Name: quantification of hypopigmentation activity in vitro
Uploaded: 2019-03-06T12:30:00
Description: Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

这项工作得到了韩国粮食、农业和林业技术规划和评价研究所 (ipet) 通过农业、粮食和农村事务部资助的农业-生物工业技术发展方案 (mafra) 的支持。) (11159-02-wt011) 和韩国大学 bk21 plus 生命科学和生物技术学院。

1. 培养基、化合物和试剂的制备
<ol><li>准备 b16f10 生长完整的介质。补充 dulbecco 的改良鹰培养基 (dmem) 与10% 的胎牛血清 (fbs) 和1% 的青霉素/链霉素 (pest)。<ol>
<li>要使500毫升的完整介质, 将 dulbecco 改性的 eagle 介质 (dmem) 的445毫升与50毫升的 fbs 和5毫升的 pest 混合在一个1升的灭菌玻璃瓶中。轻轻混合后, 通过0.2μm 的瓶盖过滤器将介质过滤到新的灭菌玻璃瓶中, 然后存放在4°c。 注意: 所有细胞培养?...

<ol>
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我们提出了评估使用培养的黑素细胞的测试化合物的低细胞化活性的方案。有代表性的结果显示了抗酪氨酸酶抑制剂 arbutin 的低细胞化作用, 它抑制酪氨酸酶活性和细胞黑色素含量。这些方法在抗黑色素活性研究中得到了广泛的应用。利用这些检测方法, 我们还成功地发现了几种生物活性化合物, 这些化合物在过去十年中对b16f10 细胞产生黑色素作用, 9、10</sup...

黑色素在包括皮肤、眼睛和大脑1在内的几个器官的生理、病理和毒理学中发挥着至关重要的作用。黑色素的主要功能是光筛选和生化效应。黑色素吸收近红外光和可见光以及紫外线 (uv) 辐射, 在较短波长的光的情况下吸收率提高;因此, 黑色素保护组织免受可见光或紫外线辐射2造成的损害.黑色素是一种抗氧化剂, 对金属和其他有毒化学品有亲和力;因此, 它可以保护组织免受氧化和化学应激3。然而, 黑色素的过度生产会导致皮肤病问题。
皮肤和虹膜中黑色素的数量和质量是虹膜和皮肤颜色的最重要决定因素。个人可能有不同的肤色偏好;有的喜欢晒黑的皮肤, 有的则喜欢较浅的皮肤颜色。根据这些消费者的情况, 低菌化妆品已经开发, 以满足个别市场的青睐的肤色4。因此, 研究低细胞和抗黑色素活性在科学和实践上都很重要。
黑色素生成是黑色素通过一系列酶性和自发化学反应在黑色素细胞中进行生物合成的复杂过程。一个黑素细胞被大约36个角质形成细胞包围, 黑素细胞是黑色素合成工厂, 将它们的产品分配给邻近的角质形成细胞。在皮肤中, 产生和储存在黑色素细胞黑色素瘤室中的黑色素通过树突输送到表皮中的角质形成细胞. 
l-tyrosine 作为黑色素生成的初始底物, 酪氨酸酶催化两种连续反应, 将 l-酪氨酸转化为 3, 4-二羟基苯基丙氨酸 (dopa), 然后转化为多巴米酮。这些反应是黑色素生成的限速步骤 5,6。因此, 低细胞分裂活性可以通过直接评估细胞酪氨酸酶活性来测量。为此, 含有酪氨酸酶的黑素细胞提取物由 dopa 孵育, 样品中产生的多巴西酮可在475纳米的分光光度法中进行测定。这些值通过样品的蛋白质浓度进行归一化, 与对照相比, 具有低渗活性的物质会导致多巴奎因的形成较少。
其次, 低细胞分裂活性可以通过直接测量培养的黑色素细胞中的黑色素来量化。用试验材料处理细胞后, 在碱性条件下提取黑色素, 用分光光度法在400纳米处测定黑色素含量。低细胞化剂会导致比对照组7更低的黑色素含量。
最后, 利用 Fontana-Masson 黑色素染色和随后的图像分析可以量化低细胞分裂活性。在 Fontana-Masson 染色中, 黑色素颗粒将硝酸铵还原为可见的黑色金属状态, 而微观图像中的黑细胞区域代表黑色素的数量。
低渗剂通常会给出与这三种方法一致和可比的结果, 这证实了该物质的活性是有效的。或者, 它可能是有用的, 以衡量关键基因和蛋白质在黑色素生成的表达, 以响应测试物质, 以检查低细胞化活性。除了酪氨酸酶外, 酪氨酸酶相关蛋白 (trp-1) 和多巴斯姆自体擦除 (trp-2) 是黑色素生成的关键酶 8.转录因子、微邻苯二甲相关转录因子 (mitf) 是黑色素生成和基因蛋白表达水平定量的主要调节剂, 也可用于评估低细胞化活性。

