Name: analyzing oxidative stress in murine intestinal organoids using reactive oxygen species-sensitive fluorogenic probe
Uploaded: 2021-09-17T14:45:00
Description: Watch this Scientific Journal Video about Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe at JoVE.com

这项工作得到了法国国家研究机构（ANR）拨款17-CE14-0022（i-Stress）的支持。

所有动物实验均在巴斯德使用委员会研究所和法国农业部批准后进行，编号为2016-0022。所有步骤都在组织培养罩内进行。
1. 肠道类器官培养试剂和材料的制备
<ol><li>为了制备生长培养基，混合先进的DMEM / F-12补充1x谷氨酰胺，1x青霉素/链霉素（P / S）溶液，10mM的HEPES，50ng / mL的小鼠EGF，20μg/ mL小鼠Noggin 500ng / mL小鼠R-Spondin1（ 见材料表）。在隐窝提取期间将培养基...

<ol>
	<li>Aviello, G., Knaus, U. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidases+and+ROS+signaling+in+the+gastrointestinal+tract+review-article.">NADPH oxidases and ROS signaling in the gastrointestinal tract review-article.</a> Mucosal Immunology. 11 (4), 1011-1023 (2018).</li><li>Holmstr&#246;m, K. M., Finkel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+and+physiological+consequences+of+redox-dependent+signalling.">Cellular mechanisms and physiological consequences of redox-dependent signalling.</a> Nature Reviews Molecular Cell Biology. 15 (6), 411-421 (2014).</li><li>vander Post, S., Birchenough, G. M. H., Held, J. 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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=ROS+production+and+NF-&#954;B+activation+triggered+by+RAC1+facilitate+WNT-driven+intestinal+stem+cell+proliferation+and+colorectal+cancer+initiation.">ROS production and NF-&#954;B activation triggered by RAC1 facilitate WNT-driven intestinal stem cell proliferation and colorectal cancer initiation.</a> Cell Stem Cell. 12 (6), 761-773 (2013).</li><li>Juhasz, 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=NADPH+oxidase+1+supports+proliferation+of+colon+cancer+cells+by+modulating+reactive+oxygen+species-dependent+signal+transduction.">NADPH oxidase 1 supports proliferation of colon cancer cells by modulating reactive oxygen species-dependent signal transduction.</a> Journal of Biological Chemistry. 292 (19), 7866-7887 (2017).</li><li>Aviello, G., Knaus, U. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+in+gastrointestinal+inflammation:+Rescue+Or+Sabotage.">ROS in gastrointestinal inflammation: Rescue Or Sabotage.</a> British Journal of Pharmacology. 174 (12), 1704-1718 (2017).</li><li>Gomes, A., Fernandes, E., Lima, J. L. F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probes+used+for+detection+of+reactive+oxygen+species.">Fluorescence probes used for detection of reactive oxygen species.</a> Journal of Biochemical and Biophysical Methods. 65 (2-3), 45-80 (2005).</li><li>Sato, 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Levy, 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=Innate+immune+receptor+NOD2+mediates+LGR5++intestinal+stem+cell+protection+against+ROS+cytotoxicity+via+mitophagy+stimulation.">Innate immune receptor NOD2 mediates LGR5+ intestinal stem cell protection against ROS cytotoxicity via mitophagy stimulation.</a> Proceedings of the National Academy of Sciences. 117 (4), 1994-2003 (2020).</li><li>Choi, H., Yang, Z., Weisshaar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell,+real-time+detection+of+oxidative+stress+induced+in+escherichia+coli+by+the+antimicrobial+peptide+CM15.">Single-cell, real-time detection of oxidative stress induced in escherichia coli by the antimicrobial peptide CM15.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (3), 303-310 (2015).</li><li>Amri, F., Ghouili, I., Amri, M., Carrier, A., Masmoudi-Kouki, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroglobin+protects+astroglial+cells+from+hydrogen+peroxide-induced+oxidative+stress+and+apoptotic+cell+death.">Neuroglobin protects astroglial cells from hydrogen peroxide-induced oxidative stress and apoptotic cell death.</a> Journal of Neurochemistry. 140 (1), 151-169 (2017).</li><li>Ahn, H. Y., 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=Two-Photon+Fluorescence+Microscopy+Imaging+of+Cellular+Oxidative+Stress+Using+Profluorescent+Nitroxides.">Two-Photon Fluorescence Microscopy Imaging of Cellular Oxidative Stress Using Profluorescent Nitroxides.</a> Journal of the American Chemical Society. 134 (10), 4721-4730 (2012).</li><li>Bidaux, 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=Epidermal+TRPM8+channel+isoform+controls+the+balance+between+keratinocyte+proliferation+and+differentiation+in+a+cold-dependent+manner.">Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a cold-dependent manner.</a> Proceedings of the National Academy of Sciences. 112 (26), 3345-3354 (2015).</li><li>Van de Bittner, G. C., Dubikovskaya, E. A., Bertozzi, C. R., Chang, C. J. <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+imaging+of+hydrogen+peroxide+production+in+a+murine+tumor+model+with+a+chemoselective+bioluminescent+reporter.">In vivo imaging of hydrogen peroxide production in a murine tumor model with a chemoselective bioluminescent reporter.</a> Proceedings of the National Academy of Sciences. 107 (50), 21316 (2010).</li><li>Rabbani, P. S., Abdou, S. A., Sultan, D. L., Kwong, J., Duckworth, A., Ceradini, D. J. <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+imaging+of+reactive+oxygen+species+in+a+murine+wound+model.">In vivo imaging of reactive oxygen species in a murine wound model.</a> Journal of Visualized Experiments. (141), e58450 (2018).</li><li>Sato, 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=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li></ol>

