我们感谢格罗斯实验室成员对手稿的批判性阅读以及他们的评论和建议。我们感谢由美国国立卫生研究院研究基础设施计划办公室（P40 OD010440）资助的Caenorhabditis遗传学中心（CGC）提供了一些菌株。这项研究得到了Vitalunga Ltd和以色列科学基金会的资助（资助号989/19）。图形抽象图（图1）是用 BioRender.com 生成的。

注意：为了方便读者，我们将协议分为两部分：一部分侧重于测量 秀丽隐杆线虫线粒体自噬的协议，另一部分侧重于测量肝细胞线粒体自噬的协议。材料清单可以在提供 的材料表 中找到。
1. 秀丽隐杆线虫 协议
<ol><li>制备线虫生长培养基（NGM）板和 大肠杆菌 OP50细菌原液 注意：作为澄清，虽然我们遵循了制备NGM板和OP50细菌储备液25，26的标准方案，但我们认识到不同实验室之间的这些方案可能存在差异。因此，我们包含了完整的实验方案，以确保实验的准确复制。<ol><li>加入 ~150 mL 的 1 M K 2 HPO 4 至 500 mL 的 1 M KH2PO4 溶液，直至达到 pH 6，制成 pH 6 M 磷酸钾缓冲液。通过0.22μm真空过滤器/存储系统对缓冲液进行灭菌。</li>
		<li>制作0.1M氯化钙（CaCl2）和硫酸镁（MgSO4），并用0.2μm注射器过滤器对其进行灭菌。</li>
		<li>在无水乙醇中制备5毫克/毫升胆固醇。 注意：由于胆固醇是在乙醇中制备的，因此请勿过滤。</li>
		<li>通过将 1.5 g 氯化钠 （NaCl）、1.25 g 蛋白胨和 8.5 g 琼脂溶解在 500 mL 双蒸水 （DDW） 中来制备 NGM-琼脂。高压灭菌并使其冷却至~55°C。</li>
		<li>在无菌条件下，加入 12.5 mL 磷酸钾缓冲液 （pH 6）、0.5 mL 0.1 M CaCl2、0.5 mL 0.1 M MgSO4 和 1 mL 5 mg/mL 胆固醇。每次添加后混合均匀。</li>
		<li>向每个 35 mm 平板中加入 4 mL 融化的 NGM-琼脂。将培养皿放置过夜，使其在室温（室温，~21°C）下凝固。</li>
		<li>要制作Luria-Bertani（LB）琼脂平板，将5g氯化钠，5g胰蛋白胨，2.5g酵母提取物和7.5g琼脂溶解在400mL蒸馏去离子水（DDW）中，将溶液的pH调节至7.0，用DDW使体积达到500mL，然后高压灭菌。一旦溶液冷却至55°C，将25mL混合物倒入每个90mm培养皿中，并使板在室温下干燥2天。接下来，从甘油原液中划出干燥的LB平板上的OP50细菌，并在37°C孵育过夜以获得单个菌落。</li>
		<li>通过将 8 g 胰蛋白胨、5 g 酵母提取物和 2.5 g 氯化钠 （NaCl） 溶解在 0.5 L DDW 中来制备 2x 酵母胰蛋白胨 （YT）。将pH值调节至7，然后高压灭菌。</li>
		<li>冷却后，将来自新鲜条纹的 LB 板中的 OP50 细菌菌落接种到 250 mL 锥形瓶中的 50 mL 2x YT 培养基中。在37°C和250rpm下摇动至光密度（OD600）约为0.6。</li>
	</ol></li>
	<li>准备载体和实验板<ol><li>将 100 μL OP50 细菌添加到每个 35 mm NGM-琼脂平板的中心。在室温（室温，21°C）下干燥过夜。</li>
		<li>在 DMSO 中制备 0.5 M VL-850，并使用 M9 缓冲液（22 mM KH 2 PO 4、42 mM Na2HPO 4、86 mMNaCl、pH 7 和 1 mM MgSO 4）稀释至 10 mM VL-850）26。确认pH值为7.0（如果不是，则用0.1M HCl滴定），并用0.22μm注射器过滤器对溶液进行过滤灭菌。如上所述准备车辆，但没有药物（在这种情况下，VL-850）。在DMSO中制备50 mM FCCP，用M9缓冲液稀释至1 mM FCCP，并用0.22 μm注射器过滤器过滤灭菌溶液。</li>
		<li>将 25 μL 载体（阴性对照）、FCCP（阳性对照;5 μM）或 VL-850（实验处理;62.5 μM）添加到细菌草坪上单独接种的 NGM 板中。</li>
