Name: sensitive measurement of mitophagy by flow cytometry using the ph-dependent fluorescent reporter mt-keima
Uploaded: 2018-08-12T14:00:00.000+00:00
Duration: 9 min 13 s
Description: Watch this Scientific Journal Video about Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima at JoVE.com

这项工作得到了韩国国家研究基金会 (2016R1D1A1B03931949) (j.u.) 和韩国政府 (MSIT) (2016R1A2B2008887, 2016R1A5A2007009) 资助的韩国国家研究基金会 (NRF) 赠款的支持。.)

1. 表达 Keima (mt-Keima) 的 HeLa 细胞的生成
<ol><li>
Keima 慢病毒北疆的制备
	<ol>
<li>用2毫升0.001% 聚 l-赖氨酸/磷酸盐缓冲盐水 (PBS) 涂上100毫米的培养基, 在室温下可以站立5分钟。</li>
		<li>使用连接到真空的玻璃吸管去除聚 l-赖氨酸溶液, 并通过添加2毫升 1x PBS 来洗涤培养皿。</li>
		<li>板 1.5 x 106 HEK293T 细胞在涂层培养皿与10毫升的 DMEM 含有10% 的血清和1% 青霉素/链霉素, 并培养细胞在37°c 在一个 CO2组织培养孵化器一天。</li>
		<li>染2µg 的 mt-Keima 慢病毒载体质粒29 DNA 连同包装脱氧核糖核酸 (psPAX 2 µg 和 pVSVG 0.25 µg) 使用的转染试剂 8 h 根据制造商的指示。</li>
		<li>去除转染介质, 加入8毫升的新鲜介质。</li>
		<li>收集含有病毒粒子的介质48小时后。通过离心 500 x g 5 分钟, 从收集的介质中去除细胞碎片, 并用0.45 µm 注射器过滤器进行过滤。 注:为长期使用, 使整除数的慢病毒载体颗粒和储存样品在-80 摄氏度。避免将病毒样本置于冰冻解冻循环中, 以有效地感染病毒。</li>
	</ol>
</li>
	<li>
Keima 慢病毒北疆感染 HeLa 细胞
	<ol>
<li>5 x 104 hela 细胞, 在60毫米的培养皿中表达, 其中4毫升的 DMEM 含有10% 个血清和1% 个青霉素/链霉素, 在37摄氏度前一天 Keima 慢病毒北疆。 注:由于在 hela 细胞中缺乏内源性的 CCCP表达, 在这里使用的 hela 细胞更清楚地显示了 mitophagy。此过程也可应用于其他主要或永生化的细胞系。</li>
		<li>第二天去除生长介质, 加入1毫升的 Keima 慢病毒载体介质。添加额外的2毫升的生长培养基, 含有3µL 的凝聚胺溶液 (8 毫克/毫升蒸馏水)。在2组织培养孵化器中孵化一天。</li>
		<li>第二天去掉 Keima 慢病毒载体混合物, 用 1x PBS 清洗两次细胞。添加4毫升的生长培养基, 并治疗细胞与2µg/毫升嘌呤霉素两天。</li>
		<li>经过两天的嘌呤霉素选择, 去除生长培养基, 并清洗细胞两次与 1x PBS。添加生长培养基, 并培养细胞在 CO2孵化器, 直到使用。 注:该方法可用于生成稳定表达 Keima 的细胞, 包括其他细胞系和主细胞。</li>
	</ol>
</li>
</ol>2. 诱导 mitophagy 使用 CCCP 处理和制备外地药物管制样品
<ol style="margin-left: 40px;"><li>板 5 x 104 HeLa 细胞表达 mt-Keima 在60毫米培养皿与4毫升的 DMEM 含有10% 个血清和1% 青霉素/链霉素和培养他们一天在37°c 在 CO2组织培养孵化器。</li>
	<li>第二天去除生长培养基, 加入4毫升新鲜 DMEM, 含10µM 羰基氰化腙 (CCCP)。在 CO2组织孵化器中孵育 HeLa-Keima 细胞, 6 小时。</li>
