Name: use of electron paramagnetic resonance in biological samples at ambient temperature and 77 k
Uploaded: 2019-01-11T13:30:00
Description: Watch this Scientific Journal Video about Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K at JoVE.com

这项工作得到了科罗拉多大学医学院院长战略研究基础设施奖 r01 hl0866868-09 和1r35hl139726-01 的支持, 颁发给 e. n. g. 和 ucd cfret 奖学金 (he)。作者感谢 sandra eaton 博士和 gareth eaton 博士 (丹佛大学)、gerald rosen 博士和 joseph p. kao 博士 (马里兰州立大学) 和 sujatha venkataraman 博士 (科罗拉多大学丹佛分校) 进行的有益讨论, 以及 joanne maltzahn, ashleytrumpie 和 ivy mcdermott (科罗拉多丹佛大学) 提供技术支持。

所有动物研究都得到了科罗拉多大学丹佛机构动物护理和使用委员会的批准。
1. 试剂的制备
<ol><li>
二乙基氨基苯乙酸 (dtpa) 库存 (150 mm)
	<ol>
<li>在去离子水中加入2.95 克 dtpa (393.35 g/mol)。</li>
		<li>要溶解 dtpa, 滴注添加 1 m naoh, 并使 ph 值为7.0。</li>
		<li>将体积带至50毫升, 水最终 dtpa 浓度为 150 mm, 并存放在4°c。</li>
	</ol>
</li>
	<li>
磷酸盐缓冲碱 (pbs) (50 mm, ph 7.4)...

