Name: assessment of cellular oxidation using a subcellular compartment-specific redox-sensitive green fluorescent protein
Uploaded: 2020-06-18T14:30:00
Description: Watch this Scientific Journal Video about Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein at JoVE.com

在西北大学弗赖伯格医学院和ViraQuest公司的实验室中，分别产生了用于细胞溶质性roGFP的构造和重组腺病毒。这项研究得到了宿主抗癌治疗研究中心的资助，授予P20GM109005，通过NIH国家生物医学研究所生物医学研究中心卓越（COBRE NIGMS），国家普通医学研究所系统药理学和毒理学培训计划赠款T32 GM106999， UAMS 基金会/医学研究捐赠奖 AWD00053956，UAMS 年终校长奖 AWD00053484。流细胞学核心设施部分由微生物发病和宿主炎症反应中心通过COBRE NIGMS授予P20GM103625支持。内容完全由作者负责，不一定代表NIH的官方观点。ATA得到土耳其科学技术研究理事会（TUBITAK）2214-A奖学金的支持。

注：此协议针对 70%-80% 的汇入 MDA-MB-231 单元进行了优化。对于其他细胞系，应重新优化细胞数量和感染数 （MOI）。
1. 细胞的制备（第1天）
<ol><li>在75厘米2瓶中保持MDA-MB-231细胞系，在5%CO2加湿的大气中补充10%的胎儿牛血清（FBS）， 补充10%的胎儿牛血清（FBS）。 注：DMEM 补充了 10% FBS、37 °C 和 5% CO2加湿气氛，用于整个协议中的所有附件和治疗孵化?...

<ol>
	<li>Sies, H. <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:+A+concept+in+redox+biology+and+medicine.">Oxidative stress: A concept in redox biology and medicine.</a> Redox Biology. 4, 180-183 (2015).</li><li>Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+Oxidative+Stress.">Redefining Oxidative Stress.</a> Antioxidants &amp; Redox Signalling. 8 (9-10), (2006).</li><li>Pizzino, G., 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:+Harms+and+Benefits+for+Human+Health.">Oxidative Stress: Harms and Benefits for Human Health.</a> Oxidative Medicine and Cellular Longevity. 20117, 8416763 (2017).</li><li>Go, Y. M., Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol/disulfide+redox+states+in+signaling+and+sensing.">Thiol/disulfide redox states in signaling and sensing.</a> Critical Reviews in Biochemistry and Molecular Biology. 48 (2), 173-191 (2013).</li><li>Hansen, J. M., Go, Y., Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+and+Mitochondrial+Compartmentation+of+Oxidative+Stress+and+Redox+Signaling.">Nuclear and Mitochondrial Compartmentation of Oxidative Stress and Redox Signaling.</a> Annual Review of Pharmacology and Toxicology. 46 (1), 215-234 (2006).</li><li>Meyer, A. J., Dick, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein-based+redox+probes.">Fluorescent protein-based redox probes.</a> Antioxidants and Redox Signaling. 13 (5), 621-650 (2010).</li><li>Dooley, C. T., 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=Imaging+dynamic+redox+changes+in+mammalian+cells+with+green+fluorescent+protein+indicators.">Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators.</a> Journal of Biological Chemistry. 279 (21), 22284-22293 (2004).</li><li>Bj&#246;rnberg, O., &#216;stergaard, H., Winther, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+intracellular+redox+conditions+using+GFP-based+sensors.">Measuring intracellular redox conditions using GFP-based sensors.</a> Antioxidants and Redox Signaling. 8 (3-4), 354-361 (2006).</li><li>Bhaskar, A., 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=Reengineering+Redox+Sensitive+GFP+to+Measure+Mycothiol+Redox+Potential+of+Mycobacterium+tuberculosis+during+Infection.">Reengineering Redox Sensitive GFP to Measure Mycothiol Redox Potential of Mycobacterium tuberculosis during Infection.</a> PLoS Pathogens. 10 (1), 1003902 (2014).</li><li>Loor, G., 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+oxidant+stress+triggers+cell+death+in+simulated+ischemia-reperfusion.">Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion.</a> Biochimica et Biophysica Acta - Molecular Cell Research. 1813 (7), 1382-1394 (2011).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Loor, G., 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=Menadione+triggers+cell+death+through+ROS-dependent+mechanisms+involving+PARP+activation+without+requiring+apoptosis.">Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis.</a> Free Radical Biology and Medicine. 49 (12), 1925-1936 (2010).</li><li>Esposito, 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=Redox-sensitive+GFP+to+monitor+oxidative+stress+in+neurodegenerative+diseases.">Redox-sensitive GFP to monitor oxidative stress in neurodegenerative diseases.</a> Reviews in the Neurosciences. 28 (2), 133-144 (2017).</li><li>Meyer, A. J., 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=Redox-sensitive+GFP+in+Arabidopsis+thaliana+is+a+quantitative+biosensor+for+the+redox+potential+of+the+cellular+glutathione+redox+buffer.">Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer.</a> Plant Journal. 52 (5), 973-986 (2007).</li><li>Galvan, D. 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=Real-time+in+vivo+mitochondrial+redox+assessment+confirms+enhanced+mitochondrial+reactive+oxygen+species+in+diabetic+nephropathy.">Real-time in vivo mitochondrial redox assessment confirms enhanced mitochondrial reactive oxygen species in diabetic nephropathy.</a> Kidney International. 92 (5), 1282-1287 (2017).</li><li>Swain, L., Nanadikar, M. S., Borowik, S., Zieseniss, A., Katschinski, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+organisms+meet+redox+bioimaging:+One+step+closer+to+physiology.">Transgenic organisms meet redox bioimaging: One step closer to physiology.</a> Antioxidants and Redox Signaling. 29 (6), 603-612 (2018).</li><li>Gutscher, 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=Proximity-based+protein+thiol+oxidation+by+H2O2-scavenging+peroxidases.">Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases.</a> Journal of Biological Chemistry. 284 (46), 31532-31540 (2009).</li><li>Morgan, B., Sobotta, M. C., Dick, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+EGSH+and+H2O2+with+roGFP2-based+redox+probes.">Measuring EGSH and H2O2 with roGFP2-based redox probes.</a> Free Radical Biology and Medicine. 51 (11), 1943-1951 (2011).</li><li>Dey, S., Sidor, A., O'Rourke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartment-specific+control+of+reactive+oxygen+species+scavenging+by+antioxidant+pathway+enzymes.">Compartment-specific control of reactive oxygen species scavenging by antioxidant pathway enzymes.</a> Journal of Biological Chemistry. 291 (21), 11185-11197 (2016).</li></ol>