	<li>去除生长培养基6小时后, 用2毫升 1x PBS 冲洗细胞。用胰蛋白酶/EDTA 溶液将细胞分离, 加入含有10% 血清的生长培养基, 使胰蛋白酶钝化。将细胞转移到15毫升锥形管中。</li>
	<li>离心细胞在 500 x g 5 分钟。</li>
	<li>通过吸入和并用重悬1毫升冷 1x PBS 的细胞, 小心地去除生长培养基。</li>
	<li>将细胞转换成适当的流式管, 放在冰上。 注:准备未感染的 HeLa 细胞作为阴性对照。 注:细胞在冰上存活数小时, Keima 荧光信号也保持稳定。</li>
</ol>3. mitophagy 的资产管制分析
注: 在本研究中, 用 405 nm 和 561 nm 激光的流式细胞仪对单元进行分析。细胞兴奋与紫激光 (405 nm) 与发射检测到 610, 10 毫微米与一个 BV605 探测器, 并与黄绿色激光 (561 nm) 与发射检测 610 @ 10 nm 由一个 PE-CF594 探测器同时 (图 1)。
<ol><li>
浇细胞
	<ol>
<li>打开流式细胞仪和计算机, 登录分析软件。</li>
		<li>生成新的实验或复制现有的实验。要激发细胞与紫罗兰 (405 毫微米) 和黄绿色 (561 nm) 激光器和检测发射在 610 @ 10 nm 探测器, 选择 BV605 和 PE-CF594, 除了在参数窗口中的正散射 (FSC) 和侧散射 (SSC), 根据需要显影。 注:应在线性模式中添加所有显影。</li>
		<li>涡旋每个细胞样品以分散细胞团聚体。</li>
		<li>将包含控制的 Keima 细胞样本的管放置在取样端口上, 不感染 mt-慢病毒北疆, 并按下 "运行" 按钮。</li>
		<li>在 fsc 与 ssc 的点图中, 调整 fsc 和 ssc 电压, 将单元格放在点图的中心。</li>
		<li>使用围绕活细胞的多边形图标绘制一个 P1 门, 并消除死细胞和细胞碎片 (图 2A)。</li>
	</ol>
</li>
	<li>
调节紫、绿激光器的电压
	<ol>
<li>将含有 Keima 慢病毒北疆感染的 HeLa 细胞的导管放置在样品注射口, 然后按下 "运行" 按钮。</li>
		<li>在 BV605 (405 nm) 与 SSC 的点图中, 调整 BV605 电压, 使表达 mt-Keima 的 HeLa 细胞明显有别于不表达 mt-Keima 的控制单元 (图 2B)。</li>
		<li>在 PE-CF594 (561 nm) 与 SSC 的点图中, 调整 PE-CF594 电压, 使表达 mt-Keima 的 HeLa 细胞明显有别于控制细胞 (图 2B)。</li>
	</ol>
</li>
	<li>
评估 mitophagy 中细胞的百分比
	<ol>
<li>利用线性尺度获取 BV605 与 PE-CF594 点图。用多边形图标在表达 mt-Keima 的细胞周围画一个门, 消除没有表达 mt-Keima 的细胞 (图 3A)。Keima 荧光阳性细胞群称为 "mt-Keima" 门。</li>
		<li>创建另一个点图 BV605 vs PE-CF594 只显示 "mt-Keima"-门控细胞。在这个点图中, 在未经处理的 mt-Keima 阳性细胞周围画一扇门。控制 HeLa 细胞的基底 mitophagy 活性低, PE-CF594/BV605 的发射率小于1。因此, 这个门被称为 "低" 门。</li>
		<li>绘制另一个包含 "低" 门区域的门。这个门被称为 "高" 门, 因为它包含高 mitophagy 活动的细胞, 即在 PE-CF594/BV605 中发射率高的细胞 (图 3B)。</li>
		<li>要计算 "高" 门中单元格的百分比, 请选择 "显示人口层次结构" 菜单。"父百分比" (%Parent) 值表示 mt-Keima 阳性人群中 "高" 门中细胞的百分比。</li>
		<li>设置至少1万事件记录在 "mt-Keima" 停止门和运行每个样本。</li>
	</ol>
</li>
</ol>

