我们感谢安迪·约翰逊在不列颠哥伦比亚大学的生物医学研究中心的协助FACS分析。巨蟹座在黄实验室的研究经费是由加拿大癌症协会研究所（经营赠款＃019250）和药学院，不列颠哥伦比亚大学医学院的研究再投资资金。 JMYW由加拿大研究主席迈克尔·史密斯基金会为卫生研究职业发展计划的支持。

1。细胞的制备<img src="/files/ftp_upload/4045/4045fig0.jpg" alt="图0" /> <ol><li>板细胞密度达到了约60％汇合。细胞必须在日志阶段在收集的时间。对于MCF7细胞，这是完成播种在5×10 5细胞/ 10厘米板，在适当的媒体。 HT29和LS180细胞接种在6×10 5细胞/ 6厘米的板。我们用于补充10％胎牛血清和1x青霉素/链霉素的DMEM培养基。要确保正确的阳性和阴性对照种子板除了您的测试样品。它们包括以下内容: </li></ol><table border="1"><tbody><tr><td>我</td><td>阳性对照</td><td>尿嘧啶只</td></tr><tr><td>二</td><td>阳性对照</td><td>有价证券投资只</td></tr><tr><td>三</td><td>阴性对照</td><td>尿嘧啶负，有价证券投资负</td></tr></tbody></table> （应当指出的是，与多个时间点，如在本协议所述的初步实验可以用来缩小的时间框架，在此期间，开展未来的集合。） <ol start="2"><li>镀细胞培养在37℃，5％的CO 2为24-48小时，从而使细胞恢复和重视。 </li><li>脉冲溴脱氧尿苷（尿嘧啶）标记细胞，培养液取代无线日含10μM的尿嘧啶的新媒体。细胞在37℃，5％CO 2的BrdU掺入到DNA的允许1小时。是一定要留下一个盘子未经处理作为阴性对照。 </li><li>脉冲标签媒体被删除，简单地用1X PBS冲洗细胞。 </li><li>添加新鲜培养基（零下的BrdU）和细胞被允许继续孵育在37℃，5％CO 2，直到收获的适当时间点到达。 </li></ol> 2。采伐和固定<ol><li>从步骤1.3未经处理的细胞被认为是零的时间点。这个范例可以一起用了1小时的时间点所收集的BrdU治疗后立即收获。 </li><li>收获的细胞，媒体被删除，板与1X PBS冲洗一次。 </li><li>是胰酶消化细胞，收集培养媒体，并在1500转离心5分钟支持随后颗粒ernatant被丢弃。 </li><li>在5毫升冰冷1X PBS冲洗1-10×10 6个细胞。在1500转离心5分钟上清被丢弃。 </li><li>然后将细胞沉淀悬浮于100μL冰冷的PBS / 1％FBS。此外，在防止细胞凝集1％FBS的PBS中艾滋病。 </li><li>添加下降到5毫升-20℃，70％的乙醇固定细胞，这些细胞下降。 </li><li>在冰上孵育30分钟或储存在4°C过夜。这是一个理想的步骤，停止从各时间点的样品采集。可以留在乙醇样品数天，让他们同时通过协议的其余部分进行处理。 </li></ol> 3。 BrdU和PI染色<ol><li> 5分钟1500转离心沉淀细胞。 </li><li>取出固定，但离开〜50μL涡旋颗粒放松。 </li><li>变性的DNA，慢慢加入1毫升2N盐酸/剂Triton X-100滴加而涡旋。室温孵育30分钟的样品。 </li><li>离心5分钟1500转的样品颗粒细胞。吸和弃上清。 </li><li>悬浮细胞沉淀在1毫升0.1M四硼酸钠，pH值8.5，以消除变性步骤。 </li><li> 5分钟1500转离心细胞。吸和弃上清。 </li><li> BrdU染色混合在75μL（50μL0.5％吐温20/1％的BSA / PBS + 20μLFITC标记的抗BrdU + 5μL10毫克/毫升核糖核酸）重悬细胞沉淀。 </li><li>在室温下孵育45分钟，避光。 </li><li>以颗粒细胞，离心5分钟1500转的样品。吸和弃上清。 </li><li> 1毫升含5μg/ ml的碘化的PBS重悬细胞沉淀。 </li></ol> 4。流式细胞仪<ol><li>分析细胞，流式细胞仪配备有一个488 nm激光和适当的过滤器是必要的。如CellQuest软件分析是必要的，以创建下面列出的地块。 </li><li>通过流式细胞仪的样品运行时，创建一个前向散射（SSC-H的）与侧散射（FSC-H的）情节，以确保适当的细胞大小分布（ 图1A）。这些参数都被画上的线性度。 </li><li>同时查看FL3-H的（有价证券染色）主场迎战的FL2 - W（ 图1B）阴谋。此图是用来创建一个门（4915）分离的部分细胞内正常的细胞周期的分布格局。在一般情况下，这种分布格局的特点是由两个2N 4N DNA含量的PI染色较G1和G2期的细胞周期，分别从细胞群。正在进行DNA复制发生在细胞周期的S期细胞位于这2个集群之间的字符串代表。 </li><li>创建一个阴谋displayi吴的门细胞从57-H的步骤4.3（FITC标记，日志图）（4915），Y轴在X轴（ 图1C）和PI（线性图）。该地块将用于设置使用1.1​​中所述的各种控件，以及收集数据进行分析的其余参数。 </li><li>将到流式细胞仪的PI只染样品。调整增益G1 x轴放置在〜200。这将是更容易在直方图的PI染色情节形象化。 </li><li>第二个控制涉及流式细胞仪上运行的只有1尿嘧啶样品。调整增益，使细胞的2种群（BrdU阳性的BrdU负）上出现的情节。理想的情况下，阴性细胞的BrdU定位出现低于10 -1。 </li><li>最终控制的BrdU / PI阴性样品。运行此阴性对照，以确保细胞不会出现在任何上限或右手象限。 </li><li>一旦各个地块的参数已经设置，流式细胞仪校准和准备BrdU和PI染色的细胞进行处理。至少10000个细胞，在正确的PI分数（请参考步骤4.3）的门必须读取每个集合。 </li><li>分析收集到的数据，如FlowJo或FacsDiva软件利用。上述地块可以重新在这些方案，并进行定量和统计分析。 </li><li>分析端点涉及几个连续的步骤。创建的FITC与PI PI PI对比的FL2 - W阴谋积极门与细胞的情节，允许一个区分单车人口。第二个门是从这个小区创建隔离BrdU阳性人口。这种独特的人口直方图情节与x轴PI表示，允许一个追踪细胞周期进程的时间过程。这可以是进一步绘制BrdU阳性细胞数量与G1或G2的内容作为时间的函数的可视化。 </li></ol> 5。代表结果通常用碘化丙啶染色循环细胞有明显的峰值，在G1和G2，对应的细胞，分别含有2N和4N DNA含量（ 图2）。 BrdU标记脉冲允许一个亚群体正在积极合成DNA（即S期）的细胞的选择性标记。拆除的BrdU试剂后不久，所有标记的细胞S期（ 图3）。限制短脉冲的标签之一，是能够按照各地大量的时间点现在不同的细胞亚群体，因为他们通过对细胞周期的后续阶段的传递。这可以是可视化，在1小时的时间点明显缺乏的G1和G2峰图3。 其他信息可从BrdU标记步骤。不仅可以积极分裂的细胞的比例来衡量，而是一种ESTimate两个样本之间的阶段，细胞周期分布以及可确定。等距的BrdU试剂去除后的时间间隔收集细胞，细胞可以追溯到作为他们继续循环，进展到G2，最终移动到细胞的原始细胞的分裂，终于新兴BrdU阳性子细胞与G1 DNA含量（ 图4）。细胞周期的量化和动力学分析的一个例子是在图5。 <img src="/files/ftp_upload/4045/4045fig1.jpg" alt="图1" /> 图1。（一）前向散射对MCF7细胞的人口有代表性的阵营散点图。 （二）PI与宽度（FL-W），剧情一个有代表性的MCF7细胞的人口。浇注被排除在最终的分析（4915）细胞的双峰。