这项工作提供了一个循序渐进的方案，以分离小鼠空肠隐窝，将其培养成3D类器官，并通过将ROS敏感的荧光探针与整个类器官的定性显微镜成像相结合，并在类器官解离后使用流式细胞术对单个细胞进行定量ROS测量来分析类器官中的ROS。
此方法中的第一个关键步骤是加密提取过程。事实上，隐窝制剂的质量是成功形成类器官的关键。因此，获得具有富集隐窝和少量细胞碎片或...

活性氧（ROS）是细胞代谢的天然副产物。它们也可以由专门的酶复合物主动产生，例如膜结合的NADPH-氧化酶（NOX）和双氧化酶（DUOX），它们产生超氧阴离子和过氧化氢1。通过表达抗氧化酶和ROS清除剂，细胞可以微调其氧化还原平衡，从而保护组织稳态2。虽然ROS可能对细胞具有剧毒并破坏DNA，蛋白质和脂质，但它们是至关重要的信号分子2。在肠上皮中，茎和祖细胞增殖需要适度的ROS水平3;高ROS水平导致其凋亡4。慢性氧化应激与许多胃肠道疾病有关，例如炎症性肠病或癌症。例如，在Wnt驱动的肠癌小鼠模型中，发现癌细胞增殖需要通过激活NADPH-氧化酶来提高ROS的产生5，6。定义肠道细胞，特别是干细胞，干细胞如何管理氧化应激以及细胞环境如何影响这种能力对于更好地了解这种疾病的病因至关重要7。
在组织中，不同的细胞类型呈现基底氧化状态，该状态可能根据其功能和代谢以及不同水平的氧化剂和抗氧化剂分子的表达而变化4，7。 在体内 监测ROS是非常具有挑战性的。已经开发出根据其氧化还原状态发射荧光的细胞渗透染料，以可视化和测量活细胞和动物中的细胞ROS。然而，它们的功效取决于它们在活组织内的扩散和快速读数，使它们难以在动物模型中使用8。
过去，化合物对ROS产生的影响的研究是使用细胞系完成的，但这可能无法反映体内情况。由Clevers9小组开发的肠道类器官模型使肠道原代细胞的离体生长成为可能。在存在确定的生长因子的情况下，在基质中培养肠隐窝，导致称为类器官（迷你肠道）的三维结构，其复制隐窝组织，来自不同上皮谱系的细胞排列在内腔内，肠干细胞驻留在小隐窝样突起中。
在这里，利用该模型，描述了一种简单的方法，通过将市售的ROS敏感染料添加到类器官培养基中，以单细胞分辨率研究原代肠细胞中的氧化应激。
读板器通常用于检测总种群中的ROS产量。该协议使用流式细胞术或成像测定来检测具有转基因细胞或特异性抗体染色的特定细胞类型中的ROS。这项工作涉及小鼠肠道类器官培养和通过共聚焦成像和流式细胞术定量的ROS可视化。使用Lgr5-GFP小鼠衍生的小肠类器官，可以特异性分析不同处理后肠干细胞中的氧化应激水平。该协议可用于测试外源分子（例如微生物群衍生的穆拉米基二肽（MDP）10）在用所选化合物刺激类器官后对ROS平衡的影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lgr5-EGFP-IRES-creERT2 (Lgr5-GFP)</td>
			<td>The Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Growth culture medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM F12 (DMEM/F12)</td>
			<td>ThermoFisher</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement, minus vitamin A</td>
			<td>ThermoFisher</td>
			<td>12587010</td>
			<td>Stock Concentration: 50x</td>
		</tr>
		<tr>
			<td>GlutaMAX (glutamine)</td>
			<td>ThermoFisher</td>
			<td>35050038</td>
			<td>Stock Concentration: 100x</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>ThermoFisher</td>
			<td>15630056</td>
			<td>Stock Concentration: 1 M</td>
		</tr>
		<tr>
			<td>Murine EGF</td>
			<td>R&#38;D</td>
			<td>2028-EG-200</td>
			<td>Stock Concentration: 500 &#181;g/mL in PBS</td>
		</tr>
		<tr>
			<td>murine Noggin</td>
			<td>R&#38;D</td>
			<td>1967-NG/CF</td>
			<td>Stock Concentration: 100 &#181;g/mL in PBS</td>
		</tr>
		<tr>
			<td>Murine R-spondin1</td>
			<td>R&#38;D</td>
			<td>3474-RS-050</td>
			<td>Stock Concentration: 50 &#181;g/mL in PBS</td>
		</tr>
		<tr>
			<td>N-2 Supplement</td>
			<td>ThermoFisher</td>
			<td>17502048</td>
			<td>Stock Concentration: 100x</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S)</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td>Stock Concentration: 100x (10,000 units/mL of penicillin and 10,000 &#181;g/mL of streptomycin)</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well round bottom</td>
			<td>Corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>ball tip scissor</td>
			<td>Fine Science Tools GMBH</td>
			<td>14086-09</td>
			<td></td>
		</tr>
		<tr>
			<td>CellROX&#174; Deep Red Reagent</td>
			<td>ThermoFisher</td>
			<td>C10422</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#8217;,6-diamidino-2-ph&#233;nylindole, dichlorhydrate) (fluorgenic probe)</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td>stock at 10 mg/mL</td>
		</tr>
		<tr>
			<td>DPBS 1x no calcium no magnesium (DPBS)</td>
			<td>ThermoFisher</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>FLuoroBrite DMEM (DMEM no phenol red)</td>
			<td>ThermoFisher</td>
			<td>A1896701</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>H3570</td>
			<td>stock at 10 mg/mL</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced, Phenol Red Free (Basement Membrane Matrix)</td>
			<td>Corning</td>
			<td>356231</td>
			<td>once received thaw o/n in the fridge, keep for 1h on ice and, make 500 mL aliquots and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>&#181;-Slide 8 Well chambers</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine (NAC)</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>tert-Butyl hydroperoxide (tBCHP)solution (70%wt. In H2O2)</td>
			<td>Sigma</td>
			<td>458139</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1X), no phenol red (trypsin)</td>
			<td>ThermoFisher</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA, pH8.0</td>
			<td>ThermoFisher</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>Rock-inhibitor to be used to minimize cell death upon tissue dissociation</td>
		</tr>
		<tr>
			<td>Programs and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Attune NxT (Flow Cytometer)</td>
			<td>ThermoFischer</td>
			<td></td>
			<td>Flow cytometer analyzer</td>
		</tr>
		<tr>
			<td>Fiji/ImageJ</td>
			<td>https://imagej.net/software/fiji/downloads</td>
			<td></td>
			<td>images generation</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>BD Bioscience</td>
			<td></td>
			<td>FACS analysis</td>
		</tr>
		<tr>
			<td>Observer.Z1</td>
			<td>Zeiss</td>
			<td></td>
			<td>confocal system</td>
		</tr>
		<tr>
			<td>Opterra (swept-field confocal)</td>
			<td>Bruker</td>
			<td></td>
			<td>confocal system</td>
		</tr>
		<tr>
			<td>high speed EMCCD Camera Evolve Delta 512</td>
			<td>Photometrics</td>
			<td></td>
			<td>confocal system</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>statistical analysis</td>
		</tr>
	</tbody>
</table>

analyzing oxidative stress in murine intestinal organoids using reactive oxygen species-sensitive fluorogenic probe

