这项工作得到了波兰国家科学中心的支持（资助/奖励编号：2018/31/D/NZ2/03901）。

1. siRNA混合物的制备
<ol><li>在实验开始前一天，将细胞（例如HeLa）接种在100毫米的培养皿上，以便第二天达到70%-90%的汇合。 注意：所有操作必须在无菌条件下在层流室中进行。</li>
	<li>在Opti-MEM培养基中制备稀释至140nM浓度的适量的siRNA（参见 材料表）。96孔板可用作储液器。</li>
	<li>向黑色 384 孔细胞培养微孔板的每个孔中加入 5 μL siRNA 溶液（或用于对照样品的 Opti-MEM 培养基）。 注意：根据测试的siRNA样品数量，可以使用电动或多通道移液器。</li>
	<li>在Opti-MEM培养基中制备适量的RNAiMAX转染试剂溶液（参见 材料表）。每 100 μL 培养基添加 1 μL RNAiMAX。</li>
	<li>向 384 孔板的每个孔中加入 10 μL 转染试剂溶液。最方便、最快捷的方法是使用试剂分配器。</li>
	<li>将siRNA与转染试剂在室温下孵育30分钟。</li>
</ol>2. 转染用细胞的制备
<ol><li>在孵育期间（步骤1.6），使用抽吸装置吸出培养基（参见 材料表），并用3mL PBS洗涤细胞。</li>
	<li>加入在PBS中以1：2稀释的1.5mL胰蛋白酶，并将细胞在37°C孵育10分钟。</li>
	<li>检查细胞是否已分离;如果是这样，请加入 3 mL 含有 10% FBS 的 DMEM 培养基。彻底悬浮细胞，并将其转移到 15 mL 管中。取至少 150 μL 悬浮液，并在细胞计数室中计数细胞（参见 材料表）。</li>
	<li>在含有 10% FBS、青霉素和链霉素的 DMEM 培养基中制备适当浓度（HeLa 系为 35，000/mL）的细胞悬液。</li>
</ol>3. 细胞转染
<ol><li>使用试剂分配器向 384 孔板的每个孔中加入 20 μL 细胞悬液。这将导致每孔接种700个细胞。</li>
	<li>将细胞在室温下孵育1小时，然后将它们置于培养箱中72小时（37°C和5%CO2）。</li>
</ol>4. 成立 BrdU
<ol><li>在含有10%FBS的DMEM培养基中制备90μM的BrdU溶液（参见 材料表）。</li>
	<li>在siRNA转染后56小时（细胞固定前16小时），向384孔板的每个孔中加入10μL的90μM BrdU溶液（最终BrdU浓度为20μM）。 注意：应尽快执行该操作;因此，最好使用试剂分配器、电动移液器或多分配器移液器。请记住准备不添加BrdU的控制孔。</li>
	<li>孵育细胞16小时（37°C和5%CO2）。</li>
</ol>5. 线粒体的标记
<ol><li>用10%FBS在DMEM中制备20μM的BrdU溶液，并向其中加入线粒体示踪染料溶液（参见 材料表）至浓度为1.1μM。</li>
	<li>在BrdU掺入开始后15小时（细胞固定前1小时），向384孔板的每个孔中加入10μL线粒体跟踪染料溶液（参见 材料表）（最终染料浓度为200nM）。 注意：使用试剂分配器、电动移液器或多分配器移液器。请记住保留对照孔，不添加BrdU，但添加线粒体跟踪染料。</li>
	<li>孵育细胞1小时（37°C和5%CO2）。</li>
</ol>6. 细胞固定
注意：所有洗涤都使用微孔板清洗机最方便地进行，而使用试剂分配器最快速地添加试剂。
<ol><li>使用微孔板清洗机（参见 材料表），用 100 μL PBS 冲洗每个孔两次。第二次洗涤后，将 25 μL PBS 留在孔中。 注意：将PBS留在孔中可降低下一步添加液体期间细胞脱离的可能性。所有的洗涤都是在PBS留下的情况下完成的;因此，下一步中添加的溶液应始终以2x浓度制备，因为它将以等体积添加到剩余的PBS中。</li>
	<li>通过在PBS中加入25μL的8%甲醛溶液，0.4%Triton X-100和Hoechst 33342，浓度为4μg/ mL（单个试剂的最终浓度分别为4%，0.2%和2μg/ mL）（参见 材料表）。</li>
	<li>将板在室温下在黑暗中孵育30分钟。</li>
</ol>7. 阻止
<ol><li>用 100 μL PBS 冲洗每个孔四次。最后一次洗涤后，将 25 μL PBS 留在孔中。</li>