<table>
	<tbody>
		<tr>
			<td>3,4-Dihydroxy-l-phenylalanine</td>
			<td>Sigma</td>
			<td>D9628</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution</td>
			<td>Sigma</td>
			<td>#320145</td>
			<td></td>
		</tr>
		<tr>
			<td>Arbutin</td>
			<td>Fluka</td>
			<td>#10960</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Yakuri Pure Chemicals</td>
			<td>#16223</td>
			<td>37%</td>
		</tr>
		<tr>
			<td>Gold chloride</td>
			<td>American MasterTech</td>
			<td>AHG0226</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Samchun chemical</td>
			<td>H0255</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Bio basic Canada Inc.</td>
			<td>PB0440</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>#60218</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>J.T.Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Duksan pure chemicals</td>
			<td>#81</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>655104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear fast red</td>
			<td>Merck</td>
			<td>100121</td>
			<td>Nuclear fast red 0.1% in 5% aluminum sulfate.</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#900</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>J.T. Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate pentahydrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#2163</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Bio Basic Canada</td>
			<td>TB0196</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Union Carbide</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosinase from mushroom</td>
			<td>Sigma</td>
			<td>T3824</td>
			<td>25 KU</td>
		</tr>
	</tbody>
</table>

quantification of hypopigmentation activity in vitro

本研究提出了体外低细胞化活性定量的实验室方法。黑色素是黑色素细胞中的主要色素, 是针对多种细胞和环境因素而合成的。黑色素保护皮肤细胞免受紫外线伤害, 但也具有生物物理和生化功能。黑色素细胞的过度产生或积累会导致皮肤病问题, 如雀斑、黑斑、黄瘤和痣。因此, 控制黑色素生成与低渗剂是重要的个人有临床或美容需求。黑色素主要是在黑色素细胞的黑色素体中合成的, 这个过程被称为黑色素生成, 它受到外部和内在因素的影响, 如激素、炎症、年龄和紫外线照射。我们描述了三种方法来确定化学物质或天然物质在黑素细胞中的低细胞化活性: 测量 1) 细胞酪氨酸酶活性和 2) 黑色素含量, 3) 染色和量化细胞黑色素与图像分析。
在黑色素生成过程中, 酪氨酸酶催化将 l-酪氨酸转化为 3, 4-二羟基苯基丙氨酸 (l-dopa), 然后转化为多巴基宁的限速步骤。因此, 抑制酪氨酸酶是一种主要的低细胞化机制。在培养的黑素细胞中, 通过添加 l-dopa 作为底物, 用分光光度法测定多巴西酮的产生, 可以量化酪氨酸酶活性。黑色素生成也可以通过定量测量黑色素含量。用 naoh 提取含黑色素的细胞分数, 用定量分光光度法提取黑色素。最后, 黑色素的图像分析可以量化黑色素的含量。虽然这些体外检测的结果可能并不总是在人体皮肤中复制, 但这些方法在黑色素生成研究中被广泛使用, 特别是作为识别潜在低细胞化活性的第一步。这些方法也可用于评估黑素细胞的活性、生长和分化。与三种不同方法一致的结果确保了效果的有效性。

具有代表性的结果, 在 b16f10 黑色素细胞的抗黑色素素化合物的低细胞分裂活性如下所示。图 1a显示, 与车辆处理的对照相比, arbutin 显著抑制了细胞酪氨酸酶活性。同样, 与对照组相比, 阿布丁刺激的细胞黑色素含量显著降低 (图 1b)。用 Fontana-Masson 染色染色的细胞的显微图像如图 1c所示。与对照相比, arbutin ...

Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

Quantification of Hypopigmentation Activity In Vitro

我们描述了三种实验方法来评估化学物质在体外的低细胞化活性: 1) 细胞酪氨酸酶活性和 2) 黑色素含量的定量, 以及 3) 细胞黑色素染色和图像分析测量黑色素。

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

该方法有助于开发具有抗黑色致病活动的功能性化妆品和皮肤剂。它是有执行和结果可以迅速获得。此外，这种方法可能有用的研究人员，谁希望确定或调查的影响或化合物或研究抗梅拉诺原或亲源性活动。测量酪氨酸酶活性，首先在细胞培养箱中24小时以完整培养基箱中，以每24个井板浓度的5倍10至第四细胞播种B16-F10黑色素杀菌剂。第二天，用DMEM更换每井的上能，辅以新鲜准备的测试化合物和抑制剂控制或阴性控制，将板再返回培养箱72小时。在治疗结束时，每口井用300微升的冷 pbs 冲洗油井两次，将盘子放在冰上。接下来，使用细胞刮刀在五分钟后收集细胞解液，将300微升的解解缓冲液添加到每井中。将产生的细胞颗粒悬浮液转移到单个 1.5 毫升微离心管中。在冰上以14，500转/小时将酸盐均质三次，两秒钟，以释放细胞间酪氨酸酶。通过离心将细胞碎屑进行分离，将超热剂转移到冰上单个1.5毫升离心管。将每个上清液的 70 微升分配到透明 96 孔聚苯乙烯微板的单个孔中，并将 140 微升的 Tyrosinase 基板溶液添加到每口上清液孔中。轻轻摇动盘子，混合井内。然后在37摄氏度下孵育板两小时。并在微孔板读卡器上测量 475 纳米的酪氨酸酶活性。测量细胞的黑色素含量后收集处理细胞的 lysate 显示。通过离心收集酸盐，将中流剂转移到新管中。在每个颗粒中加入300个普通氢氧化钠的300微升，在60摄氏度下孵育一小时。其次是溶解细胞悬浮的离心。然后将200微升的超糖转移到96井板的每个井中。以 400 纳米波长测量微孔板读取器上的黑色素含量。对于Fondna-Masson染色，用300微升的冷pbs清洗两次经过处理的细胞，用200微升10%的甲醛在4摄氏度下固定一小时。用蒸馏水冲洗厚细胞，并在每口井中加入200微升58至68摄氏度的氨化银工作溶液。在37摄氏度下孵育一小时，或直到细胞变成黄褐色。用蒸馏水冲洗染色细胞，随后在室温下以0.1%的氯化金孵育两分钟。用蒸馏水冲洗氯化金处理细胞，向细胞中加入200微升硫化钠溶液。两分钟后再次冲洗细胞。每井用200微升的核快红标记细胞5分钟。然后在光显微镜下成像之前，先用自来水清洗染色的细胞。对于阈值分析，请打开图像 J 中的图像并选择图像、调整和颜色阈值。要测量沾染丰塔纳马松的区域，请将亮度参数栏设置为零，并调整亮度到包含所有黑色或棕色染色细胞的点。选择"分析和分析粒子"并选中"分析粒子"窗口中的汇总框，然后单击"确定"以获取结果摘要，然后将数据复制并粘贴到电子表格中。与控制车辆处理的细胞相比，阿布丁治疗显著抑制细胞酪氨酸酶活性。同样，与对控组相比，使用阿布丁刺激的细胞的黑色素含量显著降低。虽然治疗组之间的细胞在形态上相似，但与对照处理的细胞相比，Arbutin会降低黑色色素的面积。此方法可用于使用我们的复合库进行活动筛选。有数百个样品需要快速测试。此外，此方法还可用于研究涉及黑色素发生的机制。在生物活性候选化合物被确定后，为研究生物机制或商业应用提供了证据。

quantification-of-hypopigmentation-activity-in-vitro

Research

JoVE Journal

Biochemistry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

科研

生物化学

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

丰度和收费水平的转移 Rna 在大肠杆菌中的量化

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

映射代谢: 直接在组织中监测乳酸脱氢酶活性

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

果蝇梅拉诺加斯特的锡酸盐合成酶活性的色度测定

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Caenorhabditis elegans中 2-阿拉希多诺诺基甘油定量的分解标准合成

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

细胞和组织中辅酶A的定量

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

mtDNA 合成和分布的高通量定量

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

作者聚焦：基于图像的高通量线粒体 DNA 合成和分布定量  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

基于图像的高通量线粒体DNA合成和分布定量

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...