活性氧（ROS）在肠道稳态中起着至关重要的作用。ROS是细胞代谢的天然副产品。它们是在粘膜水平上对感染或损伤作出反应而产生的，因为它们参与抗菌反应和伤口愈合。它们也是关键的次级信使，调节多种途径，包括细胞生长和分化。另一方面，过量的ROS水平会导致氧化应激，这对细胞有害，有利于肠道疾病，如慢性炎症或癌症。这项工作提供了一种直接的方法，通过使用市售的荧光探针，通过活体成像和流式细胞术检测肠道小鼠类器官中的ROS。在这里，该协议描述了测定调节肠道类器官氧化还原平衡并检测特定肠道细胞类型中的ROS水平的化合物的作用，此处通过分析用GFP遗传标记的肠干细胞来举例说明。该协议可与其他荧光探针一起使用。

作为所述方案的概念证明，从Lgr5-eGFP-IRES-CreERT2小鼠系获得的隐窝被用于其中肠干细胞显示由Barker等人建立的镶嵌GFP表达，以最初表征肠干细胞10 并允许基于其GFP表达绘制这些细胞。因此，提供了一个模型来比较不同处理下特定细胞类型群体中的ROS水平。使用ROS抑制剂（NAC）和诱导剂（tBHP），已知其作用于细胞ROS以可视化其水平的变化。
图1A<...

Watch this Scientific Journal Video about Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe at JoVE.com