	<li>向每个孔中加入 25 μL 6% BSA 的 PBS（最终 BSA 浓度为 3%）。</li>
	<li>将板在室温下在黑暗中孵育30分钟。</li>
</ol>8. 添加一抗
<ol><li>使用微孔板清洗机吸出BSA，并将10μL溶液留在孔中。</li>
	<li>在PBS中加入10 μL在3%BSA中制备的一抗溶液。使用0.8μg/mL的抗BrdU和0.4μg/mL的抗DNA（抗体的最终浓度分别为0.4μg/mL和0.2μg/mL）（参见 材料表）。</li>
	<li>将板在4°C的黑暗中孵育过夜。</li>
</ol>9. 二抗的添加
<ol><li>用 100 μL PBS 冲洗每个孔四次。最后一次洗涤后，将 10 μL PBS 留在孔中。</li>
	<li>在PBS中加入10 μL在6%BSA中制备的4 μg/mL二抗溶液（终浓度为2 μg/mL）。使用与荧光染料（如Alexa Fluor 488和Alexa Fluor 555）偶联的同种型特异性抗体（抗小鼠IgG1和抗小鼠IgM）（参见 材料表）。</li>
	<li>将板在室温下在黑暗中孵育1小时。</li>
	<li>用 100 μL PBS 冲洗每个孔四次。最后一次洗涤后，将 50 μL PBS 留在孔中。</li>
	<li>用粘性密封膜密封板（见 材料表），并将其储存在4°C的黑暗中。 影像学检查必须在 2 周内进行。</li>
</ol>10. 成像
注意：必须使用自动宽视场显微镜进行成像;显微镜必须配备一个电机载物台，该载物台配有控制器，以自动对板的各个区域进行成像。
<ol><li>检查板角区域（孔 A1、A24、P24、P1）和中心的自动对焦设置。 注意：对于成像，建议使用具有最高数值孔径的20倍短工作距离物镜（见 材料表）。</li>
	<li>根据成像软件在实时取景模式下生成的强度直方图，为各个荧光通道选择足够长的曝光时间，以便生成的图像不会过饱和。</li>
	<li>在 z 轴上设置适当的平面数以进行成像。 注意：20倍放大倍率下的视场足够大，并非所有细胞都在同一焦点平面上，因此必须执行z切片才能在正确的焦点平面上查看所有细胞。通常，五架飞机就足够了。根据磁盘空间的可用性，可以保存单个 z 堆栈或仅保存最大强度投影。代表性结果部分中显示的图像分析基于最大强度预测。</li>
	<li>选择每个孔要显示的适当数量的视野。 注意：根据汇合度，可以在一个视野中对大约 60-300 个细胞进行成像。通常，对于汇合度为60%-90%的HeLa细胞，对五个视场进行成像将允许分析从500个细胞到超过1，000个细胞。</li>
	<li>选择要成像的孔，然后开始成像。</li>
</ol>11. 定量图像分析
注意：采集图像的定量分析可以使用开源软件（如Cell Profiler15）进行。在本研究中，使用ScanR 3.0.0软件进行分析（见 材料表）。
<ol><li>通过对所有荧光通道的所有图像进行背景校正来开始分析。根据分析对象的大小，选择合适的过滤器尺寸：细胞核为80像素，BrdU和mtDNA斑点为4像素。</li>
	<li>通过根据荧光信号强度与对应于细胞核的通道的背景之比创建主要对象的掩模来开始分割图像。</li>
	<li>使用适合给定结构的荧光通道，分别为代表BrdU和mtDNA斑点的子对象创建掩模。 注意：每个子对象都分配给最近的主对象（细胞核）。</li>
	<li>设置分析过程中要测量的参数，并测量所有荧光通道的每个掩模内单个像素的强度。 注意：还需要计算分配给给定细胞核的子对象的数量以及每个子对象的面积。</li>
	<li>定义要根据获得的数据计算的派生参数。为了获得BrdU或mtDNA通道的总荧光强度，请汇总分配给给定细胞核的所有子对象的强度。要获得平均荧光强度，请将总荧光强度除以分配给给定细胞核的子对象的面积之和。 注意：为了确保创建的子对象（BrdU或mtDNA斑点）实际上位于线粒体中，有必要根据线粒体跟踪染料的荧光强度来门控它们。</li>
	<li>使用 R 4.2.2 统计软件16 以及以下软件包对使用 ScanR 软件获得的数据进行进一步分析：dplyr 17、data.table 18、ggplot219 和 ggpubr20。此步骤是可选的。</li>
</ol>

