Name: detection of mitochondria membrane potential to study clic4 knockdown-induced hn4 cell apoptosis in vitro
Uploaded: 2018-07-17T13:00:00.000+00:00
Duration: 13 min 20 s
Description: Watch this Scientific Journal Video about Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro at JoVE.com

我们感谢赵先生的细胞培养。这项工作得到了中国自然科学基金的资助 (81570403、81371284号赠款);安徽省自然科学基金 (批准号: 1408085MH158);安徽医科大学优秀青年研究员;安徽省高校优秀青年人才支持方案。

1. 细胞培养和转染
<ol><li>
细胞培养 注: HN4, HNSCC 细胞系, 来源于 HNSCC14的患者。<ol>
<li>Dulbecco 修饰鹰培养基中的培养 HN4 细胞 (DMEM, 4.5 克/升葡萄糖) 补充10% 胎牛血清和抗生素 (100 毫升/ml 青霉素和100µg/毫升链霉素)。孵育细胞在37°c 与 5% CO2。</li>
	</ol>
</li>
	<li>
细胞转染
	<ol>
<li>在转染前的一天, 5 x 105细胞在2毫升/井培养基中没有抗生素的 6-井板。</li>
		<li>稀释 100 pmol CLIC4 siRNA 或炒 siRNA 在250µL 减少血清介质, 轻轻地。稀释5µL 脂质体试剂250µL 减少血清培养基和孵育5分钟. 将稀释后的 siRNA 与稀释脂质体试剂结合, 轻轻搅拌, 室温下孵育20分钟。</li>
		<li>将混合物添加到每个含有细胞和培养基的井中。轻轻摇动盘子来回搅拌。在进行以下 Rh123 标记程序之前, 在 CO2孵化器中孵化37摄氏度的细胞, 24 小时。转染后用正常生长培养基4小时替换培养基。</li>
	</ol>
</li>
</ol>2. Rh123 标签
注: Rh123 用于测量 HN4 细胞中的基质金属蛋白酶1。
<ol><li>CLIC4 siRNA 治疗后 (1 节), 在6井板中使用 HN4 细胞进行以下治疗。每个井中 HN4 细胞的数量足以在盖玻片上重复实验10次。</li>
	<li>用镊子将圆形盖玻片放在新的12井板的底部。板的数量是根据实验设计 (图 1A) 设置的。</li>
	<li>把150µL 100 µg/毫升多聚赖氨酸在每个盖玻片中间的12井板和等待2分钟. 注意不要把多聚赖氨酸放在盖玻片外面。然后 pipettor 彻底删除多聚赖氨酸 (图 1B)。</li>
	<li>去除皿内 HN4 细胞的培养基, 加入1毫升0.25% 胰蛋白酶分离细胞。6分钟后, 加入2毫升培养基以中和胰蛋白酶。将 HN4 细胞通过 pipettor 轻轻混合5次, 在圆形盖玻片上种子 2-6 х 104细胞。安置在孵化器在37°c 与 5% CO2 (图 1C)。</li>
	<li>8小时孵化后, 用2µmol/升 Rh123 HN4 细胞孵化37摄氏度40分钟。轻轻地混合中上下几次, 以确保均匀分布在介质中的 Rh123 (图 1D)。</li>
	<li>准备足够的正常的生理盐水溶液 (NPSS);使 NPSS (在毫摩尔中): 140 氯化钠, 5 氯化钾, 1 氯化镁2, 1 CaCl2, 10 葡萄糖, 5 HEPES, pH 7.4)。在水浴预热到37摄氏度。</li>
	<li>使用镊子去除盖玻片和洗涤剩余的液体通过简要地安置盖玻片入预热的 NPBS 缓冲 (图 1E)。</li>
	<li>将盖玻片放在500µL 不锈钢腔的底部 (见材料表), 可更换的25毫米玻璃盖玻片底部密封到位的 o 形环。通过收紧房间的盖子来修复它 (图 1E)。</li>
	<li>将450µL 预热的 NPSS 缓冲器添加到组装腔内, 在荧光显微镜或共聚焦激光扫描荧光显微镜 (图 1E) 的视野下放置浴缸。</li>
</ol>3. 检测基质金属蛋白酶
<ol><li>
普通荧光显微镜
	<ol>
<li>按照制造商的说明打开荧光显微镜。 注: 共用荧光显微镜完成了汞灯光纤光源, 一个 FITC 过滤器设置为 Rh123 (480/40 励磁过滤器, 505 LP 分色镜和535/40 排放过滤器) 在室温下。20X. 目标被用来进行活细胞成像。</li>
		<li>打开成像系统软件, 从命令栏中选择 "新建" 按钮开始新的实验 (图 2a)。</li>
		<li>调整可视字段和焦点以在白光中查找单元格的位置。</li>
		<li>关灯, 按按钮关闭白光。单击命令栏上的 "焦点" 按钮, 然后选择 "开始聚焦" 以打开荧光。通过显微镜再发现细胞的位置与507毫微米励磁和529毫微米长的通行证发射波长 (在实验之前设置)。如果需要, 调整可视字段并重新对焦 (图 2B)。</li>
		<li>选择一个仅包含20到30个分离的 HN4 单元格的可视字段。每个细胞的细胞内荧光信号的变化将被准确记录。</li>
		<li>一旦实现了清晰的可视字段, 请单击焦点栏上的 "关闭焦点"。从命令栏中单击 "Acq 一" 以获取一组图像。单击命令栏中的 "区域", 然后单击 "确定" 按钮编辑度量区域。 注意: 从编辑区域栏, 不同的区域形状可以根据研究而使用。</li>
		<li>若要适合单个单元格的不同形状, 请选择 "跟踪区域" 工具, 并在单个单元格区域中循环并在完成后双击。重复该过程, 直到所有单元格都被圆圈 (图 2C)。单击 "完成" 并选择 "保存图像" 以查找保存的位置。</li>
