到质粒加以可视化的DNA片段，它含有约250份的乳糖操纵子序列（LACO）。我们的质粒Bacmid的是一个约170 kbp的，并包含了整个基因组的卡波济氏肉瘤相关疱疹病毒（KSHV）编码的抗潮霉素12。 </li><li>介绍法律咨询及田土转易处含有质粒DNA，脂质体2000转染哺乳动物细胞中。转染HeLa细胞和SLK细胞在60毫米菜为4小时10μg纯化的质粒DNA与25μL脂质体2000，0.5毫升OptiMEM，和3.5毫升DMEM。 </li><li>变更至5毫升的DMEM培养基中，用10％胎牛血清和孵育细胞48小时。 </li><li>板中的细胞低密度大的菜肴和培养细胞在含抗生素的选择对引进的解放军SMID;我们选择细胞与300微克/毫升潮霉素的3周，用10％的牛胎儿血清的DMEM中。 </li><li>使用无菌，胰蛋白酶浸泡过的滤纸片，潮霉素抗性菌落转移到新的培养皿中。 </li><li>当转染的克隆生长填补菜，分离DNA，限制性酶切分析和Southern印迹，以确保LACO质粒，该聚合物是均匀和预期大小。 </li><li>如果质粒并没有被设计，以表达融合的LacI的，编码乳糖阻遏（LacI的）稠合到一个红色荧光蛋白，如LacI的-tdTomato用反转录病毒感染合适的克隆。我们使用tdTomato，而不是蛋白质的荧光绿色，以尽量减少光毒性，会发生在很长的时间的多重曝光13。 </li><li>一旦被引入的LacI融合蛋白，培养细胞在200微克/毫升异丙基-BD-硫代半乳糖苷（IPTG）以阻止从结合的融合蛋白的存在下DNA。 </li><li>至少3天的培养细胞，以允许的LacI-tdTomato蛋白的表达。 </li><li>屏幕克隆（通过洗去IPTG和查看（如下文所述）的细胞）为那些与最佳水平的LacI的-tdTomato揭示不同的信号与未结合的融合蛋白的最小的背景水平。 </li></ol> 2。成像系统的发展的我们的成像系统包括配备机动化的阶段和计划Aochromat 63x/1.4油物镜的Zeiss Axiovert 200M。由“中性白光LED的科利柏系统提供荧光的激发光。发射由CascadeII检测：1024 EMCCD相机。这是一个水冷，薄型背照式的摄像头，用于放大信号的增益寄存器;每秒每像素，暗电流为0.061电子的量子效率是大于0.90。该系统是由Axiovision 4.8软件，包括SmartExperiments模块。有关检测的tdToma的我们使用的Zeiss滤波器组75HE将通过所传递的光的85％的氟和95％的发射光可激发的发射滤波器。 <ol><li>用倒置显微镜和敏感的检测信号，LED光源，用于特定的激发和快速模板，机动化阶段和焦点臂，和软件控制的自动化收购图像EMCCD相机组装的成像系统，在很长的时间间隔与多个Z-堆栈。该系统应该坐在空气表与振动隔离。 </li><li>装配一个阶段顶孵化器，以保持细胞在37℃，5〜10％CO 2的加湿室中。让系统有足够的时间来一个稳定的温度，以减少运动伪影，在实验过程中。 </li><li>使用温水领的目标，防止目标从样品虹吸热。 </li></ol> 3。标记的DNA的可视化<ol><li>在一个35毫米的板细胞玻璃底皿的密度小于10％汇合，这将允许对细胞分为8-16细胞菌落的不重叠。执行此操作前至少24-48小时的活细胞成像所以有时间来划分。 </li><li>成像开始之前的一至三个小时，冲洗该板3次，用缺乏选择性的药物和IPTG的培养基。此过程洗去非活细胞，并允许LacI的融合蛋白结合的质粒的法律咨询及田土转易处网站。 </li><li>更换用2ml的DMEM培养基中，用10％胎牛血清和25mM的HEPES。 </li><li>拉伸更换培养皿CultFoil的顶部安装环为35 mm的菜。此盖会抑制蒸发的培养基中，这将增加盐的浓度随着时间的推移和杀死细胞。 </li><li>使用微分干涉相差（DIC）的成像，以查看的细胞，并确定多个的视场中的单元格的顶部和底部的焦平面。在每个字段中，将移动到一个焦平面，约1/3的方式，从细胞的顶部。保存的x，y，z坐标列表中保存的舞台位置。 注：通过目镜观察时照明的发光二极管的波长适当的LacI融合蛋白的细胞质粒的信号可能是不可见的。弱信号可能只与EMCCD相机，它允许集成的信号随着时间的推移，从而有效地放大弱信号的使用可见。 </li><li>在的显微镜控制软件，定义堆栈的图像，来执行在每个保存的台位置的z-。选择AZ步长为0.35-0.50微米，这将允许您以近似奈奎斯特采样在z方向14。中包括的“额外”的切片平面的上方和下方，其中的细胞都位于这些片的允许检测后，在细胞周期的细胞运动，并还用在采集后的处理（去卷积）的图像栈。这包含导致堆栈40-60图像，这取决于细胞的厚度上。 </li><li>在的显微镜控制软件中，定义一个时间系列实验，以在30分钟的时间间隔（在很长的时间用于跟踪的细胞分裂和迁移）和荧光图像，在10-60分钟的时间间隔收集明亮视场图像的z栈（用于可视化的质粒在细胞内）。 DIC成像在实验过程中，不要使用，因为它需要更多的光线比标准的明场成像，可长期实验的过程中不必要的光毒性的细胞暴露。 </li><li>启动软件控制的实验。确保该系统在实验过程中不被干扰（碰撞，暴露于过量的室内光线下，等）。 </li><li>如果有必要，在实验过程中补充水的加湿系统中。 </li></ol> 4。收购后处理图像<ol><li>检查每个Z-Stack与时间点的图像。裁剪出任何不必要的领域的图像以减少计算机处理时间在下一步骤中。 </li><li>使用去卷积算法重新分配信号强度的像素的原产地每个z-堆垛15中。虽然这可以基于解卷积的理论为您的成像系统的点扩散函数，它是优选由成像荧光微球测量您的特定系统的点扩散函数，并使用此测量中去卷积算法。我们测量我们的成像系统的点扩散函数和应用约束迭代在AxioVision软件（AxioVision软件的描述，蔡司）的最大似然算法。 </li><li>检查图像跟踪的强度变化和变动的质粒在细胞周期中的分区子细胞的质粒。 </li></ol>