mtDNA 合成和分布的高通量定量

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作者没有什么可透露的。

从历史上看，通过BrdU掺入和抗体检测进行DNA标记已被用于核DNA复制和细胞周期研究14，27，28。到目前为止，所有用于检测BrdU标记DNA的方案都包括DNA变性步骤（酸性或热性）或酶消化（DNase或蛋白酶），以实现表位暴露并促进抗体渗透。这些协议是为紧密包装的核DNA开发的。然而，mtDNA的不同组织使得开发没有变性步骤的程序成...

对于大多数真核细胞来说，线粒体是必不可少的细胞器，因为它们在许多细胞过程中起着至关重要的作用。首先，线粒体是细胞1的主要能量供应商。线粒体还参与调节细胞稳态（例如，细胞内氧化还原2和钙平衡3），细胞信号传导4，5，细胞凋亡6，不同生化化合物的合成7，8和先天免疫反应9。线粒体功能障碍与各种病理状态和人类疾病有关10.
线粒体的功能取决于位于两个独立基因组中的遗传信息：核基因组和线粒体基因组。与核基因组相比，线粒体基因组编码的基因数量很少，但所有mtDNA编码的基因对人类生命都是必不可少的。维持mtDNA所必需的线粒体蛋白机制由nDNA编码。线粒体爬替体的基本成分以及一些线粒体生物发生因子已经被确定（在以前的研究中回顾了11，12）。然而，线粒体DNA的复制和维持机制仍远未被理解。与nDNA相反，线粒体基因组以多个拷贝存在，这为调节线粒体基因表达提供了额外的层。目前对细胞器内mtDNA的分布和分离知之甚少，这些过程在多大程度上受到调节，如果是，涉及哪些蛋白质13。当细胞包含野生型和突变mtDNA的混合群体时，分离模式至关重要。它们的不均匀分布可能导致产生具有有害数量突变mtDNA的细胞。
到目前为止，mtDNA维持所必需的蛋白质因子主要通过生化方法，生物信息学分析或通过疾病相关研究来确定。在这项工作中，为了确保识别以前逃脱识别的因素的高机会，描述了一种不同的策略。该方法基于在复制或修复过程中用5-溴-2'-脱氧尿苷（BrdU）标记mtDNA，BrdU是胸苷的核苷类似物。BrdU在DNA合成过程中很容易掺入新生的DNA链中，并且通常用于监测核DNA14的复制。然而，这里开发的程序已经过优化，可以使用抗BrdU抗体的免疫荧光检测掺入mtDNA中的BrdU。
该方法允许对 体外培养的人类细胞中的mtDNA合成和分布进行高通量定量。需要高通量策略才能在相对较短的时间内在不同的实验条件下进行测试;因此，在协议中建议利用多孔格式进行细胞培养和自动荧光显微镜进行成像。该方案包括用siRNA文库转染人HeLa细胞，以及随后使用BrdU对新合成的DNA进行代谢标记来监测mtDNA的复制或修复。这种方法与抗DNA抗体的帮助下对DNA进行免疫染色相结合。这两个参数都使用定量荧光显微镜进行分析。此外，线粒体用特定的染料可视化。为了证明该方案的特异性，在缺乏mtDNA的细胞（rho0细胞）上测试了BrdU染色，在沉默了众所周知的mtDNA维持因子的HeLa细胞上，以及在用mtDNA复制抑制剂处理后的HeLa细胞上测试了BrdU染色。mtDNA水平也通过一种独立的方法测量，即qPCR。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2&#8242;,3&#8242;-Dideoxycytidine (ddC)</td>
			<td>Sigma-Aldrich</td>
			<td>D5782</td>
			<td></td>
		</tr>
		<tr>
			<td>384&#160; Well Cell Culture Microplates, black</td>
			<td>Greiner Bio-One</td>
			<td>#781946</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-2&#8242;-deoxyuridine (BrdU)</td>
			<td>Sigma-Aldrich</td>
			<td>B5002-1G</td>
			<td>Dissolve BrdU powder in water to 20 mM stock solution and aliquot. Use 20 &#181;M BrdU solution for labeling.</td>
		</tr>
		<tr>
			<td>Adhesive sealing film</td>
			<td>Nerbe Plus</td>
			<td>04-095-0060</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG1 secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21121</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 goat anti-mouse IgM secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21426</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek 405 LS microplate washer</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting chamber Thoma</td>
			<td>Heinz Herenz</td>
			<td>REF:1080339</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Cytiva</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>41965-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma-Aldrich</td>
			<td>F1635</td>
			<td>Formaldehyde is toxic; please read the safety data sheet carefully.</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG1 mouse monoclonal anti-BrdU (IIB5) primary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-32323</td>
			<td></td>
		</tr>
		<tr>
			<td>IgM mouse monoclonal anti-DNA (AC-30-10) primary antibody</td>
			<td>Progen</td>