		<li>点击 "Zeroclock", 快速推 F4 开始监测荧光强度的变化。每5秒记录一次荧光强度的实时变化5分钟 (图 2D)。</li>
		<li>当基线趋于稳定时, 将50µL NPSS 与0.5 µL 100 毫摩尔/升 ATP 混合在一起, 通过 pipettor。记住不要触摸房间以避免删除视觉领域 (图 2E)。 注: 荧光图像将记录20分钟, 然后实验可以通过手动操作结束。时间期限通常被安定为20分钟为了观察整个荧光变化。然而, 当意想不到的因素影响曲线的稳定性时, 或当整个过程少于20分钟时, 就应用了手动停止捕获的操作。</li>
	</ol>
</li>
	<li>
共焦激光扫描荧光显微镜
	<ol>
<li>根据制造商的说明打开共焦激光扫描荧光显微镜, 并开启捆绑的软件。 注: 在这里, Rh123 是兴奋的氩激光设置在 488 nm 和发射的信号是记录在 545-700 nm 的范围内通过应用的 TD488/543/633 过滤器。</li>
		<li>点击 "获取" 菜单下的 "目标", 选择 63 x/1.4 油物镜。观察标本, 在光场下找到一个清晰的视野。</li>
		<li>按下 "TL/IL" 按钮切换到荧光模式, 并选择正确的荧光过滤器, 以找到在黑暗中的细胞的位置, 通过显微镜。当观察完成时, 按 "快门" 来保护标本。</li>
		<li>在 "获取" 菜单中, 调整合适的 PMT 通道并单击 "实现" 以激活已结算的光路径 (图 3A)。点击 "配置" 菜单, 选择 "激光" 来确定所需的激光类型。本协议建议使用 "氩气" (图 3B)。</li>
		<li>单击 "实时" 以获取实时图像, 并确保可视字段仅包含20到30个分离的 HN4 单元格。每个细胞的细胞内荧光信号的变化将被准确记录。</li>
		<li>在 "获取" 菜单 (图 3C) 下的 "获取" 子菜单项中, 选择 "xyt"。分别设置 5 s 和20分钟的时间间隔和工期。当所有参数都解决时, 单击 "应用"。</li>
		<li>在 "量化" 菜单下, 使用 "绘制折线" 工具环绕每个单元格的记录区域。选择 "线条轮廓", 然后选择 "开始" 进行观察。记录荧光强度的实时变化为5分钟 (图 3D E)。</li>
		<li>当基线趋于稳定时, 将50µL NPSS 与0.5 µL 100 毫摩尔/升 ATP 混合在一起, 通过 pipettor。切记不要触摸房间, 以免移除视野。 注: 荧光图像将记录20分钟, 然后完成实验。</li>
	</ol>
</li>
</ol>4. 统计分析
<ol><li>
普通荧光显微镜
	<ol>
<li>按照操作员的手册, 定期打开荧光显微镜。打开成像系统软件, 然后从命令栏中选择 "打开" 按钮以打开保存的实验。单击 "命令栏" 中的 "区域", 然后单击 "确定" 按钮编辑度量区域。</li>
		<li>选择 "跟踪区域" 工具, 然后在单个单元格区域中循环, 完成后双击。重复该过程, 直到所有单元格都已被圆圈。单击 "完成" 并选择 "F4: 向前" 按钮以获得显示荧光强度的痕迹。</li>
		<li>完成后, 单击位于图表右下角的向下箭头按钮, 然后单击 "显示图形数据" (图 2F)。单击 "确定" 两次, 然后选择 "日志数据" 按钮打开数据处理软件的接口。再次点击 "记录数据", 发现电子表格上出现荧光强度实时波动的记录。 注: 对于每个细胞, 在应用 ATP 或 TG (F1/F0) 之前, 基质的变化显示为荧光相对于强度的比值。每个荧光强度数据平均为 20-30 细胞。Rh123 的数据显示为平均值. 2 组的结果由双尾独立t检验和P值 < 0.05 被认为是统计学意义。采用统计分析软件进行统计分析。</li>
	</ol>
</li>
	<li>
共焦激光扫描荧光显微镜
	<ol>
<li>打开共焦激光显微镜, 并按照制造商的说明登录到捆绑的软件。</li>
		<li>选择 "开火" 打开录制的实验。</li>
		<li>选择 "量化" 菜单下的 "堆栈配置文件" 工具, 选择 "全部在一个" 从 "按通道和 ROIs 排序图表"。</li>
		<li>使用 "绘制多边形" 工具来圆细胞区域进行观察。</li>
		<li>选择 "图形" 菜单, 然后单击鼠标右键, 然后选择 "导出到 excel", 将荧光强度值添加到电子表格中。下面的分析与正常的荧光显微镜 (图 3F) 是一致的。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Chen, J., Liao, W., Gao, W., Huang, J., Gao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+hypoxia+protects+cerebral+mitochondrial+function+from+calcium+overload.">Intermittent hypoxia protects cerebral mitochondrial function from calcium overload.</a> Acta Neurol Belg. 113, 507-513 (2013).