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没有利益冲突的声明。

这里描述的方法可以被用来追踪跨越多个世代的随着时间的推移在活的哺乳动物细胞中的质粒。通过限制暴露于激发光，我们已经使用了这些技术，按照新划分对16-32细胞的殖民地，在至少72个小时和3个细胞分裂的细胞。这些实验提供不能获得的信息，可以从静态试验，如定量PCR，Southern杂交，和FISH。 关键是要筛选的细胞克隆具有均匀的结构的质粒，作为重排时，可能会发生...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 11965 </td><td></td></tr><tr><td> OptiMEM </td><td> GIBCO </td><td> 31985 </td><td></td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> SH30910.03 </td><td></td></tr><tr><td>青霉素</td><td>西格玛</td><td> P3032 </td><td></td></tr><tr><td>硫酸链霉素</td><td>西格玛</td><td> S9137 </td><td></td></tr><tr><td>潮霉素</td><td> Calbiochem公司</td><td> 400050 </td><td> 400微克/毫升的SLK; 300微克/毫升的HeLa细胞</td></tr><tr><td>异丙基-BD-硫代半乳糖苷（IPTG） </td><td>罗氏公司</td><td> 10 724 815 001 </td><td>最终浓度200微克/毫升</td></tr><tR&gt; <td>脂质体2000 </td><td> Invitrogen公司</td><td> 11668-027 </td><td></td></tr><tr><td> HEPES </td><td> GIBCO </td><td> 15630 </td><td></td></tr><tr><td>玻璃底菜</td><td> MatTek </td><td> P35G-1.5-10-C </td><td></td></tr><tr><td> CultFoil </td><td> Pecon </td><td> 000000-1116-08 </td><td></td></tr><tr><td> Axiovert 200M </td><td>蔡司</td><td></td><td></td></tr><tr><td>科利柏</td><td>蔡司</td><td> 423052-9500-000 </td><td></td></tr><tr><td>中性白色LED </td><td>蔡司</td><td> 423052-9120-000 </td><td></td></tr><tr><td>过滤器立方75 HE </td><td>蔡司</td><td> 489075-0000-000 </td><td></td></tr><tr><td>计划复消色差63x/1.4目标</td><td>蔡司</td><td> 440762-9904-000 </td><td></td></tr><tr><td> CascadeII：1024 EMCCD相机</td><td>光度</td><td> B10C892007 </td><td></td></tr><tr><td> Tempcontrol小</td><td>蔡司/ Pecon </td><td> 000000-1116-070 </td><td></td></tr><tr><td> Tempcontrol 37-2数字</td><td>蔡司/ Pecon </td><td> 000000-1052-320 </td><td></td></tr><tr><td> CTI控制器3700数字</td><td>蔡司/ Pecon </td><td> 411856-9903 </td><td></td></tr><tr><td>目的加热器</td><td>蔡司/ Pecon </td><td> 440760-0000-000 </td><td></td></tr><tr><td>第一阶段加热插入P </td><td>蔡司/ Pecon </td><td> 411861-9901-000 </td><td></td></tr><tr><td>舞台顶部孵化器小号</td><td>蔡司/ Pecon </td><td> 411860-9902-000 </td><td></td></tr><tr><td>加湿系统</td><td>蔡司/ Pecon </td><td> 000000-1116-065 </td><td></td></tr></tbody></table>

monitoring plasmid replication in live mammalian cells over multiple generations by fluorescence microscopy

