Name: sensitive measurement of mitophagy by flow cytometry using the ph-dependent fluorescent reporter mt-keima
Uploaded: 2018-08-12T14:00:00
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
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Keima 慢病毒北疆的制备
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<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...

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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. 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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。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">REAGENTS</td>
			<td style="width:133px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">poly-L-lysine</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:133px;">GIBCO</td>
			<td style="width:119px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">penicillin/streptomycin</td>
			<td style="width:133px;">wellgene</td>
			<td style="width:119px;">LS202-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:133px;">Hyclone</td>
			<td style="width:119px;">SH30013.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEK293T</td>
			<td style="width:133px;">ATCC</td>
			<td style="width:119px;">CRL-3216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:133px;">GIBCO</td>
			<td style="width:119px;">12800-082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">OPTI-MEM&#160;</td>
			<td style="width:133px;">GIBCO</td>
			<td style="width:119px;">31985-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Turbofect</td>
			<td style="width:133px;">Thermos scientific</td>
			<td style="width:119px;">R0531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.45 &#956;m syringe filter</td>
			<td style="width:133px;">sartorius</td>
			<td align="right" style="width:119px;">16555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HeLa</td>
			<td style="width:133px;">ATCC</td>
			<td style="width:119px;">CCL-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polybrene</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">H9268</td>
			<td>8 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">puromycin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8833</td>
			<td>2 mg/ml&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Carbonyl cyanide m-chlorophenyl hydrazine (CCCP)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:119px;">C2759</td>
			<td>10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trypsin-EDTA</td>
			<td style="width:133px;">wellgene</td>
			<td style="width:119px;">LS015-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EQUIPMENTS</td>
			<td style="width:133px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD LSRFortessa</td>
			<td style="width:133px;">BD Bioscience</td>
			<td style="width:119px;">LSRFortessa</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACSDIVA</td>
			<td style="width:133px;">BD Bioscience</td>
			<td style="width:119px;">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 ...

Research

JoVE Journal

Biology

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

IP - FCM：免疫沉淀，流式细胞仪检测

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

科研

生物学

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Flow Cytometry Purification of Mouse Meiotic Cells

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

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 ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

流式细胞仪检测盘基柄柄Macropinocytosis 的高通量

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...