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

本方案描述了一种使用定性成像和定量细胞术测定法检测肠道小鼠类器官中活性氧（ROS）的方法。这项工作可以潜在地扩展到其他荧光探针，以测试所选化合物对ROS的影响。

使用活性氧敏感荧光探针分析小鼠肠道类器官中的氧化应激

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

该方法是细胞生物学领域的一种有价值的工具，用于评估使用荧光探针在体外培养为类器官的活原代细胞中的细胞ROS水平。该技术的主要优点是ROS可以通过显微镜在完整的肠道类器官中定性地可视化，并使用96孔板的流式细胞术在解离细胞中进行定量分析。该方法可以应用于其他类器官模型，并且可以使用替代荧光传感器来分析不同的细胞途径。在首次应用该协议之前，实验者应熟悉类器官培养和传代。虽然我们的实验方案非常适合筛选方法，但初学者应该从几个样本开始。演示该程序的将是实验室的工程师Sophie Dulauroy。在牺牲一只8至10周龄的Lgr5-GFP小鼠后，收集十二指肠和回肠之间空肠周围区域的5至8厘米，并将其置于冰上补充抗生素的冷DPBS中。接下来，通过用含有抗生素的5至10毫升冷DPBS冲洗来清洁肠道内容物。然后使用球尖剪刀，纵向打开肠道，并使用镊子将组织转移到含有抗生素的冷DPBS的培养皿中。摇动培养皿内的纸巾以冲洗干净。接下来，使用塑料巴斯德移液器通过抽吸抓住肠道，并将其转移到含有10毫升冷10毫摩尔EDTA的15毫升管中。将管倒置三次，并在冰上孵育10分钟。孵育后，将组织转移到含有10毫升DPBS的管中并涡旋两分钟。立即在培养皿中加入10微升馏分，并使用显微镜评估馏分的质量。在DPBS中进行两次转移后，每次转移之间有两分钟的涡旋管，如前所述，将EDTA中的肠在冰上孵育五分钟。在含有DPBS的试管中连续转印三次后，每次转印之间涡旋三分钟，通过将选定的试管倒置三次并通过70微米细胞过滤器过滤，将最佳馏分混合到50毫升管中。然后旋转隐窝，丢弃上清液并机械破坏沉淀，然后加入五毫升冷DMEM F12。在显微镜下手动计算10微升等分试样中存在的隐窝数量。计数后，再次旋转隐窝并小心地除去上清液。然后机械地破坏沉淀并将生长培养基加入管中，以获得每微升90隐窝的浓度。接下来，加入两体积的未稀释的基底膜基质或BMM，以获得每微升30个隐窝的最终浓度，并小心地上下移液悬浮液，而不会将气泡引入混合物中。对于流式细胞术分析，将10微升的隐窝BMM板混合为预热的圆底96孔板的每个孔中心的圆顶。对于成像，将10微升的混合物作为薄层沉积在预热的微滑梯八孔室中。让BMM在室温下凝固五分钟后，将板置于37摄氏度和5%二氧化碳的培养箱中。15分钟后，将250微升生长培养基加入每个孔中，注意不要分离BMM，并将板放入培养箱中。为了通过共聚焦显微镜观察氧化应激，在镀有类器官的微滑八孔室的相应孔中加入一微升n-乙酰半胱氨酸储备溶液。孵育一小时后，在相应的孔中加入一微升叔丁基过氧化氢储备溶液，再孵育30分钟。接下来，每孔加入1.25毫摩尔稀释的荧光探针的一微升，然后加入每毫升1.25毫克Hoechst溶液的一微升。再孵育30分钟后，在不干扰BMM的情况下取出培养基，并轻轻加入250微升温热的DMEM，不含酚红。使用配备热室和气体供应的共聚焦显微镜对类器官进行成像，以检测荧光探针。使用正对照，设置ROS信号的激光强度和时间曝光，并检查该信号在负对照中是否较低。接下来，使用目镜筛选载玻片以识别表达GFP的类器官并调整激光强度。设置一个25微米的z-stack并定义位置以获得整个类器官的拼接图像，以获得显示一层细胞的类器官的一部分。在含有类器官的96孔圆形底板的阴性对照孔中加入一微升的n-乙酰半胱氨酸储备溶液。孵育一小时后，在相应的孔中加入一微升叔丁基过氧化氢储备溶液，孵育30分钟。然后使用多通道移液器，在不干扰附着的BMM的情况下取出培养基，并将其转移到另一个96孔圆形底板上。把这个盘子放在一边。接下来，加入100微升胰蛋白酶，使用多通道移液器，上下移液五次，以破坏BMM。短短的五分钟孵育后，通过第二次上下移液来解离类器官。旋转板并通过反转板来丢弃上清液。将先前在另一个96孔板中收集的培养基重新加入相应的孔中，并通过上下移液五次来重悬细胞。