Name: cellular redox profiling using high-content microscopy
Uploaded: 2017-05-14T13:00:00
Description: Watch this Scientific Journal Video about Cellular Redox Profiling Using High-content Microscopy at JoVE.com

This research was supported by the University of Antwerp (TTBOF/29267, TTBOF/30112), the Special Research Fund of Ghent University (project BOF/11267/09), NB-Photonics (Project code 01-MR0110) and the CSBR (Centers for Systems Biology Research) initiative from the Netherlands Organization for Scientific Research (NWO; No: CSBR09/013V). Parts of this manuscript have been adapted from another publication1, with permission of Springer. The authors thank Geert Meesen for his help with the widefield microscope.

对于NHDF细胞和使用材料文件中指定的多孔板，描述了下面的方案。有关工作流程的总体概述，请参见图1 。 1.试剂的制备<ol><li>通过用10％v / v胎牛血清（FBS）和100IU / mL青霉素和100IU / mL链霉素（PS）补充Dulbecco&#39;s Modified Eagle Medium（DMEM）来制备完整培养基。对于500 mL完全培养基，加入50mL FBS和5 mL PS至445 mL DMEM。 </li><li>通过用20mM HEPES补充汉克平衡盐溶液...

<ol>
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本文介绍了用于同时定量NHDF中细胞内ROS水平和线粒体形态功能的高含量显微镜方法。通过对SQV处理的NHDF的案例研究证明其性能。结果支持早期的文献证据，其中在用1型HIV蛋白酶抑制剂治疗后已经观察到ROS水平升高或线粒体功能障碍，尽管在单独的实验19,20,21,22,23,24,25中。重要的区别是所描述的测定能够在相同的活细胞中同时测量这些参数，以及形态学数据。这种方法的主要优点在于它们在空间?...

<html>通过ROS产生和ROS消除系统之间的动态相互作用，精细调节细胞内ROS的浓度。两者之间的不平衡引起氧化应激状态。线粒体的主要来源之一是线粒体1 。鉴于它们在细胞呼吸中的作用，它们负责大量的细胞内超氧化物（O 2 · - ）分子2 。这主要是由于电子传输链的复合物1在强的负内线粒体膜电位（Δψm）， 即线粒体超极化的条件下，电子泄漏到O 2 。另一方面，线粒体去极化也与增加的ROS生产相关，指向多种作用模式3,4,5 ，&gt; 6，7，8 。此外，通过裂解融合机理的蛋白质的氧化还原修饰，ROS共调节线粒体形态9。例如，碎片与增加的ROS产生和凋亡相关， 10,11 ，而丝状线粒体已经与营养物质饥饿和保护相关mitophagy 12 。鉴于细胞ROS和线粒体形态功能之间的复杂关系，两者应理想地在活细胞中同时量化。为了做到这一点，基于自动化宽视野显微镜和用荧光探针CM-H 2 DCFDA（ROS）和TMRM（线粒体Δψm和形态）染色的贴壁细胞培养物的图像分析开发了高含量成像测定。高含量成像是指提取sp关于使用多个互补标记和自动图像分析的细胞表型的信息丰富（ 即大量描述性特征）。当与自动显微镜组合时，可以并行筛选许多样品（ 即高通量），从而增加测定的统计学功能。实际上，协议的主要资产是它允许在同一个信元中同时量化多个参数，而这对于大量的信元和条件来说是可以的。 协议分为8个部分（以下协议详细描述）：1）96孔板中的播种细胞; 2）储备溶液，工作溶液和成像缓冲液的制备; 3）设置显微镜; 4）用CM-H 2 DCFDA和TMRM装载细胞; 5）首次实时成像测量基础ROS水平和线粒体形态功能; 6）加入叔丁基后进行第二次活体成像过氧化物（TBHP）测量诱导的ROS水平; 7）自动图像分析; 8）数据分析，质量控制和可视化。 该测定法最初是针对正常人皮肤成纤维细胞（NHDF）开发的。由于这些细胞是大而平坦的，它们非常适合于评估2D宽幅图像13,14 中的线粒体形态。然而，通过轻微修改，该方法适用于其他贴壁细胞类型。此外，在CM-H 2 DCFDA和TMRM的组合之后，工作流程符合各种具有不同分子特异性的荧光染料对1,15。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM)</td>
			<td>ThermoFisher Scientific</td>
			<td>T668</td>
			<td></td>
		</tr>
		<tr>
			<td>CM-H2DCFDA (General Oxidative Stress Indicator)</td>
			<td>ThermoFisher Scientific</td>
			<td>C6827</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma)Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>MatriPlate 96-Well Glass Bottom MicroWell Plate 630 &#181;L-Black 0.17 mm Low Glass Lidded</td>
			<td>Brooks life science systems</td>
			<td>MGB096-1-2-LG-L</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS w/o Phenol Red 500 ml</td>
			<td>Lonza</td>
			<td>BE10-527F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose with L-glutamine</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Bufered Saline (PBS) w/o Ca and Mg</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1M 500mL</td>
			<td>Lonza</td>
			<td>17-737F</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-Versene (EDTA) Solution</td>
			<td>Lonza</td>
			<td>BE17-161E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure F(ab&#39;)&#8322; Fragment Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson</td>
			<td>711-166-152</td>
			<td>Antibody used for acquiring flat-field image</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure F(ab&#39;)&#8322; Fragment Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson</td>
			<td>711-546-152</td>
			<td>Antibody used for acquiring flat-field image</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Ti eclipse widefield microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perfect Focus System (PFS)</td>
			<td>Nikon</td>
			<td></td>
			<td>hardware-based autofocus system</td>
		</tr>
		<tr>
			<td>CFI Plan Apo Lambda 20x objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elelements Advanced Research 4.5 with JOBS module</td>
			<td>Nikon</td>
			<td></td>
			<td>This software is used to steer the microscope and program/perform the automatic image acquisition prototocol</td>
		</tr>
		<tr>
			<td>ImageJ (FIJI) Version 2.0.0-rc-43/1.50g</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RStudio Version 1.0.44</td>
			<td>Rstudio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>R version 3.3.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

