1。细胞计数的重要性<ol><li>计数细胞是在体外细胞培养的必要条件，但繁琐的步骤。 </li><li>一致的细胞浓度，保证实验的重复性和准确性。 </li><li>细胞计数，细胞的健康和增殖率的监测，评估永生化或转化，播种随后的实验中，转染或感染的细胞，细胞检测准备的重要。 </li><li>尽管细胞计数的速度和准确性的需要，400研究人员调查了使用血球计数细胞的71％。虽然hemocytometry价格低廉，它是费力，受到用户的偏见和误用，结果不准确计数。 </li><li> hemocytometry错误的来源包括：样品中的细胞分布不均匀，太多或太少的细胞样品中，以一个给定的单元格是否落在定义的计数区域内的主观决定，血球污染，用户到用户的变化，和血球充填率的变化。 </li><li>为了减轻人工计数的单调乏味，29％的研究人员使用自动细胞计数装置计数细胞。然而，大多数研究人员，与这些大型的台式仪器相关的价格是其使用的一个障碍。 </li><li>权杖细胞计数器是一个自动化的手持设备，在一个相对较低的的成本提供了自动化和库尔特计数的准确性。 </li><li>该系统采用一个微型传感，信号处理，数据存储和图形显示，使用了模拟和数字硬件相结合的格式的阻抗为基础的粒子探测库尔特原则。 </li><li>一次性传感器的设计与一个微型的细胞感应区，使细胞大小和细胞在亚微米和子皮升的决议量歧视。 </li><li>增强与高精度液体处理渠道和电子，权杖细胞计数图形显示直方图的细胞人口统计数字。 </li></ol> 2。为计数的单细胞悬液的制备<ol><li>要使用的权杖细胞计数，开始稀释的细胞的单细胞悬液，磷酸盐缓冲。 10,000-500,000每毫升细胞浓度是最佳选择。 </li><li> 100μL稀释的细胞转移到1.5 mL离心管。 </li></ol> 3。表演细胞计数使用的权杖<ol><li>打开权杖细胞计数，按住仪器背面的切换，并等待出现在屏幕上的说明。 </li><li>提示时，附加传感器电极感应朝向仪器的前面板的权杖单位年底。 </li><li>权杖传感器包括一个精密成型的采样室，电子感应区和综合性的细胞感应电极。该传感器有可能歧视在亚微米级分辨率的细胞大小，细胞体积子皮​​升分辨率。 </li><li>一致的传感器工程，确保平等的样品量的计算和精确的尺寸测量。 </li><li>一旦传感器连接，执行每一步的点票过程中，根据屏幕上的说明如下： </li><li>当屏幕上读取“按住柱塞开始，”抑制柱塞。屏幕将读“淹没传感器。”淹没传感器进入细胞悬浮液和释放柱塞吸进传感器的细胞悬液。 </li><li>柱塞作为一个电信号，将吸引到传感器的样品加死体积。暴跌的速度或精度并不重要，因为它与吹打。这一过程将需要几秒钟。准备就绪后，仪器会发出哔声，和“样品装入”将显示在屏幕上。 </li><li>一旦样品被装入，它是通过感应区画来评估细胞的大小和细胞体积，然后通过微传感器。一旦50μL的样品计数传感器告诉仪器停止计数。 </li><li>计数完成后，仪器的屏幕上阅读“计数完成，请移除传感器和丢弃。”按照提示，并删除和丢弃的传感器。 </li><li>该仪器将在其屏幕上显示一个细胞的大小或直径的直方图，以及细胞浓度为每毫升。 </li></ol> 4。数据分析<ol><li>后计数完成，并在仪器上显示直方图，申请按下切换按钮的大门。选择“自动门控”或“使用上的”自动设置基于直方图姿态门要么，或使用从以前的计数闸门。 </li><li>无论选择，盖茨可以手动移动微调的数据。按下切换按钮，再选择左边的门，然后滚动到ggle移动门。 </li><li>然后按下切换再次激活右门和滚动切换到地方的权利门。