		<li>用铝箔覆盖板，并让它们在室温（室温，21°C）下干燥。~16小时后使用板。</li>
	</ol></li>
	<li>获得同步的年轻成年 秀丽隐杆线虫 雌雄同体<ol><li>用 22 mM KH 2 PO 4、42 mM Na2HPO4 和 86 mM NaCl 制备 1 L M9 缓冲液。通过高压灭菌灭菌，然后冷却。冷却后，加入 1 mL 的 1 mM MgSO4（0.22 μm 过滤器灭菌）。</li>
		<li>将 0.8 mL 的 2.5 N 氢氧化钠和 1 mL 的 5% 次氯酸钠溶液与 2.2 mL DDW 混合，制成 4 mL 碱性次氯酸盐溶液（氢氧化钠的终浓度为 0.5 N，次氯酸钠的终浓度为 1.25%）。</li>
		<li>用 1 mL M9 缓冲液 3x 洗涤 NGM 板，将蠕虫（妊娠雌雄同体）收集到 15 mL 锥形管中，以确保所有母体都已收集到管中。</li>
		<li>通过以500× g 离心1分钟使蠕虫沉淀，并弃去上清液直至剩余1mL的体积。</li>
		<li>加入 1 mL 碱性次氯酸盐溶液，倒置试管混合 5 倍。轻轻涡旋管3分钟以帮助释放卵子，并在解剖立体镜下观察蠕虫的状态。</li>
		<li>当大约50%的蠕虫破碎时，加入5mL的M9缓冲液，并通过以500× g离心1分钟立即使鸡蛋沉淀。</li>
		<li>小心地除去上清液，不要干扰沉淀。加入 5 mL M9 缓冲液，重复洗涤过程 2 次。</li>
		<li>除去上清液直到剩余2mL，并以20rpm旋转管（360°旋转）~16小时（RT，21°C）以获得同步的L1幼虫。从该管中，在载玻片上滴 5 μL，在立体镜下计数幼虫的数量，重复此步骤 3 次，取三个计数的平均值，并估计每微升 （μL） 的蠕虫数量。根据这些计算，每块接种有OP50细菌的NGM板添加~200个幼虫。</li>
		<li>在21°C下生长L1幼虫~48小时，直到年轻成虫阶段。</li>
	</ol></li>
	<li>药物治疗和显微镜检查程序<ol><li>在每个实验或对照板上放置100个蠕虫。确保阴性、阳性和实验板分别包含载体、5 μM FCCP 和 62.5 μM VL-850。在21°C孵育6小时。</li>
		<li>使用 1 mL M9 缓冲液将每个板上的蠕虫洗涤到 1.7 mL 微量离心管中。在小型离心机中短暂旋转试管（~3秒）。接下来，在不干扰蠕虫沉淀的情况下轻轻移出M9缓冲液来丢弃上清液。重复该洗涤步骤2x，然后轻轻除去上清液而不干扰蠕虫沉淀。</li>
		<li>将 200 μL M9 缓冲液（含有 0.1% v/v Poloxamer 188、0.1% v/v Pluronic F127 和 2 μL 染色试剂盒试剂）添加到蠕虫沉淀中。让混合物在室温（室温，21°C）下以20rpm（360°旋转）旋转1小时。为保护染料免受光照，请用铝箔覆盖管子。</li>
		<li>轻轻旋转蠕虫，如步骤 1.3.4 中所述。然后，在不干扰蠕虫颗粒的情况下去除染色溶液。接下来，如步骤1.3.2中所述洗涤蠕虫，并将它们转移到含有适当处理的种子NGM-琼脂平板中 - 例如，用FCCP处理的蠕虫转移到含有FCCP的平板中。用铝箔覆盖板，因为染色试剂对光敏感。 注意：我们将蠕虫转移到含有细菌和相应处理剂的培养板中，以最大程度地减少由于过量染料引起的背景噪音。例如，用FCCP处理的蠕虫被转移到补充FCCP的平板上，等等。</li>
		<li>使用 1 mL M9 缓冲液将平板上的蠕虫洗到新鲜的微量离心管中;以相同的方式清洗蠕虫2次。接下来，用1%甲醛将蠕虫固定在冰上30分钟，并用1mL M9缓冲液3x洗涤蠕虫以除去残留的甲醛。洗涤后，将蠕虫旋转成沉淀，并吸出最大量的上清液，使蠕虫沉淀在 10 μL M9 中保持完整。</li>
