作者要感谢德特勒夫·舒曼，詹妮弗·萨顿（赛默飞世尔科技）和纳撒尼尔·斯奈德（宾夕法尼亚大学）为质量校准和数据处理有价值的讨论。研究报告本出版物中得到了卫生部下奖号码R00CA168997国家研究院​​国立癌症研究所的支持。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

1，准备LC-MS试剂，建立一个色谱法，并建立仪器操作规程<ol><li>液相色谱溶剂的制备<ol><li>制备500mL的流动相。 A为20mM的醋酸铵和15mM的氢氧化铵的3％乙腈/水，最终pH为9.0;和B为100％乙腈。 </li><li>松散帽的瓶子，把它放在水浴超声仪，超声10分钟，无需额外加热。 （此步骤是为了确保所有的铵盐的完全溶解和不存在残留气泡。） </li><li>转让250毫升溶剂的250毫升玻璃瓶中用于LC-MS的使用，并保持剩余部分在4℃下的</li></ol></li><li>准备低质量范围的校准解决方案。使用定制的低质量范围的校准混合物代谢组学的应用，以确保准确的群众在低分子量的检测是非常重要的。 <ol><li>称取5毫克两种SODI的恩氟乙酸和高香草酸和溶解它们到5ml的水中，使其为1毫克/毫升的最终浓度。二嗪磷溶解在甲醇中，制成10毫克/毫升的最终浓度。 </li><li>为了准备加入1ml负低质量校准溶液，混匀960毫升用20毫升钠fluoroaceate和高香草酸溶液的热负校准解决方案。至1ml的正向低质量校准溶液，混匀990毫升热正校准溶液和10ml二嗪农的解决方案。 （低质量校准溶液应保存在4℃，将新鲜制备的每2个月。） </li></ol></li><li> QE-MS的校准在低质量范围<ol><li>之前执行低质量范围内的校准，进行在根据制造商的指示肯定和否定模式标准的质量校准（M / Z，150-2,000）。 </li><li>一旦常规质量校正通过，调整扫描范围为60-900 M / Z在仪器控制面板和25伏特为阳性模式和35伏特为负模式源的CID被应用。 （这将使咖啡因碎片离子和硫酸根离子的健壮信号，这里的扫描范围是固定的，因为最后的M / Z应不低于起始为m / z 15倍放大） </li><li>一旦离子源是稳定的，则执行自定义校准。注意：一个稳定的源被定义为总离子电流的变化在正模式的小于10％，并且在负离子模式下小于15％。定制的校准离子和相应的m / z的列于表1。 </li></ol></li><li>建立了LC-MS仪器的极性代谢物的分析。 LC耦合到QE-MS进行代谢物的分离和检测。 <ol><li>装备QE-MS具有加热电喷雾电离探头（H-ESI）。设置相关调谐参数的探头所列：加热器温度120℃;鞘气，30;辅助气体，10;扫气，3;喷涂电压，3.6千伏积极模式和2.5千伏负模式。于55设置在毛细管温度为320℃，S-透镜。 </li><li>建立一个完整的扫描方法如下：全扫描范围：60〜900（M / Z）;分辨率：70,000;最大注射时间：200毫秒，约50毫秒的典型注射倍;自动增益控制（AGC）：3,000,000离子。这些设置导致大约550毫秒来进行扫描的正面和负面的模式占空比。 </li><li>建立色谱方法。复方分离在室温13,15聘请酰胺柱（100×2.1 mm内径，3.5毫米）。流动相A为如上所述，并且流动相B为乙腈。使用线性梯度如下：0分钟，85％B; 1.5分钟，85％B，5.5分钟，35％B; 10分钟，35％B，10.5分钟，35％B，14.5分钟，35％B，15分钟，85％B和20分钟，85％B的流速为0.15毫升/分钟从0至10分钟和15至20分钟，和0.3毫升/分钟从10.5到14.5分钟。 </li></ol></li></ol> 2，镨代谢物样品的eparation <ol><li>准备提取溶剂。混合40ml甲醇（LC-MS）和10毫升的水（LC-MS级）在50毫升管，并将其保存在-80℃冰箱在使用前至少1小时。注意：此过程与下面的步骤可以修改为生物组织和流体样品的提取。 <ol><li>培养大肠癌细胞HCT 8细胞在310厘米菜肴或6孔板具有完全生长培养基，RPMI 1640补充有10％热灭活胎牛血清和10万个单位/ L的青霉素和100mg / L的链霉素。 </li><li>当细胞达到80％汇合，迅速 ​​取出介质，并将其放置在干冰13,15上方的盘或板。