			<td>#61014</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 System</td>
			<td>Roche</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>#13778150</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Deep Red FM</td>
			<td>Thermo Fisher Scientific</td>
			<td>M22426</td>
			<td>Mitochondria tracking dye&#160;</td>
		</tr>
		<tr>
			<td>Multidrop Combi Reagent Dispenser</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>51985-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Orca-R2 (C10600) CCD Camera</td>
			<td>Hamamatsu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P0781-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerUp SYBR Green Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>A25742</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR primer Fw B2M (reference)</td>
			<td></td>
			<td></td>
			<td>CAGGTACTCCAAAGATTCAGG&#160;</td>
		</tr>
		<tr>
			<td>qPCR primer Fw GPI (reference gene)</td>
			<td></td>
			<td></td>
			<td>GACCTTTACTACCCAGGAGA</td>
		</tr>
		<tr>
			<td>qPCR primer Fw MT-ND1&#160;</td>
			<td></td>
			<td></td>
			<td>TAGCAGAGACCAACCGAACC&#160;</td>
		</tr>
		<tr>
			<td>qPCR primer Fw POLG</td>
			<td></td>
			<td></td>
			<td>TGGAAGGCAGGCATGGTCAAACC</td>
		</tr>
		<tr>
			<td>qPCR primer Fw TFAM</td>
			<td></td>
			<td></td>
			<td>GATGAGTTCTGCCTGCTTTAT</td>
		</tr>
		<tr>
			<td>qPCR primer Fw TWNK</td>
			<td></td>
			<td></td>
			<td>GCCATGTGACACTGGTCATT</td>
		</tr>
		<tr>
			<td>qPCR primer Rev B2M (reference)</td>
			<td></td>
			<td></td>
			<td>GTCAACTTCAATGTCGGATGG&#160;</td>
		</tr>
		<tr>
			<td>qPCR primer Rev GPI (reference gene)</td>
			<td></td>
			<td></td>
			<td>AGTAGACAGGGCAACAAAGT</td>
		</tr>
		<tr>
			<td>qPCR primer Rev MT-ND1&#160;</td>
			<td></td>
			<td></td>
			<td>ATGAAGAATAGGGCGAAGGG&#160;</td>
		</tr>
		<tr>
			<td>qPCR primer Rev POLG</td>
			<td></td>
			<td></td>
			<td>GGAGTCAGAACACCTGGCTTTGG</td>
		</tr>
		<tr>
			<td>qPCR primer Rev TFAM</td>
			<td></td>
			<td></td>
			<td>GGACTTCTGCCAGCATAATA</td>
		</tr>
		<tr>
			<td>qPCR primer Rev TWNK</td>
			<td></td>
			<td></td>
			<td>AACATTGTCTGCTTCCTGGC</td>
		</tr>
		<tr>
			<td>ScanR microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>siRNA Ctrl</td>
			<td>Dharmacon</td>
			<td>D-001810-10-5</td>
			<td></td>
		</tr>
		<tr>
			<td>siRNA POLG</td>
			<td>Invitrogen</td>
			<td>POLGHSS108223</td>
			<td></td>
		</tr>
		<tr>
			<td>siRNA TFAM</td>
			<td>Invitrogen</td>
			<td>TFAMHSS144252</td>
			<td></td>
		</tr>
		<tr>
			<td>siRNA TWNK</td>
			<td>Invitrogen</td>
			<td>C10orf2HSS125597</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction device</td>
			<td>NeoLab</td>
			<td>2-9335</td>
			<td>Suction device for cell culture</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Biowest</td>
			<td>L0931-500</td>
			<td></td>
		</tr>
		<tr>
			<td>UPlanSApo 20x 0.75 NA objective</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput image-based quantification of mitochondrial dna synthesis and distribution