		<li>要制备2.5%琼脂糖，称取0.125g琼脂糖到10 mL硼硅酸盐玻璃试管中，加入5 mL M9缓冲液，然后用本生燃烧器轻轻加热管溶解琼脂糖。将熔化的琼脂糖转移到75°C的干浴中，并使用1 mL吸头将100 μL熔化的琼脂糖放入Deckgläser显微镜盖玻片（24 mm x 60 mm）上。立即，将另一张载玻片垂直放在琼脂糖滴上，形成十字形。等待~2分钟，通过（轻轻）推动上盖玻片轻轻分开载玻片，从而将琼脂糖垫留在底盖玻片上。 注意：加热管中的琼脂糖时要小心，并确保管子远离身体。切开 1 mL 吸头的边缘，以尽量减少琼脂糖的凝固。</li>
		<li>用巴斯德玻璃移液管将蠕虫转移到琼脂糖垫上（即管中的全部量，~10μL）。用实验室湿巾制成的灯芯去除多余的液体，然后用较小的盖玻片（24 mm x 40 mm）盖住蠕虫。在较小的盖玻片的外围涂抹透明的指甲油以防止蒸发。将载玻片放在暗盒中以保护其免受光照。</li>
		<li>使用共聚焦显微镜在24小时内使用60倍放大镜以适当的波长（见下文）对蠕虫进行成像。<ol><li>将载玻片放在显微镜载物台上。</li>
			<li>打开成像软件，然后右键单击软件的灰色区域。开放式 收购 |Ti2 全焊盘 |ND 收购 |LUT通过 单击右键单击后出现的弹出窗口上的选项。</li>
			<li>在 “Ti2 全焊盘”下，选择 60x。</li>
			<li>在采集下，选择目镜 DIA，然后使用显微镜细焦旋钮聚焦蠕虫。在“采集”下，选择“旋转磁盘”，然后选择“16 位 - 无分箱”选项。对于每个荧光滤光片，将曝光时间设置为 500 毫秒，明场设置为 20 毫秒。设置这些参数后，选择“立即运行”，然后等待输出图像生成为 ND2 文件。 注意：曝光时间需要通过实验确定，因为不同的成像设置具有不同的特性。使用查找表（LUT）检查每个波长的荧光强度。</li>
		</ol></li>
	</ol></li>
	<li>图像分析<ol><li>使用共定位插件在 ImageJ27 中打开共聚焦图像（此处为尼康 ND2 文件）。每个 ND 文件都包含以三种波长（使用 DAPI、GFP/FITC [绿色] 和德州红 [红色] 滤镜）和可见光拍摄的图像平面。要访问这些图像，请在 ImageJ 服务器中打开 ND 文件，然后在对话框中选择“分割图像”。处理明场 （BF）、绿色通道和红色通道图像。</li>
		<li>生成这些图像的副本，以保持原始图像不变，方法是单击 “图像 |复制 或使用键盘快捷键 Shift + D。</li>
		<li>要减少背景，请生成图像的另一个副本，如上所述。减去 滚动半径 为 100 的背景，然后选择 创建背景（不减去）选项 以生成具有给定图像背景的图像。接下来，转到 “处理”|”图像计算器，然后从第二个重复的图像中减去第一个重复的图像。使用生成的图像进行共定位分析。</li>
		<li>要使用 共定位插件，请将 绿色通道 和 红色通道图像 转换为 8 位。为此，请单击 “图像 |类型 |8 位。</li>
		<li>点击 插件 |共定位。要使用共定位插件（见上文）测量线粒体和溶酶体信号的 共定位 ，请使用以下参数： 比率 = 75%，阈值红色通道 = 80.0，阈值绿色通道 = 50.0。输出是一个 8 位二进制图像，其中包含共定位点和 RGB 图像中三个 8 位图像（绿色、红色和共定位图像）的组合。</li>
		<li>通过手动选择此区域并通过单击“ 编辑 |选拔 |创建蒙版，选择感兴趣的区域（图 2A、B）。选择性地去除其他染色实体（例如咽部肌肉）以分析头部身体壁肌肉区域。</li>
		<li>要选择感兴趣区域 （ROI） 中的粒子，请使用图像计算器选择 共定位 8 位 和 遮罩 图像。然后，使用操作 AND （图 3A）选择 ROI 中的点点。这将生成ROI中带有点点的图像（图3B）。</li>
		<li>要分析共定位线粒体和溶酶体的区域，请选择“分析”|”分析粒子，并测量点在 0.1625 μm 2 和 4 μm2 之间的总和。</li>
	</ol></li>
</ol>2. Hep-3B癌细胞方案
<ol><li>制备药物储备溶液<ol><li>在DMSO中准备100 mM VL-850。用0.5M HEPES缓冲液（pH 7.3）将其稀释至5mM，并使用0.22μm注射器过滤器对溶液进行灭菌。然后，如前所述准备车辆，但没有药物（在这种情况下，VL-850）。</li>
