这项工作是支持由美国国立卫生研究院资助1R21AG031965 - 01A1。 Mk为中老年埃利森医学基金会新学者。

第1部分：老龄化的文化准备<ol><li>到YEPD培养基琼脂平板冻结的股票（1％酵母提取物，细菌用2％的蛋白胨，2％琼脂，2％葡萄糖）利息数株。 </li><li>在30˚C的48小时或直到出现单菌落培养的细胞。 </li><li>选入YEPD液体培养基在试管中的5毫升（1％酵母提取物，细菌用的蛋白胨2％，2％葡萄糖）单菌落，接种。 </li><li>拓展文化30一夜之间˚C，而保持恒定的搅拌，使用一个振动筛或滚鼓。 </li><li> 50μL过夜培养，接种5毫升合成完整的（SC）中型（见表1）。一般有三种SC文化是为宗正每一株式三份生物复制的寿命分析每个应变准备。 </li><li>文化保持在30˚C的整个实验的辊（一般为2个或更多个星期）鼓上不断搅拌。 </li></ol>第2部分：考虑一个可行性的年龄点经过两天在SC媒体文化，细胞应在固定相和，第一年龄点是准备采取。随后的年龄应采取每2-3天至少两个星期。对于每个年龄段点： <ol><li>准备填写每孔145μLYEPD BIOSCREEN 100接种蜂窝板。只YEPD和数据分析无细胞后一定要留下，以及填补了至少一个。 </li><li>从孵化器中取出老化的文化。 </li><li>简言之涡接种到蜂窝板，同时小心，不要泄漏任何文化的第一个文化。 </li><li>取出5μL的混合文化和吸管成蜂窝板的第一口井。火焰口前和每个试管中取出后，5μL等份。 </li><li>重复此过程中，一定要注意每一种文化的相应位置以及每个老化文化。随后在整个实验的每个年龄点，应使用相同的良好阵地。 </li><li>更换成30˚彗星孵化器的文化与接种完成。 </li></ol>第3部分：载入BIOSCREEN彗星的MBR机<ol><li>揭露解除盖子孵化器舱，取出样品盘盖。 </li><li>新接种的蜂窝板插入样品盘（如果你只阅读一个板块，使用左侧的插槽）。 </li><li>更换样品盘盖和降低孵化器仓盖。 </li><li>检查，以确保传热流体上面填写最低水平。如果低，增加更多的使用了1000μL移液器提供的传热流体。 </li><li> BIOSCREEN软件“EZExperiment”的使用，设置以下参数，以获得适合酿酒酵母生长曲线： <ul><li>样品编号：输入与媒体或200井数</li><li>过滤器：420 - 580nm，宽带</li><li>温度：30˚彗星</li><li>实验时间：1天0小时0秒</li><li>测量间隔：30分钟</li><li>振动，连续晃动，高</li></ul></li><li>点击“开始”开始读数。 </li></ol>第4部分：数据分析通常情况下，6个年龄点采取了两个星期的课程。 2天，4，6，9，11和13年龄点。根据实验设计和被测试的菌株，它可能需要采取年龄点或多或少频繁或时间超过2周。重要的是要以相同的顺序加载每个年龄点，每个老化文化对应相同的立场以及在每一个年龄点，因为这将会使数据分析更容易蜂窝板。 <ol><li>获取BIOSCREEN彗星MBR机的输出文件。 EZExperiment“软件”将输出作为制表符分隔的文件，与Microsoft Excel以及其他软件兼容的BIOSCREEN数据。第一列显示的OD测量，在什么时候采取了在实验过程中，代表BIOSCREEN蜂窝板（图1）每口井的后续列。 </li><li>删除从每列的第一个外径阅读。这种阅读是“噪音”。 </li><li> OD值减去单独YEPD所有的OD值在每一列以及与标准化的数据。这将删除媒体的背景吸光度。 </li><li>绘制生长曲线。曲线将转向功能的年龄（图2）。例如，从第1列超过6个时间点（蜂窝板1）通过绘制生长曲线，随着时间的推移有一个鲜明的曲线右移。 </li><li>每口井的第2天的年龄点的生长动力学为基础，计算倍增时间（δ）。公式用于计算倍增时间，我S： <img src="/files/ftp_upload/1156/equat.png" alt="方程" /> 外径1和外径2表示连续OD测量和T 1和T 2的测量之间的时间。计算倍增至0.5 OD值0.2倍。这些值的平均值，以及倍增时间。计算倍增至0.5 OD值0.2倍。这些值的平均值，以及倍增时间。大多数野生型酵母菌株的，应给予85至90分钟之间增加一倍的时间价值。 </li><li>对于每个年龄点，计算时移（ΔT）在生长曲线的初始年龄点（2天）。一个简单的方法做到这一点是确定的时间长短不同，每口井之间的初始年龄点，每个后续的年龄点达到了0.3外径。 ，尤其是很好地达到了0.3 OD时，可以计算出相应的线性回归方程来LN（OD值）之间的时间为两个时间点包围OD = 0.3功能。 </li><li>尚存在每个年龄段点，以产生一种生存曲线（图3）计算分数。定义的初始年龄点（2天），以100％的可行性。对于每个连续的年龄点，使用公式计算％生存： <img src="/files/ftp_upload/1156/equat2.png" alt="第二个方程" width="245" height="115" /> 其中S n是生存的百分比，ΔTñ是时间的转变，δn是倍增时间。 </li><li>生成绘图岁的函数活菌比例（％）生存曲线（图3B），为每口井的井（或复制）所需。 </li><li>计算每口井的生存积分（SI）。 SI被定义为生存曲线下的面积，可以通过公式估计： <img src="/files/ftp_upload/1156/equat3.png" alt="公式3" width="400" height="79" /> 其中年龄ñ年龄点（如2，4，6，9，11和13）和S N是在这个年龄点的生存价值。 </li><li>确定从SI和生存数据复制井的统计参数。共同关心的统计参数，包括平均数，中位数，每个生物复制方差。可用于不同的实验组和对照组的SI两两比较t检验或类似的分析。它也可能是可取的，生成的生存曲线，平均生物复制为4.8。 </li></ol>第5部分：代表结果在实验完成后，你将有绘制生存曲线，并进行数据分析足以确定老化的时间为几个不同的菌株或条件的潜力。如果正确执行，从BIOSCREEN彗星MBR机获得的生长曲线看起来应该类似于图2所示的生存曲线应类似于图3所示的。一般来说，这里所描述的条件下培养的野生型细胞，将有7天的顺序中位数顺序寿命。在生存的巨大变化，观察在不同的菌株，并在某些情况下，如在0.05％的葡萄糖媒体的增长，中位生存期可超过30天。 <img src="/files/ftp_upload/1156/1156fig1.png" alt="" /> 图1。数据输出BIOSCREEN方案“EZExperiment”。 A栏显示的吸光度读数的时间。连续列表示，从老化的文化细胞接种蜂窝板的水井。 <img src="/files/ftp_upload/1156/1156fig2.png" alt="" /> 从一个单一的实验过程中的生物复制的生长曲线图2。曲线有一个独特的转变，随着时间的推移，在老龄化的文化细胞失去生存能力。初始时间点（2天）和一个连续的时间点之间的时间长度确定在那个特定的时代的可行性。 <img src="/files/ftp_upload/1156/1156fig3a.png" alt="" /><img src="/files/ftp_upload/1156/1156fig3b.png" alt="" /> 图3。 A）的生存曲线，生成从图1中使用的生长数据。第2天的时间点设置为100％的可行性点 B）最后两个菌株的存活曲线，在相同的实验测试。这些生存曲线代表三个生物复制为每一株的平均存活率。误差线表示标准偏差范围内的生物复制。下的生存曲线的阴影区域代表的生存应变1积分（SI）。 <table border="1"><tbody><tr><td colspan="2">表1。合成的定义中等年代老化研究（株backgrouND BY4743） </td></tr><tr><td>组件</td><td>浓度（g / L的） </td></tr><tr><td> D -葡萄糖</td><td> 20 </td></tr><tr><td>酵母氮基（-AA/AS） </td><td> 1.7 </td></tr><tr><td> （NH 4）2 SO 4 </td><td> 5.0 </td></tr><tr><td>腺嘌呤</td><td> 0.04 </td></tr><tr><td> L -精氨酸</td><td> 0.02 </td></tr><tr><td> L -天冬氨酸</td><td> 0.1 </td></tr><tr><td> L -谷氨酸</td><td> 0.1 </td></tr><tr><td> L -组氨酸</td><td> 0.1 </td></tr><tr><td> L -亮氨酸</td><td> 0.3 </td></tr><tr><td> L -赖氨酸</td><td> 0.03 </td></tr><tr><td> L -蛋氨酸</td><td> 0.02 </td></tr><tr><td> L -苯丙氨酸</td><td> 0.05 </td></tr><tr><td> L -丝氨酸</td><td> 0.375 </td></tr><tr><td> L -苏氨酸</td><td> 0.2 </td></tr><tr><td> L -色氨酸</td><td> 0.04 </td></tr><tr><td> L -酪氨酸</td><td> 0.03 </td></tr><tr><td> L -缬氨酸</td><td> 0.15 </td></tr><tr><td>尿嘧啶</td><td> 0.1 </td></tr></tbody></table> 注：此配方为二倍体BY4743应变auxotrophies帐户。合成完全培养基加入终浓度5倍，氨基酸auxotrophies应该得到补偿。