绝大多数细胞过程需要持续的能量供应，其中最常见的载体是ATP分子。真核细胞通过氧化磷酸化在线粒体中产生大部分ATP。线粒体是独特的细胞器，因为它们有自己的基因组，可以复制并传递给下一代细胞。与核基因组相反，细胞中线粒体基因组有多个拷贝。详细研究负责线粒体基因组复制、修复和维持的机制对于了解线粒体和整个细胞在正常和疾病条件下的正常功能至关重要。本文提出了一种允许高通量定量体外 培养的人细胞 中线粒体DNA（mtDNA）的合成和分布的方法。该方法基于对通过5-溴-2'-脱氧尿苷（BrdU）掺入标记的活性合成DNA分子进行免疫荧光检测，以及同时检测具有抗DNA抗体的所有mtDNA分子。此外，线粒体用特定的染料或抗体可视化。以多孔形式培养细胞和利用自动荧光显微镜使得在相对较短的时间内在各种实验条件下研究mtDNA的动力学和线粒体的形态变得更加容易。

For most eukaryotic cells mitochondria are essential organelles, as they play a crucial role in numerous cellular processes. First and foremost, mitochondria are the key energy suppliers of cells1. Mitochondria are also involved in regulating cellular homeostasis (for instance, intracellular redox2 and the calcium balance3), cell signaling4,5, apoptosis6, the synthesis of different biochemical compounds7,8, and the innate immune response9. Mitochondrial dysfunction is associated with various pathological states and human diseases10. 
The functioning of mitochondria depends on the genetic information located in two separate genomes: the nuclear and mitochondrial genomes. The mitochondrial genome encodes a small number of genes compared to the nuclear genome, but all the mtDNA-encoded genes are essential for human life. The mitochondrial protein machinery necessary to maintain the mtDNA is encoded by nDNA. The basic components of the mitochondrial replisome, as well as some mitochondrial biogenesis factors, have already been identified (reviewed in previous research11,12). However, mitochondrial DNA replication and maintenance mechanisms are still far from being understood. In contrast to nDNA, the mitochondrial genome exists in multiple copies, which provides an additional layer for regulating mitochondrial gene expression. Much less is currently known about the distribution and segregation of mtDNA within organelles, to what extent these processes are regulated, and if they are, which proteins are involved13. The segregation pattern is crucial when cells contain a mixed population of wild-type and mutated mtDNA. Their unequal distribution may lead to the generation of cells with a detrimental amount of mutated mtDNA.
So far, the protein factors necessary for mtDNA maintenance have been identified mainly by biochemical methods, bioinformatic analyses, or through disease-associated studies. In this work, in order to ensure a high chance of identifying factors that have previously escaped identification, a different strategy is described. The method is based on the labeling of mtDNA during replication or repair with 5-bromo-2'-deoxyuridine (BrdU), a nucleoside analog of thymidine. BrdU is readily incorporated into nascent DNA strands during DNA synthesis and, in general, is used for monitoring the replication of nuclear DNA14. However, the procedure developed here has been optimized for detecting BrdU incorporated into mtDNA using the immunofluorescence of anti-BrdU antibodies.