<ol>
	<li>Kalyanaraman, B., 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=Measuring+reactive+oxygen+and+nitrogen+species+with+fluorescent+probes:+challenges+and+limitations.">Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations.</a> Free Radical Biology and Medicine. 52 (1), 1-6 (2012).</li><li>Bobko, A. 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=In+vivo+monitoring+of+pH,+redox+status,+and+glutathione+using+L-band+EPR+for+assessment+of+therapeutic+effectiveness+in+solid+tumors.">In vivo monitoring of pH, redox status, and glutathione using L-band EPR for assessment of therapeutic effectiveness in solid tumors.</a> Magnetic Resonance in Medicine. 67 (6), 1827-1836 (2012).</li><li>Elajaili, H. B., 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=Electron+spin+relaxation+times+and+rapid+scan+EPR+imaging+of+pH-sensitive+amino-substituted+trityl+radicals.">Electron spin relaxation times and rapid scan EPR imaging of pH-sensitive amino-substituted trityl radicals.</a> Magnetic Resonance in Chemistry. 53 (4), 280-284 (2015).</li><li>Elajaili, 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=Imaging+disulfide+dinitroxides+at+250+MHz+to+monitor+thiol+redox+status.">Imaging disulfide dinitroxides at 250 MHz to monitor thiol redox status.</a> Journal of Magnetic Resonance. 260, 77-82 (2015).</li><li>Halpern, H. 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=Oxymetry+Deep+in+Tissues+with+Low-Frequency+Electron-Paramagnetic-Resonance.">Oxymetry Deep in Tissues with Low-Frequency Electron-Paramagnetic-Resonance.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (26), 13047-13051 (1994).</li><li>Epel, B., 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+thiol+redox+status+in+murine+tumors+in+vivo+with+rapid-scan+electron+paramagnetic+resonanc.">Imaging thiol redox status in murine tumors in vivo with rapid-scan electron paramagnetic resonanc.</a> Journal of Magnetic Resonance. 276, 31-36 (2017).</li><li>Legenzov, E. A., Sims, S. J., Dirda, N. D. A., Rosen, G. M., Kao, J. P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide-Linked+Dinitroxides+for+Monitoring+Cellular+Thiol+Redox+Status+through+Electron+Paramagnetic+Resonance+Spectroscopy.">Disulfide-Linked Dinitroxides for Monitoring Cellular Thiol Redox Status through Electron Paramagnetic Resonance Spectroscopy.</a> Biochemistry. 54 (47), 6973-6982 (2015).</li><li>Abbas, K., 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=Medium-throughput+ESR+detection+of+superoxide+production+in+undetached+adherent+cells+using+cyclic+nitrone+spin+traps.">Medium-throughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps.</a> Free Radical Research. 49 (9), 1122-1128 (2015).</li><li>Dikalov, S. I., 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=Distinct+roles+of+Nox1+and+Nox4+in+basal+and+angiotensin+II-stimulated+superoxide+and+hydrogen+peroxide+production.">Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production.</a> Free Radical Biology and Medicine. 45 (9), 1340-1351 (2008).</li><li>Dikalov, S. I., Kirilyuk, I. A., Voinov, M., Grigor'ev, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EPR+detection+of+cellular+and+mitochondrial+superoxide+using+cyclic+hydroxylamines.">EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines.</a> Free Radical Research. 45 (4), 417-430 (2011).</li><li>Dikalova, A. 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=Therapeutic+Targeting+of+Mitochondrial+Superoxide+in+Hypertension.">Therapeutic Targeting of Mitochondrial Superoxide in Hypertension.</a> Circulation Research. 107 (1), 106-116 (2010).</li><li>Dikalov, S. I., Polienko, Y. F., Kirilyuk, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Paramagnetic+Resonance+Measurements+of+Reactive+Oxygen+Species+by+Cyclic+Hydroxylamine+Spin+Probes.">Electron Paramagnetic Resonance Measurements of Reactive Oxygen Species by Cyclic Hydroxylamine Spin Probes.</a> Antioxidants &amp; Redox Signaling. , (2017).</li><li>Sharma, 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=L-Carnitine+preserves+endothelial+function+in+a+lamb+model+of+increased+pulmonary+blood+flow.">L-Carnitine preserves endothelial function in a lamb model of increased pulmonary blood flow.</a> Pediatric Research. 74 (1), 39-47 (2013).</li><li>Berg, K., Ericsson, M., Lindgren, M., Gustafsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+High+Precision+Method+for+Quantitative+Measurements+of+Reactive+Oxygen+Species+in+Frozen+Biopsies.">A High Precision Method for Quantitative Measurements of Reactive Oxygen Species in Frozen Biopsies.</a> PloS One. 9 (3), (2014).</li><li>Kozlov, A. V., 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=EPR+analysis+reveals+three+tissues+responding+to+endotoxin+by+increased+formation+of+reactive+oxygen+and+nitrogen+species.">EPR analysis reveals three tissues responding to endotoxin by increased formation of reactive oxygen and nitrogen species.</a> Free Radical Biology and Medicine. 34 (12), 1555-1562 (2003).</li><li>Van Rheen, 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=Lung+Extracellular+Superoxide+Dismutase+Overexpression+Lessens+Bleomycin-Induced+Pulmonary+Hypertension+and+Vascular+Remodeling.">Lung Extracellular Superoxide Dismutase Overexpression Lessens Bleomycin-Induced Pulmonary Hypertension and Vascular Remodeling.</a> American Journal of Respiratory Cell and Molecular Biology. 44 (4), 500-508 (2011).</li><li>Mouradian, G. C., 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=Superoxide+Dismutase+3+R213G+Single-Nucleotide+Polymorphism+Blocks+Murine+Bleomycin-Induced+Fibrosis+and+Promotes+Resolution+of+Inflammation.">Superoxide Dismutase 3 R213G Single-Nucleotide Polymorphism Blocks Murine Bleomycin-Induced Fibrosis and Promotes Resolution of Inflammation.</a> American Journal of Respiratory Cell and Molecular Biology. 56 (3), 362-371 (2017).</li><li>Dikalov, S. I., Li, W., Mehranpour, P., Wang, S. S., Zafari, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+extracellular+superoxide+by+human+lymphoblast+cell+lines:+comparison+of+electron+spin+resonance+techniques+and+cytochrome+C+reduction+assay.">Production of extracellular superoxide by human lymphoblast cell lines: comparison of electron spin resonance techniques and cytochrome C reduction assay.</a> Biochem Pharmacol. 73 (7), 972-980 (2007).</li><li>Kozuleva, 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=Quantification+of+superoxide+radical+production+in+thylakoid+membrane+using+cyclic+hydroxylamines.">Quantification of superoxide radical production in thylakoid membrane using cyclic hydroxylamines.</a> Free Radical Biology and Medicine. 89, 1014-1023 (2015).</li><li>Chen, K., Swartz, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+Hydroxylamines+to+Nitroxide+Spin+Labels+in+Living+Cells.">Oxidation of Hydroxylamines to Nitroxide Spin Labels in Living Cells.</a> Biochimica Et Biophysica Acta. 970 (3), 270-277 (1988).</li></ol>