<ol>
	<li>McBride, H. M., Neuspiel, M., Wasiak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+More+than+just+a+powerhouse.">Mitochondria: More than just a powerhouse.</a> Current Biology. 16 (14), R551-R560 (2006).</li><li>Zorov, D. B., Krasnikov, B. F., Kuzminova, A. E., Vysokikh, M., Zorova, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+revisited.+Alternative+functions+of+mitochondria.+Bioscience+Reports.">Mitochondria revisited. Alternative functions of mitochondria. Bioscience Reports.</a> Bioscience Reports. 17 (6), 507-520 (1997).</li><li>Taylor, R. W., Turnbull, 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=Mitochondrial+DNA+mutations+in+human+disease.">Mitochondrial DNA mutations in human disease.</a> Nature Reviews Genetics. 6 (5), 389-402 (2005).</li><li>Galluzzi, L., Kepp, O., Trojel-Hansen, C., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+control+of+cellular+life,+stress,+and+death.">Mitochondrial control of cellular life, stress, and death.</a> Circulation Research. 111 (9), 1198-1207 (2012).</li><li>Kang, D., Hamasaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+mitochondrial+DNA+in+common+diseases+and+disease+states:+aging,+neurodegeneration,+heart+failure,+diabetes,+and+cancer.">Alterations of mitochondrial DNA in common diseases and disease states: aging, neurodegeneration, heart failure, diabetes, and cancer.</a> Current Medicinal Chemistry. 12 (4), 429-441 (2005).</li><li>Sun, N., Youle, R. J., 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=The+mitochondrial+basis+of+aging.">The mitochondrial basis of aging.</a> Molecular Cell. 61 (5), 654-666 (2016).</li><li>Wallace, D. C., Brown, M. D., Melov, S., Graham, B., Lott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+biology,+degenerative+diseases+and+aging.">Mitochondrial biology, degenerative diseases and aging.</a> BioFactors. 7 (3), 187-190 (1998).</li><li>Yun, J., 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=Mitohormesis.">Mitohormesis.</a> Cell Metabolism. 19 (5), 757-766 (2014).</li><li>Ashrafi, G., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathways+of+mitophagy+for+quality+control+and+clearance+of+mitochondria.">The pathways of mitophagy for quality control and clearance of mitochondria.</a> Cell Death and Differentiation. 20 (1), 31-42 (2013).</li><li>Clark, I. 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=Drosophila+pink1+is+required+for+mitochondrial+function+and+interacts+genetically+with+parkin.">Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin.</a> Nature. 441 (7097), 1162-1166 (2006).</li><li>Park, 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=Mitochondrial+dysfunction+in+Drosophila+PINK1+mutants+is+complemented+by+parkin.">Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin.</a> Nature. 441 (7097), 1157-1161 (2006).</li><li>Narendra, D., Tanaka, A., Suen, D. F., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin+is+recruited+selectively+to+impaired+mitochondria+and+promotes+their+autophagy.">Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.</a> The Journal of Cell Biology. 183 (5), 795-803 (2008).</li><li>Zhu, J., Wang, K. Z., Chu, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=After+the+banquet:+Mitochondrial+biogenesis,+mitophagy,+and+cell+survival.">After the banquet: Mitochondrial biogenesis, mitophagy, and cell survival.</a> Autophagy. 9 (11), 1663-1676 (2013).</li><li>Um, J. H., Yun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+role+of+mitophagy+in+human+diseases+and+physiology.">Emerging role of mitophagy in human diseases and physiology.</a> BMB Reports. 50 (6), 299-307 (2017).</li><li>Al Rawi, 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=Postfertilization+autophagy+of+sperm+organelles+prevents+paternal+mitochondrial+DNA+transmission.">Postfertilization autophagy of sperm organelles prevents paternal mitochondrial DNA transmission.</a> Science. 334 (6059), 1144-1147 (2011).</li><li>Kim, M. 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=SESN2/sestrin2+suppresses+sepsis+by+inducing+mitophagy+and+inhibiting+NLRP3+activation+in+macrophages.">SESN2/sestrin2 suppresses sepsis by inducing mitophagy and inhibiting NLRP3 activation in macrophages.</a> Autophagy. 12 (8), 1272-1291 (2016).</li><li>Kim, M. J., Yoon, J. H., Ryu, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy:+A+balance+regulator+of+NLRP3+inflammasome+activation.">Mitophagy: A balance regulator of NLRP3 inflammasome activation.</a> BMB Reports. 49 (10), 529-535 (2016).</li><li>Sandoval, 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=Essential+role+for+Nix+in+autophagic+maturation+of+erythroid+cells.">Essential role for Nix in autophagic maturation of erythroid cells.</a> Nature. 454 (7201), 232-235 (2008).</li><li>Sato, M., Sato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+paternal+mitochondria+by+fertilization-triggered+autophagy+in+C.