加入1 ml提取溶剂立即（80％甲醇/水），以及所述板转移到-80℃的冰箱中。为10cm的培养皿中，添加3毫升萃取溶剂的每个孔中。 （尝试移除介质，尽可能避免因从中残留盐的离子抑制效应。）</li><li>离开该板15分钟。从冰箱中取出，并刮去细胞到在干冰上的溶剂。将溶液转移到1.7毫升的Eppendorf管中，并以20,000×g离心速度离心在4℃下10分钟。 （准备细胞代谢物从三个不同的菜肴，使三个重复样品。保持两管的目的是有一个作为备份。） </li><li>将上清转移到两个新的Eppendorf管中，并擦干他们在一个速度真空。这大约需要3-6小时，取决于所使用的速度真空。 （样品也可以一夜之间在氮气干燥。） </li><li>后在-80℃下冷冻各试样的干燥，储存管中。准备就绪后，重新组建一个样品20毫升水（LC-MS级），注射5毫升LC-QE-MS进行分析。 </li></ol></li></ol>采样序列的3。设置<ol><li>一旦校准已妥善开展了对QE-MS，平衡液相色谱柱为5分钟，85％的流量速度为0.15毫升/分钟，这是LC梯度的起始条件。 </li><li>以随机顺序设置采样序列。注意：在这种方式，分配由LC-MS引入到每个样品的波动，并确保不同的样品之间更准确的比较。每6个样品中，添加的洗涤运行，其共享相同的质谱法，除LC梯度为95％A和10分钟，然后用5分钟柱平衡在85％A为0.15毫升/分钟的流速。添加空白样品（100％水）每次清洗后运行，以评估该系统的背景和结转的水平。 </li><li>保存序列，一旦液晶栏显示稳定的压力，约400磅，启动顺序运行。如果没有其他的样品这个序列后运行，然后整理后添加的序列，其中有0毫升/分钟的渐变结束的流速月底停止运行，并选择“待机”的序列。 </li><li>重新运行相同的样本集校准后12小时。 （这是我秒校准后，以评估质量误差的波动。） </li></ol> 4。后分析仪器清洗和维护<ol><li>在序列的末尾，以0.2毫升/分钟为2小时的流速用95％A洗柱，并在必要时，洗涤前的反向柱。 </li><li>除去液相色谱柱，并直接通过一个联合的LC连接到离子源。准备清洗溶剂，水/甲醇/甲酸（体积：体积：V，90:10:0.2），待机模式设置MS和洗涤LC-MS系统以0.1的流量毫升/分钟1小时以除去残留沉淀的盐或其它杂质。降低流速，如果有太多的系统压力。 </li><li>在50℃下将毛细管温度，并取下离子笼。小心取出离子吹扫锥和离子传输管后的毛细管的温度下降到50℃。使用粗网格，诸如砂纸，除去留下的离子吹扫锥的表面上的杂质。 </li><li>将离子转移管放入含有10ml 90％水/甲醇和0.1％甲酸15毫升的Falcon管中。超声处理管在水浴超声器20分钟后，滗出溶剂内，它用10毫升纯甲醇替换和超声仪处理20分钟。 （如果需要，超声处理可以在40℃或更高的温度，以达到更好的清洁效果完成。） </li></ol> LC-MS数据的5。分析<ol><li>为确保采样序列运行平稳，完成序列中的前两个样品后，检查未知代谢物峰。使用csv文件列出的代谢物名称，中性化学分子式和检测模式（无论是正面的还是负面的），作为输入文件和输出文件包含提取的高峰，在ppm级的质量误差。如果峰形异常或质量误差是关闭的超过5ppm，则需要在序列的其余部分要被停止和故障诊断需要做的事情。 </li><li>选择“峰对齐的方法和帧提取“的市售软件。从LC-MS和他们组原始数据。挑样品中的运行顺序为峰对齐色谱法，参照样品的中间。上传一个框架种子包括已知的代谢产物为目标代谢产物分析收集到的数据和相应的帧的种子。 </li><li>在正和负模式分别执行数据分析。使用默认设置其他参数。关闭数据库的搜索功能和运行工作流。导出处理后的数据为包含每一帧的峰面积的Excel工作表。帧的第一个集合对应于在目标列表中的代谢物。注意：对于靶向代谢物的分析，基于以前的研究13,15得到的代谢信息。 </li><li>对于一个不相关的代谢产物分析，选择“成分提取”的方法。加载背景扣除空白样品。设置峰值强度阈值的T 10 5，10 ppm的和信号的3信噪比M / Z宽度。 </li><li>对未知化合物鉴定人使用代谢组数据库。使用简历过滤器重复样品中去除大简历的组件。手动通过每个组件，并挑选那些具有良好定义的峰值或在不同类型的样本数据库搜索比较大的差异。与点击导出数据的数据库。 （如果峰值比对分数太低峰值对准可被绕过。） </li></ol>

