Name: detection of total reactive oxygen species in adherent cells by 2',7'-dichlorodihydrofluorescein diacetate staining
Uploaded: 2020-06-23T13:00:00
Description: Watch this Scientific Journal Video about Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining at JoVE.com

这项工作得到了美国国家卫生研究院（K01DK114390）的部分支持，美国癌症学会的研究学者资助（RSG-18-050-01-NEC），一项研究试点项目赠款 新墨西哥大学环境卫生签名计划和超级基金（P42 ES025589），共享资源试点项目奖和UNM综合癌症中心研究项目支持试点项目奖（P30CA118100），以及新墨西哥大学医学院专门健康研究基金颁发的新研究员奖。

1. 细胞播种
<ol><li>种子 2 x 105 HCT116 结肠直肠癌细胞每孔在 24 孔板中，并在 Dulbecco 的改良鹰介质 （DMEM） 中保持细胞在 37°C 过夜。</li>
	<li>用或不使用含有介质的100μM硫酸铁（FS）或含有介质的10μM多索鲁比辛（DOX）更换培养介质，孵育24小时。</li>
</ol>2. 准备DCFH-DA解决方案
<ol><li>将4.85毫克的DCFH-DA溶解在1 mL二甲基硫氧化物（DMSO）中，以制作10mM库存溶液。</li>
	<li>在将...

<ol>
	<li>Birben, E., 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=Oxidative+stress+and+antioxidant+defense.">Oxidative stress and antioxidant defense.</a> World Allergy Organization Journal. 5 (1), 9-19 (2012).</li><li>Kim, G. H., 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=The+Role+of+Oxidative+Stress+in+Neurodegenerative+Diseases.">The Role of Oxidative Stress in Neurodegenerative Diseases.</a> Experimental Neurobiology. 24 (4), 325-340 (2015).</li><li>Sullivan, L. B., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+reactive+oxygen+species+and+cancer.">Mitochondrial reactive oxygen species and cancer.</a> Cancer &amp; Metabolism. 2, 17 (2014).</li><li>Formentini, L., 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=Mitochondrial+ROS+Production+Protects+the+Intestine+from+Inflammation+through+Functional+M2+Macrophage+Polarization.">Mitochondrial ROS Production Protects the Intestine from Inflammation through Functional M2 Macrophage Polarization.</a> Cell Reports. 19 (6), 1202-1213 (2017).</li><li>Rakotoarisoa, M., 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=Curcumin-+and+Fish+Oil-Loaded+Spongosome+and+Cubosome+Nanoparticles+with+Neuroprotective+Potential+against+H2O2-Induced+Oxidative+Stress+in+Differentiated+Human+SH-SY5Y+Cells.">Curcumin- and Fish Oil-Loaded Spongosome and Cubosome Nanoparticles with Neuroprotective Potential against H2O2-Induced Oxidative Stress in Differentiated Human SH-SY5Y Cells.</a> ACS Omega. 4 (2), 3061-3073 (2019).</li><li>Mateen, S., 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=Increased+Reactive+Oxygen+Species+Formation+and+Oxidative+Stress+in+Rheumatoid+Arthritis.">Increased Reactive Oxygen Species Formation and Oxidative Stress in Rheumatoid Arthritis.</a> PLoS One. 11 (4), (2016).</li><li>Kim, H., 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=The+interaction+of+Hemin+and+Sestrin2+modulates+oxidative+stress+and+colon+tumor+growth.">The interaction of Hemin and Sestrin2 modulates oxidative stress and colon tumor growth.</a> Toxicology and Applied Pharmacology. 374, 77-85 (2019).</li><li>Wang, S. H., 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=Sotetsuflavone+inhibits+proliferation+and+induces+apoptosis+of+A549+cells+through+ROS-mediated+mitochondrial-dependent+pathway.">Sotetsuflavone inhibits proliferation and induces apoptosis of A549 cells through ROS-mediated mitochondrial-dependent pathway.</a> BMC Complementary and Alternative Medicine. 18, 235 (2018).</li><li>Kruger, N. J., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bradford+Method+For+Protein+Quantitation.">The Bradford Method For Protein Quantitation.</a> The Protein Protocols Handbook. , 17-24 (2009).</li><li>Tetz, L. 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Z., 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=Quantitative+detection+of+hydroxyl+radical+generated+in+quartz+powder/phosphate+buffer+solution+system+by+fluorescence+spectrophotometry.">Quantitative detection of hydroxyl radical generated in quartz powder/phosphate buffer solution system by fluorescence spectrophotometry.</a> Guang Pu Xue Yu Guang Pu Fen Xi. 34 (7), 1886-1889 (2014).</li></ol>