生物体中的硫醇/二硫化物平衡反映了细胞的氧化还原状态。生物有谷胱甘肽、半胱氨酸、蛋白质三醇和低分子量三醇，所有这些都受到氧化水平的影响，并呼应细胞的氧化状态4。工程性 roGfP 允许通过其 CyS 残留物7对硫醇/二硫化物平衡进行无中断定量。roGFP 的比值特性为哺乳动物细胞提供了可靠的氧化还原测量。roGFP可以很容易地引入细胞与转染方法和/或转导...

1985年，赫尔穆特·西斯将氧化应激定义为"对前者的益氧化剂-抗氧化剂平衡的干扰"1，并进行了大量的研究，,以获得生物1、2、32的疾病、营养和衰老特异性氧化还原状态。13从那时起，对氧化应激的理解变得更加广泛。测试使用抗氧化剂对抗疾病和/或衰老的假设表明，氧化应激不仅造成伤害，而且在细胞中还有其他作用。此外，科学家已经表明，自由基在信号转导2中起着重要的作用。所有这些研究都加强了确定大分子还原氧化（氧化）比变化的重要性。酶活性、抗氧化剂和/或氧化剂以及氧化产物可以通过各种方法进行评估。其中，确定二醇氧化的方法可以说是最常用的方法，因为它们报告了细胞中的抗氧化剂和益氧化剂以及生物体4之间的平衡。具体来说，谷胱甘肽（GSH）/谷胱甘肽二硫化物（GSSG）和/或半胱氨酸（CyS）/半胱氨酸（CySS）之间的比率被用作生物标志物，用于监测生物体的氧化还原状态2。
用于测量益氧化剂和抗氧化剂之间的平衡的方法主要依赖于细胞内还原/氧化蛋白或小分子的水平。西方印迹和质谱法用于广泛评估还原/氧化大分子（蛋白质、脂质等）的比例，GSH/GSSG比率可以通过分光光度测定5来评估。这些方法的一个常见特征是细胞系统通过细胞系统化和/或组织均质化对系统的物理扰动。当需要测量不同细胞室的氧化状态时，这些分析也变得具有挑战性。所有这些扰动都会导致测定环境中的伪影。
对红氧敏感的荧光蛋白开启了一个有利的时代，用于评估氧化还原平衡，而不会在细胞6中引起干扰。它们可以针对不同的细胞内隔间，从而量化隔间特定活动（例如，检测线粒体和细胞溶酶的氧化还原状态），以调查细胞细胞器之间的串扰。黄色荧光蛋白（YFP）、绿色荧光蛋白（GFP）和HyPeR蛋白由Meyer及其同事6日审查。在这些蛋白质中，对氧化还原敏感的GFP（roGFP）是独一无二的，因为它的CyS（例如488nm/em.525nm）和CySS（例如405nm/525nm）的残留物不同，这允许比例分析，不像其他氧化还原敏感蛋白，如YFP77，8。8比例值输出是有价值的，因为它平衡了表达水平、检测敏感性和光漂白8之间的差异。细胞（细胞溶质、线粒体、细胞核）或不同生物体（细菌和哺乳动物细胞）的亚细胞隔间可以通过修改roGFP77、9、109,10来定位。
roGFP检测采用荧光成像技术进行，尤其是用于实时可视化实验。对于具有预定时间点的实验，也可以对roGFPs进行流细胞测量分析。本文描述了使用荧光显微镜和流细胞测定法，通过腺病毒转导对哺乳动物细胞中多表达的roGFP（针对细胞sol）的红氧状态进行比例评估。