对生物环境中自由基产生的评估对于了解氧化还原调节的健康和疾病信号很重要, 但由于自由基物种和技术物种的半衰期很短, 这些物种的测量极具挑战性常用方法的限制。epr 是氧化还原生物学中一个有价值和强大的工具, 因为它是检测自由基的唯一明确的方法。在本项目中, 我们演示了设计实验和准备样品的实际 epr 方法, 以便在不同的细胞隔间和体内不同的组织隔间中使用自旋探针检?...

虽然氧化应激和活性氧物种的测量对所有器官系统的各种疾病的研究都很重要, 但由于半衰期短、反应性高, 活性氧物种的检测具有挑战性。电子顺磁共振 (epr) 技术是检测自由基最明确的方法。与较常用的荧光探针相比, 旋转探针具有优势。虽然荧光探针相对便宜, 易于使用, 并提供快速, 灵敏的检测 ros, 他们确实有严重的局限性, 由于人工信号, 无法计算 ros 浓度, 以及普遍缺乏特异性 1.
为了便于 epr 用于生物研究, 合成了各种自旋探针, 可以测量一系列与生物相关的自由基物种以及po2、ph 值和氧化还原状态2,3, 4,5,6,7。自旋陷阱也被开发来捕获短命基和形成长寿的加合物, 这有利于检测 epr8。这两类 (自旋探针和自旋陷阱) 都有优点和局限性。一类常用的自旋探针是环状羟基胺, 它们是 epr 静音的, 与短命基反应, 形成稳定的亚硝基氧化物。环羟基胺与超氧化物的反应速度是自旋陷阱的 100倍, 使它们能够与细胞抗氧化剂竞争, 但它们缺乏特异性, 需要使用适当的控制和抑制剂来识别自由基的种类或来源负责亚硝基氧化物信号。虽然自旋陷阱表现出特异性, 根据被困物种的不同, 它们具有明显的光谱模式, 但它们具有超氧化物自旋诱捕的缓慢动力学, 并且容易对自由基加合物进行生物降解。自旋诱捕的应用在生物医学研究中有据可查,9、10、11、12、13.
该项目的目的是演示设计实验和准备样品的实用 epr 方法, 以便在不同的细胞隔间和体内不同的组织隔间中使用自旋探针检测超氧化物。一些手稿公布了与这些目标相关的协议, 使用细胞渗透性、细胞不渗透和线粒体靶向自旋探针, 针对不同的细胞隔间进行体外观察, 并处理组织, 以便在小鼠模型中进行分析14,15. 我们在这一文献的基础上, 验证了一种方法, 利用 1-羟基-3-甲氧基碳基 2, 2, 5, 5-四甲基吡咯烷酮 (cmh) 自旋探针在不同的细胞隔间在体外测量超氧化物, 以确保准确测量, 突出可能会扭曲结果的潜在技术问题。我们还提供了使用 cmh 自旋探针在血液、支气管肺泡灌洗液和肺组织中进行 epr 测量的方法。这些研究比较了处理组织的不同方法, 并提出了一种在收获组织之前将另一个自旋探针 cph 注入小鼠体内的方法。最后, 我们开发了一种实用的方法来储存样品在聚四氟乙烯 (ptfe) 油管, 允许存储和转移样品之前 epr 测量在 77 k。