	</ol></li>
	<li>培养用于实验、药物治疗和显微镜检查程序的 Hep-3B 细胞<ol><li>在含有 Dulbecco 改良鹰培养基 （DMEM） 的 10 cm 组织培养板中培养 Hep-3B 细胞，该培养板补充有 10% 热灭活胎牛血清 （FBS）、2% L-谷氨酰胺和 1% 四环素（以下简称完全 DMEM）。将细胞在37°C和5%CO2下孵育。</li>
		<li>选择显示70%-80%细胞汇合度（对数生长期）的Hep-3B细胞板，除去培养基，并用5mL预热的磷酸盐缓冲盐水（PBS）洗涤板。取出PBS，并在37°C下用1mL预热的0.25%胰蛋白酶/ 0.02%EDTA孵育细胞~3分钟;在组织培养显微镜（10X）下观察细胞。当细胞变圆时停止胰蛋白酶消化，并通过加入 5 mL 完全 DMEM 开始从平板上解离。将细胞以 1，000 × g 离心 5 分钟，除去上清液，然后将细胞重悬于 5 mL 完全 DMEM 中。</li>
		<li>确定细胞浓度。将 50 μL 细胞悬液与 50 μL 台盼蓝混合。使用自动细胞计数器或使用血细胞计数器5倍对细胞进行计数，并取这些计数的平均值以确保计数的准确性。</li>
		<li>在 400 μL 完整 DMEM 的每个 8 孔μ载玻片中接种 42，000 个 Hep3G 细胞（如上所述）。将细胞在37°C和5%CO2下孵育24小时。</li>
		<li>在~80%-85%汇合度下24小时后，从每个孔中取出250μL培养基，并加入50μL具有适当处理或载体的培养基。为了遵循该协议，用100μM VL-850，5μM FCCP和载体作为对照处理细胞。</li>
		<li>与化合物孵育6小时后，向含有染色试剂的每个孔中加入50μL培养基（每个孔中250μL的0.5μL染料）。用染料在37°C和5%CO2下孵育细胞30分钟。 注意：由于染色试剂对光敏感，因此请用铝箔覆盖样品并在昏暗的环境中工作（如果可能），从而最大限度地减少暴露在光线下。</li>
		<li>使用 200 μL 移液器，轻轻地从每个孔中取出所有培养基 （250 μL），然后用 200 μL 预热的 PBS 洗涤细胞。</li>
		<li>用 200 μL 含有 4% 甲醛和 2.5% 戊二醛的固定溶液在室温下用 PBS 制备 15 分钟固定细胞。</li>
		<li>倒出固定液，并用 200 μL PBS 短暂洗涤。</li>
		<li>加入 200 μL PBS，在 4 °C 下保持细胞覆盖并避光，并在 24 小时内成像。 注意：我们在四个通道中使用转盘共聚焦显微镜，包括DIC，TRITC，FITC和DAPI，如步骤1.4.8所示。我们每次处理~300个细胞。</li>
	</ol></li>
	<li>图像分析<ol><li>执行步骤 1.5.1-1.5.5。要获得细胞的ROI，请生成突出显示细胞区域的图像。为此，请选择共定位点（RGB）图像（图4A）。要选择整个单元格区域，请选择“处理”|”二进制 |创建掩码以获取二进制图像（图 4B）。若要分析单元格的区域，请选择“分析”|”分析粒子，并测量图像中从 0 到无穷大的所有粒子，这是分析粒子的默认设置。</li>
		<li>要分析共定位线粒体和溶酶体的区域，请选择共定位的 8 位图像，通过选择“过程 |二进制 |创建二进制，选择“分析”|”分析粒子，并测量点在 0.1625 μm 2 和 4 μm2 之间的总和。要测量共定位点，请将共定位线粒体和溶酶体的面积除以总细胞面积。</li>
	</ol></li>
</ol>

定量 秀丽隐杆线 虫和肝癌细胞系中的线粒体自噬

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作者没有利益冲突需要声明。

多种线粒体自噬途径涉及各种蛋白质和生物分子（例如心磷脂29）。然而，这些途径的终点是相似的——溶酶体酶12，13对线粒体的降解。事实上，有几种方法使用这个端点来量化线粒体自噬。然而，一些方法，如电子显微镜，需要昂贵的设备、训练有素的专家，以及延长标本和分析的准备时间。此外，尽管使用线粒体自噬生物传感器?...