</li><li>Gogol, P., Trzci&#324;ska, M., Bry&#322;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=Motility,+mitochondrial+membrane+potential+and+ATP+content+of+rabbit+spermatozoa+stored+in+extender+supplemented+with+GnRH+analogue+[des-Gly10,+D-Ala6]-LH-RH+ethylamide.">Motility, mitochondrial membrane potential and ATP content of rabbit spermatozoa stored in extender supplemented with GnRH analogue [des-Gly10, D-Ala6]-LH-RH ethylamide.</a> Pol J Vet Sci. 17, (2014).</li><li>Machiels, J. P., 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=Advances+in+the+management+of+squamous+cell+carcinoma+of+the+head+and+neck.">Advances in the management of squamous cell carcinoma of the head and neck.</a> F1000Prime Rep. 6, 44 (2014).</li><li>Behera, 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=Concurrent+therapy+with+taxane+versus+non-taxane+containing+regimens+in+locally+advanced+squamous+cell+carcinomas+of+the+head+and+neck+(SCCHN):+a+systematic+review.">Concurrent therapy with taxane versus non-taxane containing regimens in locally advanced squamous cell carcinomas of the head and neck (SCCHN): a systematic review.</a> Oral Oncol. 50, 888-894 (2014).</li><li>Pancari, P., Mehra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+therapy+for+squamous+cell+carcinoma+of+the+head+and+neck.">Systemic therapy for squamous cell carcinoma of the head and neck.</a> Surgical Oncology Clinics of North America. 24, 437-454 (2015).</li><li>Peretti, 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=Chloride+channels+in+cancer:+Focus+on+chloride+intracellular+channel+1+and+4+(CLIC1+AND+CLIC4)+proteins+in+tumor+development+and+as+novel+therapeutic+targets.">Chloride channels in cancer: Focus on chloride intracellular channel 1 and 4 (CLIC1 AND CLIC4) proteins in tumor development and as novel therapeutic targets.</a> Biochim Biophys Acta. 1848, 2523-2531 (2015).</li><li>Jentsch, T. J., Stein, V., Weinreich, F., Zdebik, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+structure+and+physiological+function+of+chloride+channels.">Molecular structure and physiological function of chloride channels.</a> Physiol Rev. 82, 503-568 (2002).</li><li>Ponnalagu, D., 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=Data+supporting+characterization+of+CLIC1,+CLIC4,+CLIC5+and+DmCLIC+antibodies+and+localization+of+CLICs+in+endoplasmic+reticulum+of+cardiomyocytes.">Data supporting characterization of CLIC1, CLIC4, CLIC5 and DmCLIC antibodies and localization of CLICs in endoplasmic reticulum of cardiomyocytes.</a> Data Brief. 7, 1038-1044 (2016).