+elegans+embryos.">Degradation of paternal mitochondria by fertilization-triggered autophagy in C. elegans embryos.</a> Science. 334 (6059), 1141-1144 (2011).</li><li>Geisler, 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=PINK1/Parkin-mediated+mitophagy+is+dependent+on+VDAC1+and+p62/SQSTM1.">PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.</a> Nature Cell Biology. 12 (2), 119-131 (2010).</li><li>Matsuda, N., 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=PINK1+stabilized+by+mitochondrial+depolarization+recruits+Parkin+to+damaged+mitochondria+and+activates+latent+Parkin+for+mitophagy.">PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy.</a> The Journal of Cell Biology. 189 (2), 211-221 (2010).</li><li>Allen, G. F., Toth, R., James, J., Ganley, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+iron+triggers+PINK1/Parkin-independent+mitophagy.">Loss of iron triggers PINK1/Parkin-independent mitophagy.</a> EMBO Reports. 14 (12), 1127-1135 (2013).</li><li>Chu, 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=Cardiolipin+externalization+to+the+outer+mitochondrial+membrane+acts+as+an+elimination+signal+for+mitophagy+in+neuronal+cells.">Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells.</a> Nature Cell Biology. 15 (10), 1197-1205 (2013).</li><li>Kubli, D. 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=PINK1+Is+dispensable+for+mitochondrial+recruitment+of+Parkin+and+activation+of+mitophagy+in+cardiac+myocytes.">PINK1 Is dispensable for mitochondrial recruitment of Parkin and activation of mitophagy in cardiac myocytes.</a> PloS One. 10 (6), e0130707 (2015).</li><li>Dolman, N. J., Chambers, K. M., Mandavilli, B., Batchelor, R. H., Janes, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+and+techniques+to+measure+mitophagy+using+fluorescence+microscopy.">Tools and techniques to measure mitophagy using fluorescence microscopy.</a> Autophagy. 9 (11), 1653-1662 (2013).</li><li>Kuma, A., Matsui, M., Mizushima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC3,+an+autophagosome+marker,+can+be+incorporated+into+protein+aggregates+independent+of+autophagy:+caution+in+the+interpretation+of+LC3+localization.">LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization.</a> Autophagy. 3 (4), 323-328 (2007).</li><li>Katayama, H., Kogure, T., Mizushima, N., Yoshimori, T., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+quantitative+technique+for+detecting+autophagic+events+based+on+lysosomal+delivery.">A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery.</a> Chemistry &amp; Biology. 18 (8), 1042-1052 (2011).</li><li>Lee, I. H., Yun, J., 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=The+emerging+links+between+sirtuins+and+autophagy.">The emerging links between sirtuins and autophagy.</a> Methods in Molecular Biology. 1077, 259-271 (2013).</li><li>Sun, N., 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+In+vivo+Mitophagy.">Measuring In vivo Mitophagy.</a> Molecular Cell. 60 (4), 685-696 (2015).</li><li>Denison, S. R., 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=Alterations+in+the+common+fragile+site+gene+Parkin+in+ovarian+and+other+cancers.">Alterations in the common fragile site gene Parkin in ovarian and other cancers.</a> Oncogene. 22 (51), 8370-8378 (2003).</li><li>Adan, A., Alizada, G., Kiraz, Y., Baran, Y., Nalbant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+Basic+principles+and+applications.">Flow cytometry: Basic principles and applications.</a> Critical Reviews in Biotechnology. 37 (2), 163-176 (2017).</li><li>Bingol, 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=The+mitochondrial+deubiquitinase+USP30+opposes+parkin-mediated+mitophagy.">The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy.</a> Nature. 510 (7505), 370-375 (2014).</li><li>Burbulla, L. F., Kruger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+primary+human+fibroblasts+for+monitoring+mitochondrial+phenotypes+in+the+field+of+Parkinson's+disease.">The use of primary human fibroblasts for monitoring mitochondrial phenotypes in the field of Parkinson's disease.</a> Journal of Visualized Experiments. 68, e4228 (2012).</li><li>Di Sante, G., Casimiro, M. C., Pestell, T. G., Pestell, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+video+microscopy+for+assessment+of+EYFP-Parkin+aggregation+as+a+marker+for+cellular+mitophagy.">Time-lapse video microscopy for assessment of EYFP-Parkin aggregation as a marker for cellular mitophagy.</a> Journal of Visualized Experiments. 111, e53657 (2016).</li><li>Fang, E. F., 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+vitro+and+in+vivo+detection+of+mitophagy+in+human+cells,+C.+Elegans,+and+mice.">In vitro and in vivo detection of mitophagy in human cells, C. Elegans, and mice.</a> Journal of Visualized Experiments. 129, e56301 (2017).</li></ol>