<table border="0" cellpadding="0" cellspacing="0" style="width:1993px;" width="1994">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:931px;">DMEM</td>
			<td style="width:196px;">LifeTech</td>
			<td style="width:224px;">10566-016</td>
			<td style="width:643px;">cell culture media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diethylenetriaminepentaacetic acid (DTPA)</td>
			<td>Sigma Aldrich</td>
			<td>D6518-5G</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">sodium chloride (NaCl)&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>&#160;BP358-212</td>
			<td>used to prepare 50 mM phosphate saline buffer&#160; according to Sigma aldrish&#160;&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">potassium phosphate dibasic (HK2PO4 )</td>
			<td>Fisher Scientific&#160;</td>
			<td>&#160;BP363-500</td>
			<td>used to prepare 50 mM phosphate saline buffer&#160; according to Sigma aldrish&#160;&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">potassium phosphate monobasic (KH2PO4 )</td>
			<td>Sigma Aldrich</td>
			<td>P-5379</td>
			<td>used to prepare 50 mM phosphate saline buffer&#160; according to Sigma aldrish&#160;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Krebs-Henseleit buffer (KHB)&#160;</td>
			<td>(Alfa Aesar, Hill)</td>
			<td>J67820</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine erythrocyte superoxide dismutase (SOD)</td>
			<td>Sigma Aldrich</td>
			<td>&#160;S7571-30KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phorbol 12-myristate 13-acetate (PMA)&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P1585-1MG</td>
			<td>Dissolve in DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antimycin A (AA)</td>
			<td>Sigma Aldrich</td>
			<td>A8674-25MG</td>
			<td>Dissolve in Ethanol and store in glass vials(MW used is the averaged molecular weights for four lots)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine . HCl (CMH)</td>
			<td>Enzo Life Sciences</td>
			<td>ALX-430-117-M050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine . HCl (CPH)</td>
			<td>Enzo Life Sciences</td>
			<td>ALX-430-078-M250</td>
			<td></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:931px;">1-Hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine, 1-Hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido]piperidinium dichloride ( mito-TEMPO-H)</td>
			<td style="width:196px;">Enzo Life Sciences</td>
			<td style="width:224px;">ALX-430-171-M005</td>
			<td style="width:643px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:931px;">1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium chloride . HCl (CAT1H)</td>
			<td style="width:196px;">Enzo Life Sciences</td>
			<td style="width:224px;">ALX-430-131-M250</td>
			<td style="width:643px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:931px;">Heparin&#160;</td>
			<td style="width:196px;">Sagent Pharmaceuticals</td>
			<td style="width:224px;">NDC 25021-400-10</td>
			<td style="width:643px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Diphenyliodonium chloride&#160;</td>
			<td style="width:196px;">Sigma Aldrich</td>
			<td>43088</td>
			<td style="width:643px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:931px;">Deferoxamin mesylate salt</td>
			<td>Sigma Aldrich</td>
			<td style="width:224px;">D9533-1G</td>
			<td style="width:643px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Critoseal</td>
			<td>Leica</td>
			<td>39215003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BRAND disposable BLAUBRAND&#160;micropipettes, intraMark</td>
			<td>Sigma Aldrich</td>
			<td>708733</td>
			<td>Capillaries</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:931px;">PTFE FRACTIONAL FLUOROPOLYMER TUBING 
			3/16&#8221; OD x 1/8&#8221; ID</td>
			<td>NORELL</td>
			<td>1598774A</td>
			<td>Teflon tubing&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">SILICONE RUBBER STOPPERS FOR NMR SAMPLE TUBES&#160; FOR THIN WALL TUBES HAVING AN OD OF 4mm-5mm (3.2mm TO 4.2mm ID) TS-4-5-SR</td>
			<td>NORELL</td>
			<td>94987</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">EMXnano Bench-Top EPR spectrometer&#160;</td>
			<td>Bruker BioSpin GmbH</td>
			<td>E7004002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EMX NANO TISSUE CELL</td>
			<td>Bruker BioSpin GmbH</td>
			<td>E7004542</td>
			<td></td>
		</tr>
	</tbody>
</table>

use of electron paramagnetic resonance in biological samples at ambient temperature and 77 k