The approach allows for the high-throughput quantification of mtDNA synthesis and distribution in human cells cultured in vitro. A high-throughput strategy is necessary to conduct tests under different experimental conditions in a relatively short time; therefore, it is proposed in the protocol to utilize a multi-well format for cell culturing and automated fluorescence microscopy for imaging. The protocol includes the transfection of human HeLa cells with an siRNA library and the subsequent monitoring of mtDNA replication or repair using the metabolic labeling of newly synthesized DNA with BrdU. This approach is combined with immunostaining of the DNA with the help of anti-DNA antibodies. Both parameters are analyzed using quantitative fluorescence microscopy. Additionally, mitochondria are visualized with a specific dye. To demonstrate the specificity of the protocol, BrdU staining was tested on cells devoid of mtDNA (rho0 cells), on HeLa cells upon the silencing of well-known mtDNA maintenance factors, and on HeLa cells after treatment with an mtDNA replication inhibitor. The mtDNA levels were also measured by an independent method, namely qPCR.

作者聚焦：基于图像的高通量线粒体 DNA 合成和分布定量  

基于图像的高通量线粒体DNA合成和分布定量

The protocol aims to identify proteins with dysfunction leads to changes in the number of mitochondrial DNA molecules. This research may also reveal the factors involving the distribution of mitochondrial DNA within the mitochondria. Mitochondrial replication and maintenance mechanisms are still not fully understood.Much less is known about regulating the distribution of mitochondrial genomes within organoids and the proteins involved in it. As a result, identifying and characterizing some new players will be of great importance. The main advantage of the technique is its high-throughput and high content protocol.We can simultaneously test many experimental conditions and measure various parameters during a single experiment. In genome-wide studies, the proposed protocol provides an opportunity to outline a global picture of how nuclear encoded genetic information regulates its mitochondrial counterpart. Additionally, it has the potential to identify proteins whose dysfunction causes mitochondrial DNA stress and activates the interferon response pathway.

图1显示了mtDNA合成和分布动力学的高通量研究程序方案。使用多孔板形式可以同时分析许多不同的实验条件，例如使用siRNA文库沉默不同的基因。用于用BrdU标记新合成的DNA分子的条件允许检测HeLa细胞线粒体中的BrdU标记DNA（图2A），但也可以用作建立其他细胞类型测定的起始条件。应根据时程实验为单个细胞系选择BrdU孵育时间（补充图1）?...