作者没有什么可透露的。

在这里, 我们提出了一个快速和灵敏的方法, 使用流式细胞仪来测量细胞 mitophagy 活动在表达 mt Keima。接受高水平 mitophagy 的细胞表现为 PE-CF594 (561 nm)/BV605 (405 nm) 激发的比例增加。因此, mitophagy 活动可以表示为高561/405 比例的细胞百分比。我们计算了 PE-CF594 (561 nm) 与 BV605 (405 nm) 的斑点图中 "高" 门区域内细胞的百分比, 结果表明 CCCP 的治疗导致了 HeLa 细胞 mitophagy 的增加。我们以前证明, 慢病毒北疆?...

线粒体是细胞增殖和生理的基本器官。线粒体负责产生超过80% 的 ATP 供应通过氧化磷酸化, 他们也提供了各种代谢中间体的生物合成和新陈代谢1,2。除了它们在能源供应和新陈代谢中的作用, 线粒体在许多其他重要过程中扮演中心角色, 包括活性氧 (ROS) 生成、细胞死亡调节和胞内 Ca2 +动力学3.线粒体功能的改变与人类疾病有关, 包括癌症、糖尿病和各种神经退行性疾病4、5。增加的线粒体功能障碍和线粒体 DNA 突变也被认为有助于正常衰老过程6,7,8。因此, 质量控制是线粒体中的一个关键问题。线粒体是高度动态的细胞器, 可以在拉长的网络和短而零碎的形态之间不断改变它们的形状。线粒体动力学在维持线粒体功能以及通过 mitophagy9的降解过程中起着重要的作用。
Mitophagy 是一种机制, 它涉及利用自噬机械对整个线粒体进行选择性降解。Mitophagy 是线粒体更替和去除受损或功能失调的线粒体的原理机制。在这个过程中, 线粒体首先被膜包围, 导致自噬体的形成, 然后与溶酶体融合水解降解9。以往果蝇的基因研究表明, 两种帕金森病相关基因, PTEN 诱导的激酶 1 (PINK1) (PARK6) 和 PARK2, 功能在同一途径10,11。子序列研究表明, PINK1-Parkin 通路是负责消除受损的线粒体和缺陷的这一途径导致功能失调的 mitophagy, 可能导致人类疾病12,13。最近在各种人类疾病中发现了 mitophagy 过程的缺陷, 包括癌症、心脏病、肝病和神经退行性疾病14。此外, 最近的研究也表明, mitophagy 是关键的许多生理过程, 如分化, 发展和免疫反应15,16,17,18 ,19, 表明 mitophagy 可能在控制细胞功能方面发挥更积极的作用。
尽管最近证实 mitophagy 在线粒体和人类疾病的质量控制中起着重要作用, 但 mitophagy 的分子机制仍然不太清楚。虽然在酵母中系统地研究了 mitophagy 相关的机制, 但旨在探索哺乳动物细胞 mitophagy 相关机制的研究主要集中在 PINK1-Parkin 途径上。以前的研究已经证实, PINK1-Parkin 途径主要负责选择性地去除受损的线粒体通过 mitophagy12,20,21。然而, 最近的研究报告说, 在某些模型中, 即使在没有功能 PINK122、23、24的情况下, mitophagy 也可以被激活。这些结果表明, 除了 PINK1-Parkin 通路, 有一个额外的不明途径, 可以激活 mitophagy。
缺乏一种方便的评估 mitophagy 活动的方法是探索调节 mitophagy 及其在病理生理事件中作用的途径的主要障碍。电子显微术是直接可视化 autophagosome 吞噬的线粒体的有力工具。然而, 电子显微镜在量化 mitophagy 活动方面有局限性。虽然使用微管相关的 protein-1 光链 3 (LC3)-共轭荧光探针 (如 GFP-LC3) 的策略是目前应用最广泛的方法25, LC3-based 信号的瞬态性及其高速率假阳性信号限制了其检测26细胞 mitophagy 的灵敏度。结合西方印迹测量线粒体蛋白水平, 线粒体 DNA 的量化, 和荧光显微分析 colocalize 线粒体与自噬体或溶酶体将是一个很好的方法来评估mitophagy。然而, 量化限制和现有方法的缺乏方便要求采取新的办法。引入一种新的 pH 依赖性荧光蛋白, 线粒体靶 Keima (mt-Keima), 大大提高了检测 mitophagy27的能力。将细胞色素 c 氧化酶亚基 viii (COX viii) 的线粒体靶向序列融合成 Keima, Keima 被定向到线粒体基质。Keima 的励磁峰的大位移从440毫微米到 586 nm (分别对应于 ph 7 和 ph 4) 可以用来评估 mitophagy在体外和体内实验28,29.更重要的是, Keima 对溶酶体降解的抗性导致了 Keima 信号在溶体蛋白中的整合, 使得 mitophagy 活性的定量测量变得容易。在 Keima 中发生的荧光变化可以用共焦显微镜或流式细胞术28,29进行分析。然而, 目前还没有详细提供基于流式细胞仪的测量 mitophagy 活动的方法--Keima。
在这里, 我们描述了一个详细的协议, 以流式细胞术为基础的技术, 以测量细胞 mitophagy 活动, 使用 mt-Keima。虽然我们在这里表明, 流式细胞术分析成功地检测到 CCCP 诱导 mitophagy 在 HeLa 细胞表达, 这一技术可以应用于多种细胞类型。