此处描述的实验协议易于重现，可测量细胞总 ROS。关键步骤包括使 DCFH-DA 解决方案新鲜，避免光线照射，最大限度地减少细胞状态干扰和大量 PBS 清洗，然后再拍摄图像。为准备DCFH-DA工作解决方案，在加入24井板之前，应将库存溶液添加到预热的DMEM中。原因是，产生高背景荧光或轻暴露的旧解决方案将导致光漂白。大多数研究使用1xPBS或1x汉克斯的平衡盐溶液（HBSS）稀释DCFH-DA，并将其用作反应缓...

细胞代谢产生的三种具有生理意义的主要活性氧种（ROS）是超氧化物类动物、羟基和过氧化氢1。在低浓度时，它们参与生理细胞过程，但在高浓度时，它们对细胞信号通路1有不利影响。我们的身体已经开发了抗氧化系统，这是有效的对抗过量的ROS。然而，当ROS压倒我们身体的排毒能力时，可能会出现氧化应激，这会导致许多病理状况，包括炎症、癌症和神经退行性疾病22，3，4。3,4该方法的目的是使用2'，7'-二氯二氢氟酸二乙酸（DCFH-DA）染色来确定粘附细胞中的总细胞ROS。其原理是DCFH-DA氧化为2'-7'二氯氟素（DCF）已被广泛用于总ROS检测，包括羟基基（+OH）和二氧化氮（#NO2）。从机械上讲，DCFH-DA被细胞所占用，细胞酯酶从乙酰组脱落，导致DCFH。ROS对DCFH的氧化将分子转化为DCF，DCF在485nm的激发波长和530nm的发射波长下发出绿色荧光。与用流细胞测定和其他替代方法5检测荧光相比，该方法采用荧光显微镜和板读取器的优点是产生清晰可见的荧光图像，易于执行、高效且经济高效。该方法已广泛用于检测细胞ROS，用于研究各种条件66，7，8。7,8该协议用于检测粘附细胞中的总 ROS。使用此方法检测悬浮细胞中的 ROS 可能需要一些修改。

<table border="0" cellpadding="0" cellspacing="0" style="width:900px;" width="899">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">2&#39;,7&#39;-Dichlorofluorescein diacetate</td>
			<td style="width:229px;">Cayman Chemical, Ann Arbor, MI</td>
			<td style="width:93px;">20656</td>
			<td style="width:300px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Doxorubicin hydrochloride</td>
			<td style="width:229px;">TCI America, Portland, OR</td>
			<td style="width:93px;">D4193-25MG</td>
			<td style="width:300px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Corning, Corning, NY</td>
			<td>45000-304</td>
			<td style="width:300px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Ferrous Sulfate Heptahydrate</td>
			<td style="width:229px;">VWR, Radnor, PA</td>
			<td style="width:93px;">97061-542</td>
			<td style="width:300px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Invitrogen EVOS FL Auto Imaging System</td>
			<td style="width:229px;">Thermo Fisher Scientific Waltham, MA</td>
			<td>AMAFD1000</td>
			<td>or any other fluorescence microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein assay Bradford solution</td>
			<td>Bio-Rad, Hercules, CA</td>
			<td style="width:93px;">5000001</td>
			<td style="width:300px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpectraMax M2 Microplate Reader</td>
			<td>Molecular Devices, Radnor, PA</td>
			<td>89429-532</td>
			<td>or any other fluorescence microplate reader</td>
		</tr>
	</tbody>
</table>

detection of total reactive oxygen species in adherent cells by 2',7'-dichlorodihydrofluorescein diacetate staining