Watch this Scientific Journal Video about High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution at JoVE.com

描述了使用多孔板格式和自动免疫荧光成像来检测和量化mtDNA合成和分布的细胞中线粒体DNA（mtDNA）代谢动力学的程序。这可以进一步用于研究各种抑制剂，细胞应激和基因沉默对mtDNA代谢的影响。

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

该协议旨在识别功能障碍导致线粒体DNA分子数量变化的蛋白质。这项研究也可能揭示线粒体内线粒体DNA分布的因素。线粒体复制和维持机制仍未完全了解。关于调节类器官内线粒体基因组的分布以及参与其中的蛋白质，人们知之甚少。因此，识别和描述一些新参与者将非常重要。该技术的主要优点是其高通量和高内涵的协议。我们可以在单个实验中同时测试许多实验条件并测量各种参数。在全基因组研究中，拟议的方案提供了一个机会来概述核编码遗传信息如何调节其线粒体对应物的全球图景。此外，它还有可能识别其功能障碍导致线粒体DNA应激并激活干扰素反应途径的蛋白质。

high-throughput-image-based-quantification-mitochondrial-dna

Research

JoVE Journal

Biochemistry

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

1.5K 次观看.  University of Warsaw. 该协议旨在识别功能障碍导致线粒体DNA分子数量变化的蛋白质。这项研究也可能揭示线粒体内线粒体DNA分布的因素。线粒体复制和维持机制仍未完全了解。关于调节类器官内线粒体基因组的分布以及参与其中的蛋白质，人们知之甚少。因此，识别和描述一些新参与者将非常重要。该技术的主要优点是其高通量和高内涵的协议。我们可以在单个实验中同时测试许多实...

视频: 作者聚焦：基于图像的高通量线粒体 DNA 合成和分布定量  

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

科研

生物化学

Preparation of siRNA Mixture for Mitochondrial DNA Synthesis

To begin, prepare a 140 nano molar mix of small interfering RNA or siRNA by diluting the siRNA in optimum medium. Add the five microliters of the diluted mix to the wells of a black 384 well cell culture microplate. Prepare the appropriate amount of RNAiMAX transfection reagent solution in optimum medium and add 10 microliters to each well of the 384 well plate with a reagent dispenser.

用于线粒体DNA合成的siRNA混合物的制备

Cell Transfection and 5-Bromo-2&#39;-Deoxyuridine (BrdU) Incorporation in HeLa Cells

Cell Transfection and 5-Bromo-2&#039;-Deoxyuridine (BrdU) Incorporation in HeLa Cells  

To begin the cell transfection, add 20 microliters of the HeLa cell suspension to the 384-well plate wells containing diluted siRNA to seed 700 HeLa cells per well. After one hour of incubation at room temperature, place the well plate in an incubator for 72 hours at 37 degrees Celsius and 5%carbon dioxide. To incorporate BrdU in the transfected cells, add 10 microliters of 90-micromolar BrdU solution to each well after 56 hours of siRNA transfection.Incubate the cells for 16 hours at 37 degrees Celsius and 5%carbon dioxide. The specificity of BrdU incorporation into the mitochondrial DNA of HeLa cells was studied using rho0 cells lacking mitochondrial DNA.

细胞转染和5-溴-2&#39;-脱氧尿苷（BrdU）在HeLa细胞中的掺入

5-Bromo-2&#39;-Deoxyuridine (BrdU) Labeling of Mitochondrial DNA in HeLa Cells

5-Bromo-2&#039;-Deoxyuridine (BrdU) Labeling of Mitochondrial DNA in HeLa Cells  

To begin the mitochondria labeling prepare a 20 micromolar solution of BrdU in DMEM with 10%fetal bovine serum or FBS. Then add the mitochondria tracking dye to adjust the final concentration to 1.1 micromolar. After 15 hours add 10 microliters of the mitochondria tracking dye solution to the HeLa cell suspension containing wells.Incubate the cells for one hour. The conditions used to label newly synthesized DNA molecules with BrdU allowed the detection of BrdU labeled DNA in the mitochondria of HeLa cells. The signal obtained with anti-BrdU antibodies was specific and only observed in cells treated with BrdU.Regardless of BrdU treatment no anti-BrdU or anti-DNA antibody signal was detected in the mitochondria of row zero cells. Quantifying the fluorescent signal from the anti-BrdU antibodies indicated that row zero cells treated with BrdU showed the same low level of fluorescence as the BrdU untreated cells, while the signal for the parental lines A549 and HeLa were 50 fold higher.