线粒体对所有有氧动物（包括人类）都是必不可少的。它们通过氧化磷酸化1将生物分子的化学能转化为三磷酸腺苷（ATP），合成血红素2，通过β氧化降解脂肪酸3，调节钙4和铁5稳态，通过细胞凋亡控制细胞死亡6，并产生活性氧（ROS），在氧化还原稳态中起至关重要的作用7.两个互补和相反的过程保持线粒体的完整性和正常功能：新线粒体成分的合成（生物发生）和通过线粒体自噬选择性去除受损成分（即线粒体自噬）8。
几种线粒体自噬途径由酶介导，例如PINK1 / Parkin和受体，包括FUNDC1，FKBP8和BNIP / NIX9，10。值得注意的是，线粒体成分的选择性降解可以独立于自噬体机制（即，通过线粒体衍生的囊泡）发生11。然而，不同选择性线粒体自噬途径的终点是相似的（即溶酶体酶的线粒体降解）12，13。出于这个原因，识别和测量线粒体自噬的各种方法依赖于线粒体和溶酶体标记物14，15，16，17的共定位以及线粒体蛋白/线粒体DNA水平的降低18。
以下是使用荧光显微镜测量动物细胞线粒体自噬的现有实验方法的简要描述，强调线粒体自噬终点阶段。
线粒体自噬生物传感器 线粒体降解发生在溶酶体19的酸性环境中。因此，线粒体成分，包括蛋白质，在线粒体自噬过程的终点经历了从中性pH值到酸性pH值的转变。这种模式支撑了几种线粒体自噬生物传感器的作用机制，包括mito-Rosella18 和串联mCherry-GFP-FIS114。这些传感器含有pH敏感的绿色荧光蛋白（GFP）和pH不敏感的红色荧光蛋白（RFP）。因此，在线粒体自噬的终点，由于GFP荧光团的猝灭，绿红荧光比显着下降。这些传感器的主要限制是（1）荧光团之间可能存在的Förster共振能量转移（FRET）;（2）GFP和RFP的差异成熟率;（3）由于连接它们的多肽的蛋白水解裂解，GFP和RFP之间的解离;（4）荧光-发射重叠;（5）差异荧光团亮度和淬灭15，16。
克服其中一些限制的传感器是Keima线粒体传感器17。mt-Keima传感器（源自珊瑚蛋白Keima）显示单个发射峰（620 nm）。然而，其激发峰对pH敏感。结果，当从高pH值转变为酸性pH 16，17时，从绿色激发（440 nm）过渡到红色激发（586 nm）。最近的线粒体自噬传感器Mito-SRAI通过允许在固定生物样本中进行测量来推进该领域20。然而，尽管遗传传感器具有许多优点，例如能够在特定的组织/细胞中表达它们并将它们靶向不同的线粒体区室，但它们也有局限性。一个限制是遗传传感器需要在细胞或动物中表达，这可能是耗时和资源密集型的。
此外，线粒体本身中传感器的表达可能会影响线粒体功能。例如，在 秀丽隐杆线虫 体壁肌肉中表达线粒体GFP（mtGFP）可扩展线粒体网络21。该表型取决于应激激活转录因子ATFS-1的功能，该因子在线粒体中未折叠蛋白质反应的激活（UPRmt）21中起重要作用。因此，尽管基因编码的线粒体/线粒体自噬生物传感器对于监测 体内线粒体稳态非常有用，但它们可能会影响它们旨在测量的过程。
线粒体/溶酶体特异性抗体和染料 测试线粒体/溶酶体共定位的另一种策略是使用针对线粒体/溶酶体蛋白的抗体，例如线粒体外膜蛋白TOM20和溶酶体相关膜蛋白1（LAMP1）22。在大多数情况下，与荧光团偶联的二抗用于 通过显微镜检测 荧光信号。另一种策略是将遗传构建体与线粒体/溶酶体染料相结合，例如在细胞中表达LAMP1：：GFP融合构建体，同时用红色线粒体染料（例如，Mitotracker Red）染色16。这些方法虽然有效，但需要特异性抗体，并且通常涉及使用固定标本或产生表达荧光标记线粒体/溶酶体的细胞/转基因动物。
在这里，我们概述了利用商业溶酶体/线粒体/核染色试剂盒来评估合成二胺O，O（辛烷-1，8-二基）双（羟胺），以下简称VL-85023，在 秀丽隐杆线虫 和人类癌细胞系Hep-3B（图1）。染色试剂盒包含溶酶体/线粒体/核靶向染料的混合物，可特异性染色这些细胞器23。我们之前使用该试剂盒证明了 秀丽隐杆线虫23 中 1，8 二氨基辛烷（以下简称 VL-004）的线粒体自噬活性。重要的是，我们使用丝线粒体：核DNA含量的qPCR测量验证了染色试剂盒结果23。该染色试剂盒具有以下优点。首先，不需要产生表达线粒体生物传感器的转基因动物或细胞。因此，我们可以研究未经修饰的野生型动物或细胞，从而节省大量时间、金钱和劳动力。此外，如前所述，表达线粒体生物传感器可以改变线粒体功能。其次，该套件具有成本效益，易于使用且快速。第三，尽管我们在 秀丽隐杆线虫 和人类细胞中展示了该方法，但它可以针对其他细胞类型和生物体进行修饰。
话虽如此，与任何方法一样，染色试剂盒方案也有缺点。例如，蠕虫与试剂的孵育是在没有食物的情况下进行的（我们已经看到，即使是死细菌也会显着降低染色效率）。虽然孵育时间相对较短，但即使在这个时间范围内，稳态反应也可能改变，包括线粒体自噬。此外，染料与ER/线粒体/核蛋白和其他生物分子的结合可能会影响这些细胞器的活性。此外，与使用遗传传感器测量线粒体自噬不同，我们使用经过化学固定的蠕虫和细胞。因此，不可能在不同时间继续监测相同的蠕虫/细胞。因此，我们建议结合不同的方法来验证线粒体自噬在特定生理过程中的功能。下面，我们提供了新的数据，表明VL-850在秀丽隐杆线虫和Hep-3B细胞中诱导强大的线粒体自噬。因此，这些数据进一步支持了VL-850延长秀丽隐杆线虫寿命并通过诱导健康线粒体自噬保护秀丽隐杆线虫免受氧化损伤的假设。我们使用质子离子载体羰基氰化物4-（三氟甲氧基）苯腙（FCCP），这是一种有效的线粒体自噬诱导剂24，作为阳性对照。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent or resource</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Mettler-Toledo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD-Difco</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>BD-Difco</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Tryptone</td>