细胞双峰脉冲宽度大于一个单细胞，因为它们需要更长的时间，通过激光束和theref通过矿石可以从分析中排除。 （三）有价证券与FITC（尿嘧啶）绘制一个有代表性的MCF7细胞的人口。浇注包括只有FITC（尿嘧啶）（R4）的阳性细胞。 <img src="/files/ftp_upload/4045/4045fig2.jpg" alt="图2" /> 图2。代表总人口的细胞通常骑自行车直方图情节。 <img src="/files/ftp_upload/4045/4045fig3.jpg" alt="图3" /> 图3。已收集后拆除的BrdU脉冲后1h门FITC（尿嘧啶）阳性细胞，细胞直方图的情节。在早期的时间点BrdU阳性细胞，去除标签后显示的PI型材，对应的样本显示DNA含量与细胞，在细胞周期的S期，DNA合成的细胞，只有在确认成功标签一致。 <img src="/files/ftp_upload/4045/4045fig4.jpg" a<html> LT ="图4"/&gt; 图4。细胞周期的肿瘤细胞株，大肠癌HT29（A）和LS180（二），以及乳腺癌MCF7（c）之间的进展动力学比较。收集细胞，每隔一小时，8小时后拆除的BrdU脉冲。在这个实验中，我们发现在大肠癌细胞株LS180通过细胞周期G2期的加速细胞周期进程的轮廓清晰。动力学，大肠癌细胞的两个剖面之间的比较是明显的G1峰出现在t = 4H后的BrdU在LS180细胞相比，HT-29细胞缺乏此峰与相应的时间点。要么两个大肠癌细胞株相比，MCF7细胞是骑自行车在率显着降低。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4045/4045fig4large.jpg">点击这里查看大图</a> 。 <html>重5"/&gt; 图5（一）定量细胞周期的BrdU标记的癌细胞相分析。院长/杰特/福克斯算法应用于HT29，LS180和MCF7（绿色所示）。表示为％的总BrdU阳性每个阶段的细胞产生的细胞周期相分布，从每个样品。只能选择时间点，每个细胞株显示，一些较早的时间点的分析结果无效。在这些较早的时间点，所有的BrdU阳性细胞在S期，因此，缺乏鲜明的G1和G2的高峰，这是算法的应用所必需的。 （二）癌细胞的直方图显示动力学的进展，通过对细胞周期的G2 / M期。 LS180细胞，HT29和MCF7通过G2 / M期的进展是最快的。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4045/4045fig5large.jpg">点击这里查看大图</a> 。 <html> JPG"ALT ="图S1"/&gt; 图S1。流式细胞仪控制。 （一）阴性对照细胞既不是PI也不尿嘧啶染色显示MCF7细胞。 （乙）有价证券仅染色。 （三）尿嘧啶-FITC标记的样品。 <img src="/files/ftp_upload/4045/4045figS2.jpg" alt="图S2" /> 图S2示意图显示的PI与FITC（尿嘧啶）的荧光发射光谱之间的关系。荧光通道1（FL1，FITC标记）和荧光通道3（FL3，PI）的光谱收集窗口显示在相应的框中。有没有与FL1和FL3检测的荧光光谱重叠。由此可见，荧光补偿是没有必要的PI和FITC-尿嘧啶的实验数据共同标记细胞分别在FL1/FL3渠道收集。荧光光谱获得了BD公司网站:目标="_blank"&gt; http://www.bdbiosciences.com/research/multicolor/spectrum_viewer。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4045/4045figS2large.jpg" target="_blank">点击这里查看大图</a> 。