<table><tbody><tr><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>poly-L-lysine</td><td>Sigma-Aldrich</td><td>P2636</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>16000-044</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>wellgene</td><td>LS202-02</td><td></td></tr><tr><td>PBS</td><td>Hyclone</td><td>SH30013.02</td><td></td></tr><tr><td>HEK293T</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>DMEM</td><td>GIBCO</td><td>12800-082</td><td></td></tr><tr><td>OPTI-MEM&amp;nbsp;</td><td>GIBCO</td><td>31985-070</td><td></td></tr><tr><td>Turbofect</td><td>Thermos scientific</td><td>R0531</td><td></td></tr><tr><td>0.45 &amp;mu;m syringe filter</td><td>sartorius</td><td>16555</td><td></td></tr><tr><td>HeLa</td><td>ATCC</td><td>CCL-2</td><td></td></tr><tr><td>polybrene</td><td>Sigma-Aldrich</td><td>H9268</td><td>8 mg/ml</td></tr><tr><td>puromycin</td><td>Sigma-Aldrich</td><td>P8833</td><td>2 mg/ml&amp;nbsp;</td></tr><tr><td>Carbonyl cyanide m-chlorophenyl hydrazine (CCCP)</td><td>Sigma-Aldrich</td><td>C2759</td><td>10 mM</td></tr><tr><td>trypsin-EDTA</td><td>wellgene</td><td>LS015-01</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BD LSRFortessa</td><td>BD Bioscience</td><td>LSRFortessa</td><td></td></tr><tr><td>FACSDIVA</td><td>BD Bioscience</td><td>FACSDIVA (v8.0.1)</td><td></td></tr></tbody></table>

sensitive measurement of mitophagy by flow cytometry using the ph-dependent fluorescent reporter mt-keima

Mitophagy 是一种选择性去除受损或不必要的线粒体使用自噬机械的过程。有缺陷的 mitophagy 和各种人类疾病 (包括神经退行性疾病、癌症和代谢性疾病) 之间存在着密切的联系。此外, 最近的研究表明, mitophagy 参与正常的细胞过程, 如分化和发展。然而, mitophagy 的确切作用和分子机制需要进一步研究。因此, 建立一种稳健、方便的测量 mitophagy 活动变化的方法至关重要。在这里, 我们描述了一个详细的协议, 以定量评估 mitophagy 活动, 通过流式细胞术使用线粒体靶向荧光蛋白 Keima (mt-Keima)。这种流式细胞仪可以比常规显微术或免疫印迹法方法更快速、灵敏地分析 mitophagy 活动。该协议可应用于分析各种细胞类型中的 mitophagy 活动。

如图 4所示, CCCP 诱导的 mitophagy 细胞的流式细胞术分析。应用上述流式细胞仪分析方法, 可以检测出 "高" 门 mitophagic 细胞的急剧增加。与未经处理的控制细胞相比, "高" 门细胞的百分比增加了10倍以上 (图 4A)。mitophagy 活动的增加完全被取消了与 bafilomycin A (论坛) 的合作 (图 4A和4B)。
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Watch this Scientific Journal Video about Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima at JoVE.com