氧化应激是生理和病理条件下的一个重要事件。在这项研究中，我们演示了如何通过使用2'，7'-二氯二氢氟酸二乙酸（DCFH-DA）染色结在结肠直肠癌细胞系中测量总活性氧种类（ROS）来量化氧化应激。该协议描述了详细的步骤，包括制备DCFH-DA溶液、使用DCFH-DA溶液孵育细胞以及测量标准化强度。DCFH-DA 染色是检测细胞中 ROS 的一种简单且经济高效的方法。它可用于测量化学处理或基因修饰后产生的ROS。因此，它对于确定细胞氧化应力对环境压力的确定非常有用，为机械学研究提供了线索。

HCT116结肠直肠癌细胞用100μM FS或10μM DOX治疗，诱导氧化应激7。如图1所示，FS 和 DOX 都按预期增加绿色荧光。为了量化相对强度变化，细胞在拍摄图像后被分化，并与蛋白质浓度进行规范化。HCT116细胞中的FS或DOX显著提高了定量荧光强度。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60682/60682fig1.jpg"> 图1?...

Watch this Scientific Journal Video about Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining at JoVE.com

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

在这里，我们提出了一个协议，使用2'，7'-二氯二氢氟辛二乙酸酯（DCFH-DA）检测细胞活性氧物种（ROS）。2'，7'-二氯二氢氟酸酯（DCFH-DA）。该方法使用荧光显微镜可视化粘附细胞中的细胞ROS定位，并用荧光板读取器量化ROS强度。该协议简单、高效且经济高效。

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

该方法可用于分析附着细胞中的细胞活性氧物种，仅使用荧光显微镜，并用荧光板读卡器量化活性氧种强度。该协议简单、高效且经济高效。首先在DMEM中将两次10至第五个HCT116结肠直肠癌细胞播种到24井板的每个井中。在37摄氏度下孵育细胞过夜。第二天，用100微摩尔硫酸铁或10微摩尔多索鲁比辛基基代替培养基，再孵育24小时。通过将 4.85 毫克的 DCFH-DA 溶解在一毫升的 DMSO 中，以制造 10 毫摩尔库存溶液，准备 DCFH-DA 解决方案。在将库存添加到井中之前，用预热的 DMEM 稀释库存，使 10 微摩尔工作溶液，并旋转 10 秒。要弄脏细胞，请取出含药物的介质，用DMEM洗涤一次。将 500 微升 DCFH-DA 工作溶液添加到每个井中，并在 37 摄氏度下孵育板 30 分钟。孵育后，从井中去除DCFH-DA，用DMEM洗涤两次，用PBS清洗一次。然后，在每井中加入500微升PBS。使用荧光显微镜上的绿色荧光蛋白通道拍摄细胞的代表性图像。然后取出PBS，并在每井中加入200微升的放射性免疫沉淀测定缓冲液。在冰上孵育盘子五分钟，将细胞落酸收集到1.5毫升管中。在 4 摄氏度下将管在 21，130 次 G 下离心 10 分钟。然后将 100 微升的上一液转移到黑色 96 井板中。使用 485 纳米的激发波长和 530 纳米的发射波长的微孔板读取器测量每孔的荧光。接下来，使用布拉德福德测定测量蛋白质浓度，将一微升的上清液转移到一个带100微升蛋白质测定溶液的透明96井板中。使用蛋白质浓度使荧光强度正常化。HCT116结肠直肠癌细胞用100微摩尔硫酸铁或10微摩尔多索鲁比辛治疗，以诱导氧化应激。不出所料，这两种治疗方法都显著增加了绿色荧光。为了量化相对强度的变化，细胞被分析，用蛋白质浓度进行规范化。硫酸铁或多索鲁比辛相对荧光强度显著增加。执行此过程时，重要的是使 DCFH-DA 解决方案新鲜，避免光线照射，以及尽量减少细胞状态干扰，并在拍摄图像之前执行广泛的 PBS 洗涤。除了通过本协议检测总 ROS 外，还可以执行过氧化物等 ROS。

Research

JoVE Journal

Biochemistry

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

基于蓝光照射诱发甲沉淀的 DNA 染色方法

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

科研

生物化学

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

用两个稳定 Isotopically 标记鞘磷脂种鞘磷脂的定量和定性方法

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

用最小组织量测定果蝇透纤维中线粒体耗氧量

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

聚丙烯酰胺凝胶中蛋白质的荧光银染色

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

利用高含量成像对增强细胞中的目标参与进行量化

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...