准确、特异地检测不同细胞和组织间的活性氧 (ros) 是研究生物环境中氧化还原调节信号的关键。电子顺磁共振波谱 (epr) 是唯一明确评估自由基的直接方法。它的优点是, 它检测特定物种的生理水平具有较高的特异性, 但它确实需要专门的技术, 仔细的样品制备, 并适当的控制, 以确保准确的解释数据。循环羟胺自旋探针与超氧化物或其他自由基选择性地反应, 产生可通过 epr 光谱学量化的亚氧化物信号。细胞渗透性自旋探针和自旋探针设计在线粒体中快速积累允许测定不同细胞隔间中的超氧化物浓度。
在培养的细胞中, 使用细胞渗透性 1-羟基-3-甲氧基碳酸-2, 2, 5, 5-四甲基吡咯烷酮 (cmh) 以及和不具有细胞不透水超氧化物歧化酶 (sod) 预处理, 或使用细胞渗透性 peg-sod, 允许细胞外与细胞质超氧化物的分化。线粒体 1-羟基-4-[2-三苯基磷酰磷)-乙酰胺多]-2, 2, 6, 6-四甲基-哌啶, 1-羟基 2, 2, 6, 6-四甲基-4-[2-(三苯基磷磷) 乙酰胺]线粒体 ros (主要是超氧化物)。
自旋探针和 epr 光谱也可应用于体内模型。超氧化物可以检测到细胞外液体, 如血液和肺泡液, 以及组织, 如肺组织。提出了几种处理和储存组织进行 epr 测量的方法, 并在体内提供静脉注射 1-羟基-3-羧基 2, 2, 5, 5-四甲基吡咯烷酮 (cph) 自旋探针。虽然测量可以在室温下进行, 但从体外和体内模型中获得的样品也可以储存在-80°c, 并由 epr 在 77 k 进行分析。样品可以存储在稳定在-80°c 的专用管材中, 并在 77 k 下运行, 以实现实用、高效和可重复的方法, 从而便于存储和转移样品。

利用 x/xo 超氧化物生成系统验证了 cmh 的超氧化物检测, 证明 sod 完全抑制了亚氧化物(cm) 信号, 而过氧化氢酶无效应 (图 1a)。然后用细胞渗透 cmh 自旋探针 +/-sod 预处理培养细胞, 在 raw 264.7 细胞中对细胞外超氧化物进行评价。在细胞悬浮液和缓冲液中都测定了硝基氧化物浓度, 这表明由于自旋探针的渗透性和快速平衡性, 这两种样品类型的值相?...

Watch this Scientific Journal Video about Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K at JoVE.com

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

电子顺磁共振 (epr) 光谱是一种明确的方法来测量自由基。使用选择性自旋探针可以检测不同细胞隔间中的自由基。我们提出了一个实用、有效的方法来收集生物样本, 方便处理、储存和转移用于 epr 测量的样品。

电子顺磁共振在生物样品环境温度和 77 k 的应用

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of ...