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作者宣称没有利益冲突。

在使用这种协议的细胞成功代谢物图谱中最关键的步骤是：1）控制所述生长培养基中并小心萃取的细胞; 2）调整液相色谱方法基于MS方法设置，以确保有足够的（通常至少为10）跨数据进行定量峰点; 3）做一个低质量运行校准样品前; 4）注入不超过5毫升到避免保留时间偏移和峰展宽引起的水; 5）准备和运行样品在同一批次，以减少批效果比较。 标准（ 表1）选择?...

代谢组学，定义为同时测量多个代谢物的试验，一直是激烈感兴趣的领域。代谢组学提供了分子生理学的一个直接读出，并提供了见解发育和疾病如癌症1-4。核磁共振（NMR）和气相色谱-质谱（GC-MS）是最常用的工具5-9之间。 NMR，尤其是已自重同位素标记的化合物，如13 C标记的代谢物用于通量的实验中，都NMR-活性10,11。然而，这种策略要求比较高的样品纯度和大样本量，这限制了它的应用代谢组学。同时，从核磁共振收集的数据密集型需求分析和复杂的NMR谱复合赋值是困难的。 GC-MS已广泛用于极性代谢物和脂质的研究，但它需要的挥发性化合物 -ds和因此往往衍生的代谢物，这有时会涉及复杂的化学反应，可以是耗时的，并引入实验噪音。 液相色谱（LC），耦合到三重四极杆质谱法使用第一个四极杆选择的完整母离子，然后将其分段中的第二四极，而第三个四极杆被用来选择特征的片段或子离子。该方法中，其中记录了从母体离子，以特定的子离子的过渡，被称为多反应监测（MRM）。 MRM是为小分子和蛋白质定量12-15,21非常敏感，特异，可靠的方法。然而，MRM也有其局限性。为了实现高特异性的MRM方法需要为每个代谢物兴建。该方法包括确定一个特定的片段和相应的最佳碰撞能量，这就要求PROPE的预先知识感兴趣的代谢物，如化学结构信息的rties。因此，与同一片段的中性丢失一些例外，它是不可能的，以确定未知代谢物与该方法。 在最近几年，高分辨率质谱（HRMS）的仪器已被释放，如LTQ-轨道阱和Exactive系列，QuanTof技术，和的TripleTOF 5600 16-18,22。 HRMS可以提供​​质量到几个ppm的误差内充完好离子的比（m / z）表示。因此，通过检测所有的前体离子（ 即全扫描模式）操作的HRMS的仪器可以从精确质量和分析物所产生的元素组成直接获得的结构信息，并且该信息可用于确定潜在的代谢物。确实，可以用一个精确质量来获得关于一种化合物的所有信息，至多结构异构体的水平。此外，全扫描方法不需要的代谢产物以前的知识，也不需要方法的优化。此外，由于m / z为落入扫描范围的所有离子进行分析，HRMS具有几乎无限的容量可量化在单次运行相比，MRM方法代谢物的数量而言。人力资源管理系统也相当于定量容量的三重四极杆MRM由于短的工作周期造成的，可以在一个完整的MS将扫描获得的数据点的数量相媲美。因此，人力资源管理系统提供了定量的代谢组学的另一种方法。近日，人力资源管理系统的改进版本称为Q-Exactive质谱（QE-MS）可以足够快的周期正，负模式在一个单一的方法，扩大了检测范围19之间的切换下操作。在这里，我们将介绍使用的QE-MS代谢组学我们的策略。