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我们什么都没有透露。

结合流式细胞仪BrdU掺入，我们有必要的工具来研究细胞周期动力学。运作作为胸苷类似物的BrdU的独特属性是允许单车​​细胞的DNA含量定量。是什么让一个按照循环细胞亚群，通过S期的DNA复制到G2期的增长，并最终以细胞分裂纳入到越来越多的女儿在合成阶段的细胞周期的DNA链的BrdU 。女儿BrdU阳性细胞可连续之后，直到在时间点的BrdU标签稀释细胞分裂的信号降低到背景水平的程度。 <p class="j...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>溴</td><td>碧迪</td><td> 55089 </td><td></td></tr><tr><td>碘化</td><td>西格玛</td><td> 287075 </td><td> 1mg/ml的股票</td></tr><tr><td> FITC标记的抗-尿嘧啶</td><td>碧迪</td><td> 347583 </td><td></td></tr><tr><td>四硼酸钠</td><td>费舍尔</td><td> S80172 </td><td> 0.1M，pH值8.5 </td></tr><tr><td>流式细胞仪口径</td><td>碧迪</td><td></td><td></td></tr></tbody></table>

measuring cell cycle progression kinetics with metabolic labeling and flow cytometry

通过细胞周期的各个阶段的启动和后续进展的精确控制细胞增殖至关重要。细胞周期分裂，生长和繁殖和细胞周期的关键组成部分的放松管制已在诱发癌变1,2事件有牵连的一个组成部分。分子药物抗癌疗法经常针对负责监管和协调细胞周期分裂3的生物学途径。虽然细胞周期动力学往往根据不同细胞类型，细胞之间的细胞周期的四个阶段的分布是一致而不是在一个特定的细胞株，由于一贯的有丝分裂和生长因子的表达。遗传毒性事件和其他细胞的压力可导致细胞周期进程的一个临时块，在逮捕或在一个特定的细胞周期阶段的暂时停止导致允许instigation适当的反应机制。 实验观察与参考其细胞周期的进展阶段的细胞群的行为的能力是在细胞生物学的重要进展。常见的程序，如丝分裂摆脱，差速离心法或流式细胞仪为基础的排序是用于分离细胞，细胞周期的特定阶段4-6。这些分馏，细胞周期，丰富的人口，然后试验性治疗。产量，纯度和分离组分的可行性，可以经常使用这些物理的分离方法受到损害。同时，分离的细胞群，并开始实验性治疗，使分馏细胞可以进步，从选定的细胞周期阶段之间的时间间隔，可以构成重大的挑战在这些实验的成功实施和解释。 ST其他方法的UDY细胞周期阶段包括化学品的使用同步细胞。治疗的细胞，每个细胞周期阶段的关键代谢过程中的化学抑制剂阻止细胞周期进展到下一阶段的有用。例如，核苷酸还原酶抑制剂羟基脲停止在G1 / S期交界处的细胞限制供应脱氧核苷酸，DNA的积木。其他著名的化学品包括用aphidicolin治疗，G1期阻滞聚合酶阿尔法抑制剂，与秋水仙碱和诺考达唑治疗，这两种干扰有丝分裂纺锤体的形成，以停止在M期细胞，最后，治疗与DNA链终止5-fluorodeoxyridine的启动S期阻滞7-9。这些化学品的处理是同步在​​某个特定阶段的一个细胞的整个人口的有效手段。去除与化学，细胞归队齐声细胞周期。测试代理以下释放的治疗从细胞周期的阻断化学，确保引起的药物反应是从一个统一的，细胞周期的特定阶段的人口。然而，由于许多化学同步器是已知的基因毒性化合物，戏弄除了参与各种反应途径（与测试代理的同步器），是具有挑战性的。 在这里，我们描述了通过他们从DNA复制阶段的进展，通过他们的女儿细胞的分裂和分离的积极骑自行车细胞亚群，代谢标记方法。再加上流式细胞仪定量测量细胞周期动力学进展，该协议使在没有任何机械或化学诱导细胞强调，通常与其他细胞周期同步方法10。在下面的章节中，我们将讨论的方法，以及一些在生物医学研究中的应用。

Watch this Scientific Journal Video about Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry at JoVE.com

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

在细胞周期各阶段的进展和动力学跟踪微妙的变化，可以实现通过碘化丙啶与BrdU和总DNA染色核酸代谢标记的组合使用。这种方法避免了化学循环细胞同步的需要，从而防止非特异性的DNA损伤，这反过来又影响细胞周期进程的介绍。

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 ...

measuring-cell-cycle-progression-kinetics-with-metabolic-labeling

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

21.0K 次观看.  University of British Columbia. 在细胞周期各阶段的进展和动力学跟踪微妙的变化，可以实现通过碘化丙啶与BrdU和总DNA染色核酸代谢标记的组合使用。这种方法避免了化学循环细胞同步的需要，从而防止非特异性的DNA损伤，这反过来又影响细胞周期进程的介绍。

视频: Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

测量中使用FM标签的神经元的胞外分泌

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

科研

生物学

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

研究与基于荧光素酶的记者基因检测膜生源

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

测量细胞内Ca<sup&gt; +</sup在人类精子的变化采用4项技术:传统的荧光，停流荧光，流式细胞术及单细胞成像

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED热流量 - 结合光遗传学与流式细胞仪

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

代谢标记和流式细胞仪测定细胞周期进程的动力学

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