<table>
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco by Life Sciences</td>
			<td>25200-056</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>4-well chamber slide</td>
			<td>Thermo Scientific</td>
			<td>154526</td>
			<td>Cell seeding material for fluorescent imaging</td>
		</tr>
		<tr>
			<td>5 ml tubes with cell strainer cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td>Single cell suspension tube for flow cytometry analysis</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td>Cell seeding material for flow cytometry analysis</td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>MidSci</td>
			<td>C15B</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>75 cm2 ventilated cap tissue culture flasks</td>
			<td>Corning</td>
			<td>4306414</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Adenoviral cytosol specific roGFP</td>
			<td>ViraQuest</td>
			<td>VQAd roGFP</td>
			<td>roGFP construct kindly provided by Dr. Schumaker</td>
		</tr>
		<tr>
			<td>Class II, Type A2 Safety Hood Cabinet</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>Invitrogen</td>
			<td>C10227</td>
			<td>Cell counting</td>
		</tr>
		<tr>
			<td>Countess cell counter chamber slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td>Cell counting</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco by Life Sciences</td>
			<td>11995-065</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Filtered pipette tips, sterile, 20 &#181;l</td>
			<td>Fisherbrand</td>
			<td>02-717-161</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Filtered pipette tips, sterile, 1000 &#181;l</td>
			<td>Fisherbrand</td>
			<td>02-717-166</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>LSRFortessa</td>
			<td>Instrument equipped with FITC and BV510 bandpass filters for flow cytometry analyses</td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Advanced Microscopy Group (AMG)</td>
			<td>Evos FL</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide 30%</td>
			<td>Fisher Scientific</td>
			<td>H325-100</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Light Cube, Custom</td>
			<td>Life Sciences</td>
			<td>CUB0037</td>
			<td>Fluorescent imaging of roGFP expressing cells (ex 405 nm)</td>
		</tr>
		<tr>
			<td>Light Cube, GFP</td>
			<td>Thermo Scientific</td>
			<td>AMEP4651</td>
			<td>Fluorescent imaging of roGFP expressing cells (ex 488 nm)</td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>American Tissue Culture Collection</td>
			<td>HTB-26</td>
			<td>Human epithelial breast cancer cell line</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 2 ml</td>
			<td>Grenier Bio-One</td>
			<td>623201</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco by Life Sciences</td>
			<td>10010-023</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Pipet controller</td>
			<td>Drummond</td>
			<td>Hood Mate Model 360</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Serologycal pipet, 1 ml</td>
			<td>Fisherbrand</td>
			<td>13-678-11B</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Serologycal pipet, 5 ml</td>
			<td>Fisherbrand</td>
			<td>13-678-11D</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Serologycal pipet, 10 ml</td>
			<td>Fisherbrand</td>
			<td>13-678-11E</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Tissue Culture Incubator</td>
			<td>Thermo Scientific</td>
			<td>HERACell 150i</td>
			<td>CO2 incubator for cell culture</td>
		</tr>
		<tr>
			<td>Trypan blue stain 0.4%</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td>Cell counting</td>
		</tr>
	</tbody>
</table>

assessment of cellular oxidation using a subcellular compartment-specific redox-sensitive green fluorescent protein

测量细胞内氧化/减少平衡提供了一个有机体的生理和/或病理性红氧状态的概述。硫醇对于通过降低二恶英和氧化二硫化物比来照亮细胞的氧化还原状态尤为重要。工程中含半胱氨酸的荧光蛋白为对氧化还原敏感的生物传感器开启了一个新时代。其中之一，对氧化还原敏感的绿色荧光蛋白（roGFP），可以很容易地引入具有腺病毒转导的细胞中，从而在不中断细胞过程的情况下评估亚细胞室的氧化还原状态。减少的半胱氨酸和氧化的囊肿的roGFP有激发最大值分别在488nm和405nm，发射在525nm。评估这些还原和氧化形式的比率，可以方便地计算细胞内的氧化平衡。在这种方法中，利用不朽的人类三阴性乳腺癌细胞（MDA-MB-231）来评估活细胞内的红氧状态。协议步骤包括MDA-MB-231细胞线转导与腺病毒，以表达细胞状roGFP，治疗与H2O2，并评估半胱氨酸和半胱氨酸比与流动细胞学和荧光显微镜。

CyS/CySS 的氧化还原状态可通过转导的 roGFP 轻松测定。荧光探针量化还原和氧化形式之间的比率（激励波长分别为 488 nm 和 405 nm）。荧光数据可以通过流细胞学和显微镜获得。
使用流细胞测定，可以一致、方便地获得大量细胞。分析包括3个主要步骤：1）使用FSC区域滤波器选择感兴趣的细胞群（图1A）;2） 用前 488/em 为 roGFP 表达细胞进行门。525 nm 带带通?...