</li><li>Fernandez-Salas, 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=mtCLIC/CLIC4,+an+Organellular+Chloride+Channel+Protein,+Is+Increased+by+DNA+Damage+and+Participates+in+the+Apoptotic+Response+to+p53.">mtCLIC/CLIC4, an Organellular Chloride Channel Protein, Is Increased by DNA Damage and Participates in the Apoptotic Response to p53.</a> Mol Cell Biol. 22, 3610-3620 (2002).</li><li>Suh, K. S., Mutoh, M., Gerdes, M., Yuspa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIC4,+an+intracellular+chloride+channel+protein,+is+a+novel+molecular+target+for+cancer+therapy.">CLIC4, an intracellular chloride channel protein, is a novel molecular target for cancer therapy.</a> J Invest Derm Symp P. 10, 105-109 (2005).</li><li>Zhong, J., Kong, X., Zhang, H., Yu, C., Xu, Y., Kang, J., Yu, H., Yi, H., Yang, X., Sun, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+CLIC4+Enhances+Autophagy+and+Triggers+Mitochondrial+and+ER+Stress-Induced+Apoptosis+in+Human+Glioma+U251+Cells+under+Starvation.">Inhibition of CLIC4 Enhances Autophagy and Triggers Mitochondrial and ER Stress-Induced Apoptosis in Human Glioma U251 Cells under Starvation.</a> PloS one. 7, 39378 (2012).</li><li>Ponnalagu, D., Singh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anion+Channels+of+Mitochondria.">Anion Channels of Mitochondria.</a> Handbook of Experimental Pharmacology. , (2016).</li><li>Leanza, 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=Intracellular+ion+channels+and+cancer.">Intracellular ion channels and cancer.</a> Front Physiol. 4, 227 (2013).</li><li>Kim, S. Y., Chu, K. C., Lee, H. R., Lee, K. S., Carey, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+nine+new+head+and+neck+cancer+cell+lines.">Establishment and characterization of nine new head and neck cancer cell lines.</a> Acta Oto-Laryngologica. 117, 775-784 (1997).</li><li>Jia, L., Liu, W., Guan, L., Lu, M., Wang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Calcium-Activated+Chloride+Channel+ANO1/TMEM16A+Suppresses+Tumor+Growth+and+Invasion+in+Human+Lung+Cancer.">Inhibition of Calcium-Activated Chloride Channel ANO1/TMEM16A Suppresses Tumor Growth and Invasion in Human Lung Cancer.</a> PloS one. 10, 0136584 (2015).</li><li>Xu, 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=Suppression+of+CLIC4/mtCLIC+enhances+hydrogen+peroxide-induced+apoptosis+in+C6+glioma+cells.">Suppression of CLIC4/mtCLIC enhances hydrogen peroxide-induced apoptosis in C6 glioma cells.</a> Oncol Rep. 29, 1483-1491 (2013).