			<td>BD-Difco</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Yeast extract</td>
			<td>BD-Difco</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)</td>
			<td>Sigma</td>
			<td>C2920</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cholestrol</td>
			<td>Thermo Fisher</td>
			<td>C/5360/48</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Biological Industries</td>
			<td>01-055-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Double distilled water (DDW)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td>Biological Industries</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS heat inactivated</td>
			<td>Invitrogen</td>
			<td>M7514</td>
			<td></td>
		</tr>
		<tr>
			<td>Gluteradehyde (25%)</td>
			<td>Sigma</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer 1 M</td>
			<td>Biological Industries</td>
			<td>03-025-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>L-gluatamine</td>
			<td>Biological Industries</td>
			<td>03-020-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysosome/Mitochondria/Nuclear Staining Cytopainter Reagent</td>
			<td>Abcam</td>
			<td>ab139487</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P 40</td>
			<td>Sigma</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformalydehyde (16%)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15720</td>
			<td></td>
		</tr>
		<tr>
			<td>Poloxamer 188 Solution</td>
			<td>Sigma</td>
			<td>P5556</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Millipore</td>
			<td>1.04873.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr>
			<td>SeaKem LE Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Bio-Lab</td>
			<td>1903059100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Gadot</td>
			<td>1310732</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic dodecahydrate</td>
			<td>Sigma</td>
			<td>4273</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline hydrochloride</td>
			<td>Sigma</td>
			<td>87128-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Biological Industries</td>
			<td>03-052-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>VL-850: 1,8-diaminooxy-octane</td>
			<td>Patented</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass/Plastic Disposables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m syringe filter</td>
			<td>Millex GV</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL Micro Centrifuge Tubes</td>
			<td>Lifegene</td>