Watch this Scientific Journal Video about Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein at JoVE.com

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

该协议描述了对细胞内亚细胞室特异性氧化还原状态的评估。对氧化还原敏感的荧光探针可在完整细胞中方便地进行比例分析。

使用亚细胞室特异性红氧敏感绿色荧光蛋白评估细胞氧化

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...

当前纺织品协议评估员，氧化还原状态变化在细胞内隔间内特定的方式。我们的协议使更好的理解和监测氧化应激的所有生理或病理生理机制。该技术允许使用比率公制输出对完整细胞中的平铺眼硫化物比率进行无中断定量，以平衡信号表达水平、照片漂白和检测敏感性之间的差异。该协议可应用于各种生物体，包括植物中的细菌。实验的第一天，从汇合乳癌细胞系治疗细胞两分钟，在EDTA中两毫升0.2，5%的行程。当细胞开始分离时，用六毫升的完整介质阻止反应，通过离心沉淀细胞。将颗粒重新悬浮在五毫升新鲜、完整的介质中，数数细胞，并稀释至每毫升完整介质的1.5倍至第五细胞。然后在6个井中每毫升细胞中播种1.5倍至第5个细胞，并在细胞培养培养箱中孵育细胞。或添加无病毒行 GFP 转导。第二天早上，加入12.5、25和50微升的1至100微升稀释6倍10至第10个斑块形成单位每毫升添加无病毒侵蚀GP溶液，在完全介质中。将其添加到适当的井或6孔板中，分别以50、100和200倍的感染率对细胞进行转导。当所有样本Well被病毒转导时，将细胞返回细胞培养箱16至24小时。第二天，通过荧光显微镜将细胞可视化，用介质替换上一代，没有病毒，使细胞恢复24小时。要确定最佳转导效率，通过流式细胞测量评估细胞的荧光强度和形态。请务必在生物安全二级生物安全柜内执行所有可能导致气溶胶或飞溅感染的程序。为了评估细胞的氧化还原状态，第二天早上，用10微摩尔过氧化氢孵育6井板的细胞和适当的孔，然后分离细胞与750微升0.2 5%滴入EDTA每井，如证明。停止反应与两毫升完整的中等每孔和拉上一种条件在个别15毫升锥形管的超级物。通过离心沉淀细胞，通过离心两次将颗粒洗净到每井500微升PBS中。第二次洗涤后，通过40微米网格过滤细胞悬浮液，进入冰上流动细胞测量兼容管，防止光线。在流式细胞仪软件中，运行感染控制样本的零多重性。然后分析过氧化氢和未经处理的细胞。它将允许使用指示公式计算氧化与减少的行 GFP 之间的平均荧光强度比。以下是通过流式细胞学选择大量细胞的浇注策略。流式细胞仪还可用于确定行GP分析的剂量响应曲线和最有效的感染输入的多重性。根据感染的多重性，剂量反应曲线在此代表性分析中，200多重感染给出了最高的道路GP表达，但细胞形态的影响表明细胞毒性。因此，确定最佳转导效率为100的多重感染。在该氧化还原状态分析中，从车辆流式细胞学分析和四个10微摩尔正控过氧化氢处理中获得氧化和还原行GP平均荧光强度。叠加的直方图表示 10 微摩尔过氧化氢和车辆处理组或还原和氧化侵蚀 GFP 中细胞数的变化。本分析的氧化排和还原行GP之间的比率计算表明，与车辆处理相比，10微摩尔过氧化氢导致行GP氧化增加三倍。拥有准确的细胞数对于 MRI 计算和可重复性非常重要。为了获得可重复的结果，研究人员应彻底混合抗病毒溶液，以获得均匀的悬浮液。该协议允许研究人员研究快速氧化还原变化，作为广泛正常细胞过程和疾病进展的一个组成部分的实时。

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

用 [NiFe] 氢对 H2氧化的研究表明, 蛋白质膜红外电化学

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

体外支持性脂质双层自组织蛋白模式的重组

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

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

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

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 ...

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

利用网格和收集荧光蛋白陶瓷的结构解

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological ...

Extraction and Analysis of Taiwanese Green Propolis

台湾绿蜂胶的提取与分析

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

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 ...