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:156px;">Positive calibration mix</td>
			<td style="width:117px;">Thermo Scientific</td>
			<td style="width:120px;">#88323</td>
			<td style="width:207px;">It is light sensitive. Store at 4 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Negative calibration mix</td>
			<td>Thermo Scientific</td>
			<td>#88324</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Diazinon</td>
			<td style="width:117px;">Sant Cruz Biotechnology</td>
			<td>#C0413</td>
			<td style="width:207px;">It causes eyes irritation, so work in hood. Store at 4 &#176;C</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">H-ESI needle insert</td>
			<td>Fisher Scientific</td>
			<td>#1303200</td>
			<td style="width:207px;">This could be replaced or cleaned with 5 % Formic acid/water (remove rubber ring) if clogged.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Xbridge amide column</td>
			<td>Waters</td>
			<td>#186004860</td>
			<td style="width:207px;">Guard column is recommend to increase column lifetime.</td>
		</tr>
	</tbody>
</table>

a strategy for sensitive, large scale quantitative metabolomics

代谢产物分析一直是宝贵的资产在代谢在健康和疾病的研究。然而，当前的平台上有不同的限制因素，如劳动密集型样品制剂，低检测限，扫描速度很慢，对于每种代谢物密集的方法优化，以及无法积极地同时测量和在单一实验中带负电荷的离子。因此，一种新的代谢协议可以促进代谢组学研究。基于酰胺的亲水色谱法使极性代谢物的分析，而无需任何化学衍生。高分辨质谱使用Q-Exactive（QE-MS）有所改善离子光学系统，提高了扫描速度（256毫秒的分辨率70,000），并具有进行正/负切换的能力。使用冷甲醇提取策略，并与耦合QE-MS酰胺色谱柱能够稳健检测168有针对性的极性代谢物的附加功能和成千上万的同时进行。 DAT的处理是用市售的软件在一个高度有效的方法，并从质谱图中提取未知功能可以在数据库中进行查询。

代谢组数据的精度在很大程度上取决于对LC-QE-MS仪器的性能。评估状况良好仪器是否正在运行，并且应用该方法是否是正确的，几个已知代谢物的LC峰被从总离子色谱图（TIC）萃取， 如图1。极性代谢物，包括氨基酸，糖酵解中间体，三氯乙酸中间体，核苷酸，维生素，ATP，NADP +等有很好的保留在目前的LC条件下的酰胺列的列和良好的峰形。同时，大容量误差测试低质量校准后2...

Watch this Scientific Journal Video about A Strategy for Sensitive, Large Scale Quantitative Metabolomics at JoVE.com

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

代谢产物分析一直是宝贵的资产在代谢在健康和疾病的研究。利用正常阶段的液相色谱和高分辨率质谱与极性切换和快速的工作周期，我们描述了一个协议分析生物材料，具有灵敏度高，准确度和分辨率的极性代谢成分。

敏感，大规模定量代谢组学的策略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

a-strategy-for-sensitive-large-scale-quantitative-metabolomics

Research

JoVE Journal

Biology

20.9K 次观看.  Cornell University. 代谢产物分析一直是宝贵的资产在代谢在健康和疾病的研究。利用正常阶段的液相色谱和高分辨率质谱与极性切换和快速的工作周期，我们描述了一个协议分析生物材料，具有灵敏度高，准确度和分辨率的极性代谢成分。 

视频: A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

宏基因组库的大型屏幕

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

科研

生物学

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

在整个猴脑进行大规模蛋白质检测的批次免疫染色

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

斑马鱼为基础的大型在体内小分子屏幕

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Infinium检测的大型SNP基因分型应用

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY：用于酵母生长多参数定量的大规模方法

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

用于大规模表观遗传学研究的半自动ChIP-Seq程序

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

3D生物反应器培养大规模制备滑液间充质干细胞来源外泌体

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.