</li><li>Zhu, W., Wang, X., Zhou, Y., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C2-ceramide+induces+cell+death+and+protective+autophagy+in+head+and+neck+squamous+cell+carcinoma+cells.">C2-ceramide induces cell death and protective autophagy in head and neck squamous cell carcinoma cells.</a> Int J Mol Sci. 15, 3336-3355 (2014).</li><li>McFarland, R., 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=A+neurological+perspective+on+mitochondrial+disease.">A neurological perspective on mitochondrial disease.</a> Lancet Neurol. 9, 829-840 (2010).</li><li>Alston, C. L., Rocha, M. C., Lax, N. Z., Turnbull, D. M., Taylor, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+and+pathology+of+mitochondrial+disease.">The genetics and pathology of mitochondrial disease.</a> J Pathol. 241, 236-250 (2017).</li><li>Zhang, W., Wang, X., Chen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resveratrol+induces+mitochondria-mediated+AIF+and+to+a+lesser+extent+caspase-9-dependent+apoptosis+in+human+lung+adenocarcinoma+ASTC-a-1+cells.">Resveratrol induces mitochondria-mediated AIF and to a lesser extent caspase-9-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells.</a> Mol Cell Biochem. 354, 29-37 (2011).</li><li>Bernardi, P., Rasola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+cell+death:+the+mitochondrial+connection.">Calcium and cell death: the mitochondrial connection.</a> Subcell Biochem. 45, 481-506 (2007).</li><li>Perveen, S., Yang, J. S., Ha, T. J., Yoon, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyanidin-3-glucoside+Inhibits+ATP-induced+Intracellular+Free+Ca(2+)+Concentration,+ROS+Formation+and+Mitochondrial+Depolarization+in+PC12+Cells.">Cyanidin-3-glucoside Inhibits ATP-induced Intracellular Free Ca(2+) Concentration, ROS Formation and Mitochondrial Depolarization in PC12 Cells.</a> Korean J Physiol Pharmacol. 18, 297-305 (2014).</li><li>Paddock, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+practices+of+laser+scanning+confocal+microscopy.">Principles and practices of laser scanning confocal microscopy.</a> Mol Biotechnol. 16, 127-149 (2000).</li><li>Paddock, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+boldly+glow...+applications+of+laser+scanning+confocal+microscopy+in+developmental+biology.">To boldly glow... applications of laser scanning confocal microscopy in developmental biology.</a> BioEssays : news and reviews in molecular, cellular and developmental biology. 16, 357-365 (1994).</li></ol>