保持在哺乳动物细胞中很少有天然存在的质粒。其中γ-疱疹病毒的基因组，包括Epstein-Barr病毒（EBV）和卡波济氏肉瘤相关疱疹病毒（KSHV），这会导致多种人类恶性肿瘤的1-3。在有执照的方式，每使用一个单一的病毒蛋白和细胞复制机制，这两个基因组复制传递到子代细胞在细胞分裂过程中，尽管他们缺乏传统的着丝粒4-8。 已经做了许多工作，这些质粒基因组的复制特性，使用方法，如Southern杂交和荧光原位杂交（FISH）。是有限的，虽然这些方法。定量PCR和Southern杂交提供的每个细胞质粒细胞的人口平均数的信息。 FISH技术是一种单细胞的检测，揭示的平均数量和分布的质粒<html> s的细胞的细胞群，但是是静态的，允许任何信息的父母或研究细胞的后代。 在这里，我们描述了一个可视化的质粒在活细胞中的方法。这种方法是基于上的荧光标记的景点9的质粒中的多个站点的乳糖阻遏蛋白的结合。感兴趣的DNA的设计，以约250包括乳糖运算符（LACO）序列的串联重复序列。 LACO特异性结合由乳糖阻遏蛋白（LacI的），它可以是稠合的荧光蛋白质。的融合蛋白可以从工程质粒或引进由逆转录病毒载体表达。在这种方式中，该DNA分子是荧光标记的，并因此成为可见的，通过荧光显微镜。的融合蛋白被阻止质粒是通过在IPTG存在下培养细胞中的质粒DNA的绑定，直到准备好以进行查看。 ontent“&gt;此系统允许被监视的质​​粒通过几代人在活细胞中，揭示其合成的属性和分区子细胞。理想的细胞粘附，容易转染，并具有大的细胞核。这种技术已经被使用，以确定84％的EBV-来源质粒合成每一代和88％的新合成的质粒分区忠实地对这些EBV质粒在HeLa细胞中的子细胞。看到被拴或与姐妹染色单体在S之后所合成阶段，直到他们被视为分开的姐妹染色单体分离，后期10的方法目前正在研究的KSHV在HeLa细胞中的基因组和SLK细胞的复制。HeLa细胞的永生化人上皮细胞，SLK细胞永生化人脐静脉内皮虽然SLK细胞最初来自一个KSHV病变的细胞。，本HeLa细胞也SLK细胞系自然哈布口服补液盐KSHV基因组的11。除了研究病毒的复制，该可视化技术可用于调查其他重组质粒DNA的合成，定位，和分区的各种DNA序列上的元素的添加，删除，或突变的影响。

<html>在图3中示出在一个有代表性的实验中获得的z-堆栈的最大强度投影。典型的质粒的信号都出现在3-8片的z-堆栈，这取决于它们是否代表单或簇的质粒。在EBV-和KSHV衍生质粒的情况下，在细胞内信号慢慢地移动，直到电池达到的有丝分裂。细胞本身迁移的实验过程。他们也变成球形，并在有丝分裂过程中脱离的菜。健康的HeLa细胞和SLK细胞的细胞周期约24小时，;这些类型超过30小时，?...

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Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

观察单个活细胞中的DNA分子的方法，进行说明。该技术是基于对一个荧光标记的lac阻遏物蛋白质设计到感兴趣的DNA结合位点的结合。此方法可适于后续许多随着时间的推移在活细胞中的重组DNA。

在Live哺乳动物细胞中质粒复制多代荧光显微镜监测

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

monitoring-plasmid-replication-live-mammalian-cells-over-multiple

Research

JoVE Journal

Biology

11.7K 次观看.  University of Wisconsin - Madison. 观察单个活细胞中的DNA分子的方法，进行说明。该技术是基于对一个荧光标记的lac阻遏物蛋白质设计到感兴趣的DNA结合位点的结合。此方法可适于后续许多随着时间的推移在活细胞中的重组DNA。

视频: Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

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 ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

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

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

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

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

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

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...