<ol>
	<li>Steinkraus, K. A., Kaeberlein, M., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+aging+in+yeast:+the+means+to+the+end.">Replicative aging in yeast: the means to the end.</a> Annu Rev Cell Dev Biol. 24, 29 (2008).</li><li>Kaeberlein, M., Conn, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of models for human aging. , 109 (2006).</li><li>Fabrizio, P., Longo, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chronological+life+span+of+Saccharomyces+cerevisiae.">The chronological life span of Saccharomyces cerevisiae.</a> Aging Cell. 2 (2), 73 (2003).</li><li>Murakami, C. J., Burtner, C. R., Kennedy, B. K., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+high-throughput+quantitative+analysis+of+yeast+chronological+life+span.">A method for high-throughput quantitative analysis of yeast chronological life span.</a> J Gerontol A Biol Sci Med Sci. 63 (2), 113 (2008).</li><li>Piper, P. W., Harris, N. L., MacLean, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preadaptation+to+efficient+respiratory+maintenance+is+essential+both+for+maximal+longevity+and+the+retention+of+replicative+potential+in+chronologically+ageing+yeast.">Preadaptation to efficient respiratory maintenance is essential both for maximal longevity and the retention of replicative potential in chronologically ageing yeast.</a> Mech Ageing Dev. 127 (9), 733 (2006).</li><li>Fabrizio, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+is+a+mediator+of+an+altruistic+aging+program+in+Saccharomyces+cerevisiae.">Superoxide is a mediator of an altruistic aging program in Saccharomyces cerevisiae.</a> J Cell Biol. 166 (7), 1055 (2004).</li></ol>