作者没有什么可透露的。

有充分的文献表明, Cl−通道是必不可少的保持止血的内部环境, 并发挥重要作用, 细胞增殖和凋亡15,16。因此, 了解离子通道靶向干预与细胞凋亡之间的关系, 对于寻找一种更好的治疗方法对17种癌症有很大的必要性和意义。线粒体维持细胞正常的生物状态, 其功能与细胞膜通透性和跨膜电位密切相关。除了癌症, 积累病理调查记?...

Rh123 是一种阳离子荧光染料, 可作为跨膜电位的指标。Rh123 能够穿透细胞膜, 进入线粒体基质, 这取决于细胞膜内外电位的差异1。细胞凋亡导致线粒体膜完整性受损。线粒体通透性过渡孔隙 (MPTP) 将打开并导致基质细胞的崩溃, 从而使 Rh123 释放到线粒体的外部。最后, 在荧光显微镜下检测出更强的绿色荧光信号。有充分的证据表明, 细胞凋亡和高膜通透性的减少是2的早期迹象。因此, Rh123 可用于检测基质金属蛋白酶的变化和细胞凋亡的发生。
头颈癌作为世界上第六大常见的癌症, 严重恶化了一个人的健康3。虽然近年来发展了许多方法, 但治疗头颈部鳞状细胞癌 (HNSCC) 患者的临床疗效仍不理想4。探索新的治疗方法可以提高 HNSCC5的治疗效果。涉及许多生物过程的离子通道在不同的癌症的发展中显示了重要的作用6。氯离子通道的部分或全部参与高度参与了肿瘤转化的各种性质, 包括主动迁移、高增殖率和侵袭性。有鉴于此, 新的蛋白质家族点击已被列为癌症治疗6、7治疗靶点。最近的研究表明, 点击家族的成员, 包括 CLIC1, CLIC4 和 CLIC5, 定位到心脏线粒体和活性氧 (ROS) 水平上调的 CLIC5, 表明线粒体定位 Cl的功能作用-在凋亡反应中的通道8。CLIC4, 点击家族的一个成员 (也称为 mtCLIC, P64H1 和 RS43), 已被广泛研究, 其凋亡调节性质的癌细胞和亚细胞位置, 包括高尔基, 内质网和线粒体的人角质细胞7,9,10。CLIC4 的表达谱由肿瘤坏死因子α (TNF α)、P53 和外部刺激调节。CLIC4 的过度表达和下调引起的凋亡反应主要是通过线粒体通路伴随 Bcl-2 家族成员的不平衡, 活化 caspase 叶栅和释放细胞色素 C11,12,13. 因此, 基质金属蛋白酶的测量对探讨 CLIC4-related 细胞凋亡至关重要, Rh123 是理想的荧光指标。
本研究描述了一个详细的协议, 以检测基质干细胞研究 CLIC4 击倒诱导凋亡的 HN4 细胞。Rh123 用作荧光探针观察基质金属蛋白酶的变化。在普通荧光显微镜和共聚焦激光扫描荧光显微镜下, 可解决该基质金属蛋白酶的实时波动。本文详细讨论了共焦激光扫描荧光显微镜的优点和局限性, 并与其他方法进行了比较。该协议也可应用于其他与凋亡相关的研究。