调节性氧化还原信号与各种疾病的发病机制有关。氧化还原生物学的研究要求准确检测不同细胞和组织区中的活性氧物种。EPR光谱是明确测量自由基的唯一方法。该协议演示了一种处理和存储EPR光谱生物样品的实用方法。这些协议说明了在两个代表性模型中使用EPR和自旋探头。虽然它们可以适应在其他生物环境中评估活性物种。实验的设计以及样品的处理对于其可重复性至关重要。我们建议考虑细胞密度，并确保适当的时间匹配控制。首先用每井一毫升的克雷布斯-亨塞利特缓冲液或KHB轻轻清洗生的264.7个细胞。用500微升KHB，每井补充100微米DTPA。要预处理超氧化物脱木酶，请在每个井中加入15微升的超氧化物脱化酶储存或车辆，并将板在37摄氏度下放置10分钟。在超氧化物去液化预处理结束时，将12.5微升10毫摩尔CMH和40微升125微摩尔PMA工作溶液加入到适当的井中，并在37摄氏度下孵育50分钟。立即将盘子放在冰上，将每个井的缓冲液收集到冰上单独的1.5毫升管中。接下来，将100微升新鲜KHB加DTPA添加到每一个井中，然后轻轻地从每一个井的底部刮取细胞。然后用移液重新悬浮细胞悬浮液，将细胞转移到冰上单独的1.5毫升管中。在室温下进行超氧化物检测，首先将一个缓冲液或细胞样品的50微升装入毛细管并密封管。立即将管放入电子顺磁共振（EPR）光谱仪中，并设置室温测量的EPR采集参数。始终测试含有与对照器相同的实验条件下处理的相同浓度的探针的空白样品。对于在 77 度开尔文的超氧化物检测，将一个样品的 150 微升装入一到两英寸的直聚四氟乙烯（PTFE）管中，一端有橡胶塞。用第二个塞子密封管的另一端，并在液氮中闪冻样品。然后快速拆下塞子，将 PTFE 管放在标有低温管中，以储存液氮。对于 EPR 测量，用液氮填充 EPR 光谱仪的手指脱华，并将含有样品的 PTFE 管放在液氮中。将手指 dewar 放入 EPR 光谱仪中，将 77 开尔文的测量的 EPR 采集参数设置为 77 度。在冷冻肺组织中进行ROS检测时，将组织放在干冰上，用钳子将闪冻组织稳定在干冰上。将样品切成五块15毫克，并在焦油1.5毫升管中称重。在样品中加入196微升KHB加DTPA和4微升CMH，在37摄氏度的水浴中孵育肺样本一小时，然后通过短暂的离心对组织片段进行造粒。然后将样品放在冰上，然后将 150 微升的上一液液转移到一块 PTFE 管中进行闪光冷冻，以准备 EPR 测量，正如刚刚演示的。如证明，对用PMA刺激刺激的原始264.7细胞的总超氧化物产生情况的评估表明，由于自旋探针的渗透性质和快速平衡，在细胞悬浮液和PMA处理细胞缓冲液中，硝基氧化物浓度相似。与对照细胞相比，硝基基在受PMA刺激的原始264.7细胞中增加，但在使用细胞不渗透超氧化物不变性酶的预处理细胞中则不增加。使用PMA刺激后，在原始264.7细胞中低温获得的硝基氧化物浓度在与室温数据一致的超氧化物脱化酶的存在下也衰减。与PBS治疗的对症小鼠和受伤动物相比，在血鼠和经过治疗的小鼠的血液和支气管中，以及从血小鼠和受伤动物中收获的肺组织片段的超级盐中，硝基氧化物浓度增加。此外，在逆轨道旋转探针注射后，与对控装置相比，蓝鼠和经过治疗的动物表现出更高的旋转探针信号。这些实用方案将允许研究人员通过EPR测试不同细胞隔间和生物样品中的自由基生产，包括低温测量。

Research

Education

JoVE Core

JoVE Journal

Physics

Biochemistry

Unit 1

Sources of Magnetic Fields

SCIENTISTS IN ACTION

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

Sheathless Capillary ElectrophoresisÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry for Metabolic Profiling of Biological Samples

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

科研

生物化学

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

制备及<em&gt;在体内</em&gt;使用基于活动的探针对<em&gt;ñ</em&gt; -acylethanolamine酸酰胺酶

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

多聚-PAGE：生物样品中的用于拍摄方法和解决蛋白复合体

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Analysis of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

使用微尺度热泳测量蛋白质-脂质相互作用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

从脂质绑定蛋白和过敏原中去除和替换内源性利根

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

Microcrystal Electron Diffraction of Small Molecules

小分子的微晶电子衍射

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been ...

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

使用双光束扫描电子显微镜从玻璃体生物样品制备薄片进行冷冻电子断层扫描

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...