再次点击，将显示一个新的细胞浓度，平均细胞体积和直径。 </li><li>切换也可以移动到任何一方的开关之间的日期显示，时间，平均细胞大小，平均细胞体积。 </li><li>仪器本身可以存储多达72直方图。要上传到个人电脑的数据，将通过在显示屏幕上的USB线接口的仪器。 </li><li>在电脑上，双击权杖软件应用程序，以启动该软件，然后点击“连接”。所有的罪名将自动端口的软件。 </li><li>权杖应用软件显示直方图，详细介绍了人口分布的文化，无论是体积或直径。下载的文件，然后可以导出到Microsoft Excel中，原始数据将显示的特定的细胞直径或细胞卷箱。 </li></ol> 5。代表性的成果<ol><li>十九个不同的细胞系，准备计数使用库尔特计数器和理论出发浓度进行了测定。这些细胞悬浮液，然后进一步在理论的细胞浓度稀释至细胞悬浮液。 </li><li>然后用权杖细胞计数，以确定细胞浓度。图1显示了5 5代表性的细胞系，以及与理论的细胞浓度测量的稀释。 </li><li>的R2值表示高度的线性，表明权杖计数是一种可靠的方法测试，在很宽的线性工作范围，细胞株。 </li><li>图2表明，其中权杖计数所得的数据与库尔特计数所得的计数相比整体权杖计数的相对精度。 </li><li>权杖细胞计数的日志暗算库尔特计数的日志，并显示，所有单元测试线，权杖计数匹配库尔特计数。 </li><li>使用一个Z2的库尔特计数器，权杖细胞计数，一个自动化的基于视觉VI -细胞或无数的系统，如计数器，一个hemocytemeter，19个细胞株的计数。 </li><li>根据制造商的指示进行计数，使用相同的单元格开始悬挂和相同的稀释。在图3所示的结果表明权杖计数密切配合，具有高线性理论的细胞浓度，细胞浓度。 </li><li> ％的变异系数，或CV，计算出每个点票系统的每个细胞系的每个稀释。图4显示了超过19细胞株的细胞浓度和计算方法方面的平均％的CV。 </li><li>经营范围内，权杖细胞计数超过基于视觉的血球计数精确，显示较小的标准偏差和平均系数的变化较小。 </li><li>权杖计数比hemocytometry快十倍，速度比其他自动计数器。在图5，SF9，MCF7细胞，HEK293细胞计数使用此影片中所描述的方法。 </li><li>录得的平均所需时间来衡量细胞浓度。请注意，hemocytometry每个样本进行一次。 </li><li>权杖使得基于细胞大小的测量。因此，在细胞形态变化，可以捕获在直方图。健康的文化展示一个漂亮的钟形高斯分布，而不健康的细胞，溶解，并导致在直方图左移（图6，左侧面板）。 </li><li>同样，当了50％的死细胞人口与50％的活细胞混合，直方图的变化，也观察到（图6，右图）。 </li></ol><img src="/files/ftp_upload/2204/2204fig1.jpg" alt="图1" /><img src="/files/ftp_upload/2204/2204fig2.jpg" alt="图2" /><img src="/files/ftp_upload/2204/2204fig3.jpg" alt="图3" /><img src="/files/ftp_upload/2204/2204fig4.jpg" alt="图4" /><img src="/files/ftp_upload/2204/2204fig5.jpg" alt="图5" /><img src="/files/ftp_upload/2204/2204fig6.jpg" alt="图6" /><img src="/files/ftp_upload/2204/2204fig7.jpg" alt="图7" />