<table><tbody><tr><td>HNSCC cells</td><td>ATCC</td><td>CRL-3241</td><td></td></tr><tr><td>Polylysine</td><td>Thermo Fisher Scientific</td><td>P4981</td><td></td></tr><tr><td>Specific siRNA for human CLIC4</td><td>Biomics</td><td>NM_013943</td><td>(accession numbers, NM_013943; corresponding to the cDNA sequence&lt;br/&gt; 5-GCTGAAGGAGGAGGACAAAGA-3) and scrambled siRNA (5 ACGCGUAACGCGGGAAUUU-3) were designed and obtained from Biomics Company</td></tr><tr><td>Lipofectamine 2000 Transfection Reagent&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>L3000-015</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium, GlutaMAX</td><td>Thermo Fisher Scientific</td><td>51985-042</td><td></td></tr><tr><td>Rhodamine 123, FluoroPure grede</td><td>Thermo Fisher Scientific</td><td>R22420</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;smodified Eagle medium (DMEM, 4.5 g/L glucose)</td><td>Gibco</td><td>11965-084</td><td></td></tr><tr><td>Fetal Bovine Serum, Qualified, Australia Origin</td><td>Gibco</td><td>10099141</td><td></td></tr><tr><td>Trypsin-EDTA Solution</td><td>Beyotime</td><td>C0201</td><td></td></tr><tr><td>Antibiotic-Antimycotic, 100X</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>Laser Scanning Confocal Microscopy</td><td>Leica Microsystems GmbH</td><td>LEICA.SP5-DMI6000-DIC</td><td></td></tr><tr><td>Nikon Eclipse TE300 Inverted Microscope</td><td>Nikon</td><td>N/A</td><td></td></tr><tr><td>Metaflour, V7.5.0.0</td><td>Universal Imaging Corporation</td><td>N/A</td><td></td></tr><tr><td>Leica application suite, v2.6.0.7266</td><td>Leica Microsystems GmbH</td><td>N/A</td><td></td></tr><tr><td>Microsoft office Excel 2007</td><td>Microsoft</td><td>N/A</td><td></td></tr><tr><td>Sigma Plot 12.5</td><td>Systat Software</td><td>N/A</td><td></td></tr><tr><td>Attofluor Cell Chamber</td><td>Thermo Fisher Scientific</td><td>A7816</td><td></td></tr></tbody></table>

detection of mitochondria membrane potential to study clic4 knockdown-induced hn4 cell apoptosis in vitro

线粒体膜电位的耗竭 (基质金属蛋白酶, ΔΨm) 被认为是凋亡级联的最早事件。它甚至发生在核凋亡特征之前, 包括染色质凝结和 DNA 断裂。一旦基质细胞崩溃, 癌细胞的凋亡就会不可逆转地启动。一系列的亲脂阳离子染料可以通过细胞膜和聚集在线粒体基质内, 作为荧光标记来评价基质金属蛋白酶的变化。CLIC4 作为 Cl 胞内通道 (点击)家族的六名成员之一, 主要通过线粒体通路参与细胞凋亡过程。在这里, 我们描述了一个详细的协议, 通过监测罗丹明 123 (Rh123) 的荧光涨落来测量基质, 通过它, 我们研究 CLIC4 的凋亡诱导。本文详细讨论了共焦激光扫描和正常荧光显微镜的优点和局限性, 并与其它方法进行了比较。

在本研究中, Rh123 用于检测基质金属蛋白酶。最初, HN4 细胞被培养为以下荧光染色实验。用镊子将圆形盖玻片放在6井板的底部 (图 1A)。盖玻片涂上多聚赖氨酸5分钟, 然后通过 pipettor 去除多聚赖氨酸 (图 1B)。然后, HN4 细胞被 trypsinized, 并在被放置在6井板底部的盖玻片上播种。接下来, 添加适量的 Rh123, 混?...

Watch this Scientific Journal Video about Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro at JoVE.com

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

在这里, 我们提出了一个详细的协议, 应用罗丹明123识别线粒体膜电位 (基质) 和研究 CLIC4 击倒诱导 HN4 细胞凋亡的体外。在普通荧光显微镜和共聚焦激光扫描荧光显微镜下, 记录了基质金属蛋白酶的实时变化。

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

detection-mitochondria-membrane-potential-to-study-clic4-knockdown

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

10.3K 次观看.  Anhui Medical University. 在这里, 我们提出了一个详细的协议, 应用罗丹明123识别线粒体膜电位 (基质) 和研究 CLIC4 击倒诱导 HN4 细胞凋亡的体外。在普通荧光显微镜和共聚焦激光扫描荧光显微镜下, 记录了基质金属蛋白酶的实时变化。