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy, 线粒体的选择性降解, 与线粒体稳态有关, 在各种人类疾病中被解除管制。然而, 方便的实验方法测量 mitophagy 活动是非常有限的。在这里, 我们提供了一个敏感的检测 mitophagy 活动使用流式细胞仪。

pH 依赖性荧光 Keima 对流式细胞术 Mitophagy 的敏感性测定

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

sensitive-measurement-mitophagy-flow-cytometry-using-ph-dependent

Research

JoVE Journal

Biology

14.7K 次观看.  Dong-A University. Mitophagy, 线粒体的选择性降解, 与线粒体稳态有关, 在各种人类疾病中被解除管制。然而, 方便的实验方法测量 mitophagy 活动是非常有限的。在这里, 我们提供了一个敏感的检测 mitophagy 活动使用流式细胞仪。

视频: Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

科研

生物学

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

免疫与感染

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

医学

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

时间推移视频显微镜对EYFP-Parkin的聚集性评估作为蜂窝Mitophagy一个标记

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

通过使用多光谱成像流式细胞术测量LC3，p62和LAMP1共定位评估自噬通量

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

通过抑制Efflux泵,提高造血干细胞和造血细胞中线粒体膜电位流细胞测定的准确性

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. Mitophagy is the primary mechanism of mitochondrial quality control and is generally studied using microscopic observation, however in vivo mitophagy assays are difficult to perform. Evaluating mitophagy by imaging live organelles is an alternative and necessary method for mitochondrial research. This protocol describes the procedures for using the cell-permeant green-fluorescent mitochondria dye MitoTracker Green and the red-fluorescent lysosome dye LysoTracker Red in live cells, including the loading of the dyes, visualization of the mitochondria and the lysosome, and expected outcomes. Detailed steps for the evaluation of mitophagy in live cells, as well as technical notes about microscope software settings, are also provided. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. In addition, it can be used to quantify mitochondria and lysosomes and assess mitochondrial morphology.

Mitophagy is the primary mechanism of mitochondrial quality control. However, the evaluation of mitophagy in vivo is hindered by the lack of reliable quantitative assays. Presented here is a protocol for the observation of mitophagy in living cells using a cell-permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye.

用荧光染料可视化线粒体和溶酶体的线粒体自噬

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. ...

定量 秀丽隐杆线 虫和肝癌细胞系中的线粒体自噬

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. 
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

作者聚焦：使用细胞器特异性染料检测秀丽隐杆线虫和哺乳动物细胞中的线粒体自噬  

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

使用细胞器特异性染料检测 秀丽隐杆 线虫和哺乳动物细胞中的线粒体自噬

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...

pH 依赖性荧光 Keima 对流式细胞术 Mitophagy 的敏感性测定

摘要

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此视频中的章节

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

时间推移视频显微镜对EYFP-Parkin的聚集性评估作为蜂窝Mitophagy一个标记

通过使用多光谱成像流式细胞术测量LC3，p62和LAMP1共定位评估自噬通量

通过抑制Efflux泵,提高造血干细胞和造血细胞中线粒体膜电位流细胞测定的准确性

用荧光染料可视化线粒体和溶酶体的线粒体自噬

使用细胞器特异性染料检测秀丽隐杆线虫和哺乳动物细胞中的线粒体自噬

使用细胞器特异性染料检测秀丽隐杆线虫和哺乳动物细胞中的线粒体自噬

pH 依赖性荧光 Keima 对流式细胞术 Mitophagy 的敏感性测定

摘要

探索更多视频

此视频中的章节

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

时间推移视频显微镜对EYFP-Parkin的聚集性评估作为蜂窝Mitophagy一个标记

通过使用多光谱成像流式细胞术测量LC3，p62和LAMP1共定位评估自噬通量

通过抑制Efflux泵,提高造血干细胞和造血细胞中线粒体膜电位流细胞测定的准确性

用荧光染料可视化线粒体和溶酶体的线粒体自噬

使用细胞器特异性染料检测 秀丽隐杆 线虫和哺乳动物细胞中的线粒体自噬

使用细胞器特异性染料检测 秀丽隐杆 线虫和哺乳动物细胞中的线粒体自噬

使用细胞器特异性染料检测秀丽隐杆线虫和哺乳动物细胞中的线粒体自噬

使用细胞器特异性染料检测秀丽隐杆线虫和哺乳动物细胞中的线粒体自噬