cellular redox profiling using high-content microscopy

活性氧（ROS）调节基本细胞过程，包括基因表达，迁移，分化和增殖。然而，过量的ROS水平诱导氧化应激状态，伴随着对DNA，脂质和蛋白质的不可逆的氧化损伤。因此，ROS的定量为细胞健康状况提供了直接依据。由于线粒体是ROS的主要细胞来源和靶标，所以在同一细胞中线粒体功能和ROS产生的联合分析对于更好地了解病理生理条件的互连至关重要。因此，开发了基于高含量显微镜的策略，用于同时定量细胞内ROS水平，线粒体膜电位（ΔΨm）和线粒体形态。它是基于自动化的宽场荧光显微镜和生物贴壁细胞的图像分析，生长在多孔板和staine中d与细胞可渗透的荧光报告分子CM-H 2 DCFDA（ROS）和TMRM（ΔΨm和线粒体形态）。与荧光测定法或流式细胞术相反，该策略允许在实验刺激前后具有高时空分辨率的个体细胞水平的亚细胞参数的定量。重要的是，该方法的基于图像的性质允许除了信号强度之外还提取形态学参数。组合特征集用于探索和统计多变量数据分析，以检测亚群，细胞类型和/或治疗之间的差异。在此，提供了测定的详细描述，以及证明其在化学扰动之后在细胞状态之间明确区分的潜力的示例性实验。

该测定已经使用几个对照实验进行了基准测试，其结果在Sieprath et al。 1 。简而言之，CM-H 2 DCFDA和TMRM的荧光响应分别定量分析了细胞内ROS和Δψm的变化，以确定其动态范围。对于CM-H 2 DCFDA，当用10μM至160μM范围内的增加的TBHP浓度处理时，NHDF显示出荧光信号的线性增加。同样，对于TMRM，当用1〜10μg/μL范围内增加浓度的寡霉素（其诱?...

Watch this Scientific Journal Video about Cellular Redox Profiling Using High-content Microscopy at JoVE.com

Cellular Redox Profiling Using High-content Microscopy

本文提出了一种高含量显微镜工作流程，用于同时定量细胞内ROS水平，以及线粒体膜电位和形态 - 共同称为线粒体形态功能 - 使用细胞透过性荧光报告分子5-（和 - 6） - 氯甲基-2&#39;，7&#39;-二氯二氢荧光素二乙酸酯，乙酰酯（CM-H 2 DCFDA）和四甲基罗丹明甲酯（TMRM）。

使用高含量显微镜的细胞氧化还原分析

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Research

JoVE Journal

Biology

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

一个高含量的成像流程研究Grb2的信号复合体的表达克隆

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

高内涵筛选分析法评价有害和潜在有害成分的毒性效应（HPHC）

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

开源高内涵分析利用自动荧光寿命成像显微镜

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...