视频: Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

科研

环境科学

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

监测劈开caspase - 3活性和钾诱导膜去极化使用活细胞成像和Cleaveable荧光染料基板的永生的少突胶质细胞凋亡

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

神经科学

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

FAM83A 作为宫颈癌靶基因的验证

Knockdown of FAM83A to Verify Its Role in Cervical Cancer Cell Growth and Cisplatin Sensitivity

The exploration of tumor target genes holds paramount importance for the prevention and treatment of cervical cancer. In this study, we outline the steps involved in the identification of a tumor target gene FAM83A in cervical cancer. First, the Cancer Genome Atlas dataset was employed to validate the expression and prognostic significance of FAM83A in women. A small interfering RNA (siRNA) was used for knockdown of the FAM83A gene in HeLa and C33a cells. Next, 5-ethynyl-2&#39;-deoxyuridine (EdU) staining was conducted to determine the effects on the proliferation capabilities of the tumor cells. Wound healing and porous membrane insert assays were performed to evaluate tumor cell migration and invasion abilities.
Western blotting was used to quantify apoptosis-related protein levels. JC-1 staining was employed to evaluate mitochondrial function alterations. Furthermore, cisplatin (diaminedichloroplatinum, DDP) intervention was used to assess the therapeutic potential of the target gene. Flow cytometry and colony formation assays were conducted to further validate the anticancer characteristics of the gene. As a result, FAM83A knockdown was shown to inhibit the proliferation, migration, and invasion of cervical cancer cells and sensitize these cells to cisplatin. These comprehensive methodologies collectively validate FAM83A as a tumor-associated target gene, holding promise as a potential therapeutic target in the prevention and treatment of cervical cancer.

作者聚焦：探索 FAM83A 在宫颈癌中的作用 

Here, we show the procedures for FAM83A knockdown; the assays to detect its effects on proliferation, migration, and invasion of cervical cancer cells; and the sensitization of these cells to cisplatin. This study provides a promising target gene for cervical cancer and a reference for further drug research.

敲低 FAM83A 以验证其在宫颈癌细胞生长和顺铂敏感性中的作用

The exploration of tumor target genes holds paramount importance for the prevention and treatment of cervical cancer. In this study, we outline the steps ...

癌症研究

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

生物学

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

E4orf4诱导的细胞死亡的击倒效果的基因表达和测量条件击倒细胞系的制备

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

流式细胞仪测定缺氧/复氧后心肌细胞线粒体膜电位的实验研究

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

摘要

探索更多视频

此视频中的章节

检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

监测劈开caspase - 3活性和钾诱导膜去极化使用活细胞成像和Cleaveable荧光染料基板的永生的少突胶质细胞凋亡

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

敲低 FAM83A 以验证其在宫颈癌细胞生长和顺铂敏感性中的作用

敲低 FAM83A 以验证其在宫颈癌细胞生长和顺铂敏感性中的作用

敲低 FAM83A 以验证其在宫颈癌细胞生长和顺铂敏感性中的作用

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的

E4orf4诱导的细胞死亡的击倒效果的基因表达和测量条件击倒细胞系的制备

流式细胞仪测定缺氧/复氧后心肌细胞线粒体膜电位的实验研究

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

摘要

探索更多视频

此视频中的章节

检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

监测劈开caspase - 3活性和钾诱导膜去极化使用活细胞成像和Cleaveable荧光染料基板的永生的少突胶质细胞凋亡

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

敲低 FAM83A 以验证其在宫颈癌细胞生长和顺铂敏感性中的作用

敲低 FAM83A 以验证其在宫颈癌细胞生长和顺铂敏感性中的作用

敲低 FAM83A 以验证其在宫颈癌细胞生长和顺铂敏感性中的作用

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

E4orf4诱导的细胞死亡的击倒效果的基因表达和测量条件击倒细胞系的制备

流式细胞仪测定缺氧/复氧后心肌细胞线粒体膜电位的实验研究

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的