高通量的时间顺序寿命试验，这里描述的是一个量化的菌株具有高准确度和精密度的大量老化潜力的有效方法。这种方法比传统方法主要事先确定计数菌落形成单位（如见3）的生存是一个摇床/孵化器/板读取装置，如使用的BIOSCREEN彗星MBR机获取高分辨率的生长曲线每个年龄点。在直接比较低吞吐量，按时间顺序寿命试验，这种方法已被证明，以达到可比性（或更好）的精度，同时可以...

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		<th>Comment</th>
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<td>Bacto Peptone</td>
<td>Reagent</td>
<td>BD</td>
<td>211677</td>
<td>&nbsp;</td>
<tr>
<td>Bacto Yeast Extract</td>
<td>Reagent</td>
<td>BD</td>
<td>288620</td>
<td>&nbsp;</td>
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<td>Difco Agar</td>
<td>Reagent</td>
<td>BD</td>
<td>214530</td>
<td>&nbsp;</td>
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<td>Yeast Nitrogen Base w/o A.A. and A.S.</td>
<td>Reagent</td>
<td>MidSci</td>
<td>J630-500G</td>
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<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
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<td>Fisher</td>
<td>D16-10</td>
<td>&nbsp;</td>
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<td>Growth Curves USA</td>
<td>5101370</td>
<td>&nbsp;</td>
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<td>Bioscreen 100-well Honeycomb plate</td>
<td>Tool</td>
<td>Growth Curves USA</td>
<td>9502550</td>
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quantifying yeast chronological life span by outgrowth of aged cells

芽殖酵母<em&gt;酿酒酵母</em&gt;已被证明是一个重要的模式生物，在衰老研究领域<sup&gt; 1</sup&gt;。复制和时间顺序寿命是用来研究在酵母老化的两个既定的范式。复制性衰老定义为一个单一的酵母母细胞在衰老之前产生的子细胞的数量，按时间顺序老化时间细胞的长度定义能够生存在一个不分裂的，静止的状态<sup&gt; 2</sup&gt;。我们已经开发出一种高通量的方法按时间顺序寿命的定量测量。此方法涉及定义介质在搅拌下，在恒定温度和老化细胞。每个年龄点，一个细胞亚群是从老龄化的文化，并接种到丰富的生长介质。一个高分辨率的生长曲线，然后获得这种使用BIOSCREEN彗星的MBR机的老化细胞亚群。一个算法，然后应用，确定可行的细胞在每个分人口的相对比例，在每个年龄点的生长动力学。这种方法需要更少的时间比其他时间顺序寿命检测和资源，同时保持重现性和精密度。这个实验的高通量的性质，应允许进行大规模的遗传和化学的屏幕，在更复杂的生物进一步检测，以确定新型长寿修饰符。

Watch this Scientific Journal Video about Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells at JoVE.com

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

在酵母老化的年代，是指与固定相相关的细胞活力的损失。在这里，我们描述了一个高通量的方法，定量确定酵母按​​时间顺序寿命。

量化老化细胞生长的酵母年谱跨度

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

quantifying-yeast-chronological-life-span-by-outgrowth-of-aged-cells

Research

JoVE Journal

Biology

16.5K 次观看.  University of Washington. 在酵母老化的年代，是指与固定相相关的细胞活力的损失。在这里，我们描述了一个高通量的方法，定量确定酵母按​​时间顺序寿命。

视频: Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

一个现场的固定化酵母细胞可视化的快速技术

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

科研

生物学

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

测量线虫固体介质的寿命

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

在芽殖酵母中的自我复制寿命测量

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

测量线虫寿命在96孔板

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

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 ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

在单酵母细胞MAPK诱导表达的时空定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

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) ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

人外周血代文化血液的产物内皮细胞

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.