HeLa细胞中线粒体DNA的5-溴-2&#39;-脱氧尿苷（BrdU）标记

HeLa Cell Fixation with Hoechst 33342 and Blocking

To begin the HeLa cell fixation, rinse the HeLa cell-containing 384-well plate two times with 100 microliters of PBS using the microplate washer. Leave 25 microliters of PBS in the well after the second wash and add 25 microliters of an 8%formaldehyde solution. Incubate the plate in the dark at room temperature for 30 minutes.Then, rinse each well four times with 100 microliters of PBS and leave 25 microliters of PBS in the well after the last wash. Add 25 microliters of 6%bovine serum albumin, or BSA, in PBS to each well and incubate the plate in the dark for 30 minutes.

HeLa细胞固定与Hoechst 33342和阻断

Addition of Primary and Secondary Antibodies to the Transfected HeLa Cells to Monitor the mtDNA Distribution

To begin the addition of the primary antibody, aspirate the bovine serum albumin or BSA from the fixed and blocked transfected HeLa cells leaving 10 microliters of BSA solution in the well. Then add 10 microliters of the primary antibody solution prepared in 3%BSA in PBS and incubate the plate in the dark at four degrees Celsius overnight. After the incubation, rinse each well four times with 100 microliters of PBS, leaving 10 microliters after the last wash.Next, add 10 microliters of secondary antibody solution to the 384 well plate. Use isotype specific antibodies conjugated to fluorochromes, such as Alexa Fluor 488 and Alexa Fluor 555. After incubating the plate in the dark for one hour, rinse each well four times with 100 microliters of PBS and leave 50 microliters of PBS in the well after the last wash.The use of anti-DNA antibodies allowed the monitoring of the mitochondrial DNA distribution in the cell under the given experimental conditions. The downregulation of mitochondrial transcription factor A TFAM led to drastic changes in the mitochondrial DNA distribution with fewer mitochondrial DNA spots than in control cells. Quantifying the mean fluorescent signal from the anti-DNA antibody showed a several fold increase upon TFAM silencing compared to the other conditions tested.

向转染的HeLa细胞中添加一抗和二抗以监测mtDNA分布

Quantitative Image Analysis to Measure the mtDNA and Gene Expression Levels in the Transfected HeLa Cells

To begin the imaging of transfected HeLa cells select a sufficiently long exposure time for the individual fluorescence channels in live view mode based on the intensity histograms generated by the imaging software. Set the appropriate number of planes for imaging on the Z axis. Select the appropriate number of fields of view to display per well.To start the quantitative analysis of acquired images, perform background correction for all the images from all the fluorescence channels. Depending on the size of the analyzed objects, select the appropriate filter size. Next, start the image segmenting by creating a mask of the main object based on the ratio of the intensity of the fluorescent signal to the background for the channel corresponding to the cell nuclei.Create masks for the sub objects representing BrdU and mitochondrial DNA spots using the fluorescence channels appropriate for the given structures. Set the parameters and measure the intensity of the individual pixels within each mask for all the fluorescence channels. To obtain the total fluorescence intensities for the BrdU or mitochondrial DNA channel sum up the intensities of all the sub objects assigned to a given cell nucleus.To obtain the mean fluorescence intensity, divide the total fluorescence intensity by the sum of the area of the sub objects assigned to a given cell nucleus. The imaging results were verified by measuring the mitochondrial DNA and gene expression levels using quantitative real-time PCR. Treatment with dideoxycytidine or DDC resulted in a very strong decrease in the mitochondrial DNA levels.A weaker effect was observed in TFAM and TWINKLE helicase silencing, while the transfection of cells with mitochondrial DNA polymerase gamma or POLG siRNA had a modest effect on the mitochondrial DNA copy number. Quantifying the TFAM and TWINKLE helicase expression confirmed an efficient reduction in their mRNA expression to approximately 15 to 20%of the control levels. In POLG, the mRNA expression was reduced only to 30%which explains the unexpected modest effect on the mitochondrial DNA copy number.