<ol>
	<li>Barghshoon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Counting Survey. , (2009).</li><li>Tucker, K. G., Chalder, S., al-Rubeai, M., Thomas, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+hybridoma+cell+number,+viability,+and+morphology+using+fully+automated+image+analysis.">Measurement of hybridoma cell number, viability, and morphology using fully automated image analysis.</a> Enzyme Microb Technol. 16 (1), 29-35 (1994).</li><li>Houwen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fifty years of hematology innovation: the Coulter principle. , (2003).</li></ol>

凯瑟琳Ongena，Chandreyee达斯，珍妮特史密斯索尼亚吉尔和格雷斯庄士敦受聘微孔，产生了本文中使用的仪器。

权杖细胞计数的性能比较从其他计数方法的结果，我们得出结论，这种新的手持设备，自动细胞计数在较宽的工作范围内提供精确，快速和可靠的细胞计数。权杖计数的功能，是一个嵌入到传感器头和库尔特原则为基础的复杂的计数仪表精密工程技术的结果。的性能质量的建议权杖方法来提高速度和提高细胞检测的可重复性的计数。手持格式允许在细胞培养罩快速单调乏味的免费票的权利。

<table border="1"><tbody><tr><td> 材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>手持权杖2.0 40微米权杖传感器自动细胞计数器</td><td> EMD的微孔</td><td> PHCC20040 </td><td></td></tr><tr><td>手持权杖2.0 60微米权杖传感器自动细胞计数器</td><td> EMD的微孔</td><td> PHCC20060 </td><td></td></tr><tr><td>权杖传感器，60微米</td><td> EMD的微孔</td><td> PHCC60050 </td><td> 50包的传感器</td></tr><tr><td>权杖传感器，40微米</td><td> EMD的微孔</td><td> PHCC40050 </td><td> 50包的传感器</td></tr><tr><td>通用电源适配器</td><td> EMD的微孔</td><td> PHCCPOWER </td><td> </td></tr><tr><td>权杖O型圈试剂盒</td><td> EMD的微孔</td><td> PHCCOCLIP </td><td> 2 O形圈和一个过滤器盖</td></tr></tbody></table>

determining cell number during cell culture using the scepter cell counter

计数细胞， 在体外细胞培养往往是必要的，但繁琐的步骤。一致的细胞浓度，保证实验的重复性和准确性。细胞计数，细胞的健康和增殖率的监测，评估永生化或转化，播种随后的实验中，转染或感染的细胞，细胞检测准备的重要。重要的是准确，一致和快速的，特别是对于细胞反应的定量测量，细胞计数。 尽管这种细胞计数的速度和准确性的需要，71％接受调查的400 研究人员 1人使用血球计数细胞。虽然hemocytometry价格低廉，它是费力，受到用户的偏见和误用，结果不准确计数。特种光学玻璃，装在指定的卷，并在显微镜下计数细胞悬液Hemocytometers。 hemocytometry错误的来源包括：样品中的细胞分布不均匀，太多或太少的细胞样品中，以一个给定的单元格是否落在定义的计数区域内的主观决定，血球污染，用户到用户的变化和血球变化充填率2。 为了减轻人工计数的单调乏味，29％的研究人员使用自动细胞计数装置计数细胞，其中包括基于视觉的计数器，检测细胞使用库尔特原则的系统，或流式细胞仪 1 。对于大多数研究人员，使用一个自动化系统的主要障碍是与这些大型的台式仪器 1相关价格。 权杖细胞计数器是一个自动化的手持设备，在一个相对较低的的成本提供了自动化和库尔特计数的准确性。该系统采用阻抗为基础的粒子探测，在一个小型化的格式3库尔特原则使用的模拟和数字硬件相结合的传感，信号处理，数据存储，图形显示。一次性提示是设计一个微型的细胞感应区，使细胞大小和细胞在亚微米和子皮升的决议量歧视。精密液体处理渠道和电子增强，权杖的细胞的计数器报告细胞群统计图形直方图显示。

Watch this Scientific Journal Video about Determining Cell Number During Cell Culture using the Scepter Cell Counter at JoVE.com

Determining Cell Number During Cell Culture using the Scepter Cell Counter

权杖细胞计数是一种手持式的自动化设备，可用于计数细胞，细胞的直径和体积的监测，用于检查从一种文化到下一个细胞群的健康和质量。

确定在细胞培养的细胞数量，使用权杖细胞计数

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

determining-cell-number-during-cell-culture-using-scepter-cell

Research

JoVE Journal

Biology

37.8K 次观看.  Millipore Inc. 权杖细胞计数是一种手持式的自动化设备，可用于计数细胞，细胞的直径和体积的监测，用于检查从一种文化到下一个细胞群的健康和质量。

视频: Determining Cell Number During Cell Culture using the Scepter Cell Counter

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

人类胚胎干细胞：启动文化冷冻细胞

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

科研

生物学

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

测定哺乳动物细胞计数，细胞的大小和使用末席ž迷你自动细胞计数细胞的健康

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

的三种不同方法对乳腺癌细胞株确定细胞增殖的比较

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

活体细胞荧光显微镜观察微生物细胞生长过程中的基本过程

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.