接下来，加入一微升荧光探针并孵育30分钟。然后再次旋转板并用250微升DAPI溶液重悬细胞。将样品转移到流式细胞术管中，并将试管保持在冰上。使用单色样品优化未染色控制和激光电压的正向和侧面散射电压设置，适用于每个荧光团。然后使用适当的门控策略，收集至少 20， 000 个事件。ROS染色和类器官的代表性共聚焦图像显示，在NAC抑制剂存在下，只有来自类器官管腔中含有的死细胞的信号是可见的。在未经处理的类器官中，可以看到基础ROS水平，特别是在GFP阳性细胞中，证明干细胞产生的ROS高于分化细胞。GFP阳性细胞在荧光探针存在下通过诱导剂呈现更显着的细胞质信号，表明ROS水平在治疗后特别在干细胞中增加。肠道类器官中ROS产生的流式细胞术分析表明，用抑制剂刺激后基础ROS水平降低，用诱导剂激发后增加。用抑制剂预处理然后用诱导剂刺激的细胞比单独用诱导剂刺激的细胞存在更低的水平。对门控为GFP阳性的干细胞的类似分析显示，与非刺激细胞相比，抑制剂处理后ROS水平降低3.5倍，诱导剂处理后增加4倍。当尝试这个过程时，用户应该记住在类器官操作和解离期间限制细胞应激。此外，在分析 ROS 时，应始终包括阳性和阴性对照。按照该程序，分析可以通过对固定的完整类器官进行免疫荧光染色，细胞分选和基因表达分析来补充，以获得有关ROS调控的其他见解。观看此视频后，您应该知道如何生长小鼠肠道类器官，对完整的类器官进行实时成像分析，并处理肠道器官以进行流式细胞术分析。

Research

JoVE Journal

Biology

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

检测，可视化和定量活性氧的生成在变形虫模型系统

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

科研

生物学

Analysis of Oxidative Stress in Zebrafish Embryos

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

测量氧化应激性<em&gt;秀丽隐杆线虫</em&gt;在96孔微量滴定板

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

彗星试验系统性氧化应激的间接测量

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

肠道维利表皮的器官3D培养进行分化

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

In Vitro Analysis of E3 Ubiquitin Ligase Function

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

用化学发光法实时检测水稻免疫反应中活性氧的产生

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...

3D Culture of Organoids from Murine Intestinal Crypts and Single Stem Cell

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research (Video) | JoVE 

小鼠肠隐窝和单个干细胞的类器官的3D培养用于类器官研究

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...

Epithelial Effects of Intestinal Inflammation on Established Murine Colonoids

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids (Video) | JoVE 

研究体 外 肠道炎症对已建立的小鼠结肠样的上皮影响

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...