			<td>LMCT1.7B-500</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Petri plates</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL Erlenmeyer Flask</td>
			<td>IsoLab, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Sterile Polypropylene tube</td>
			<td>Lifegene</td>
			<td>LTB15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri dishes</td>
			<td>Bar Naor</td>
			<td>BN9015810</td>
			<td></td>
		</tr>
		<tr>
			<td>500&#160;mL vacuum filter/storage bottle system, 0.22&#160;&#956;m</td>
			<td>Lifegene</td>
			<td>LG-FPE205500S</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Sterile Polypropylene tube</td>
			<td>Lifegene</td>
			<td>LTB50-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Deckgl&#228;ser Microscope cover glass 24 x 60 mm</td>
			<td>Marienfeld</td>
			<td>101152</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass test tubes (10 mL- 13 x 100 mm) Borosilicate glass</td>
			<td>Pyrex</td>
			<td>99445-13</td>
			<td></td>
		</tr>
		<tr>
			<td>iBiDi 8 well &#956;-slides</td>
			<td>iBiDi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass 24 x 40 mm</td>
			<td>Bar Naor</td>
			<td>BN1052421ECALN</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum iridium 0.25 mM wire</td>
			<td>World Precision Instruments</td>
			<td>PT1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter CellDrop BF</td>
			<td>DeNovix</td>
			<td>CellDrop BF-UNLTD</td>
			<td></td>
		</tr>
		<tr>
			<td>Microspin FV-2400</td>
			<td>Biosan</td>
			<td>BS-010201-AAA</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Yokogawa W1 Spinning Disk confocal microscope with DAPI, FITC, and TRITC filters and bright-field, with a 60x CFI Plan-Apochromat Lambda type lens (air lens) and NIS-Elements software</td>
			<td>Nikon</td>
			<td>CSU-W1</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus SZ61 stereo microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Mettler-Toledo</td>
			<td>MT30019032</td>
			<td></td>
		</tr>
		<tr>
			<td>Revolver Adjustable Lab Rotator</td>
			<td>Labnet</td>
			<td>H5600</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of mitophagy in caenorhabditis elegans and mammalian cells using organelle-specific dyes

线粒体对于各种生物学功能至关重要，包括能量产生、脂质代谢、钙稳态、血红素生物合成、调节细胞死亡和活性氧 （ROS） 的产生。ROS对于关键的生物过程至关重要。然而，如果不加以控制，它们会导致氧化损伤，包括线粒体损伤。受损的线粒体释放更多的ROS，从而加剧细胞损伤和疾病状态。一种称为线粒体自噬（线粒体自噬）的稳态过程选择性地去除受损的线粒体，然后被新的线粒体取代。有多种线粒体自噬途径，共同的终点是溶酶体中受损线粒体的分解。 
包括遗传传感器、抗体免疫荧光和电子显微镜在内的几种方法使用该终点来量化线粒体自噬。每种检查线粒体自噬的方法都有其优点，例如特定的组织/细胞靶向（使用遗传传感器）和出色的细节（使用电子显微镜）。然而，这些方法通常需要昂贵的资源，训练有素的人员，以及在实际实验之前漫长的准备时间，例如用于创造转基因动物。在这里，我们提出了一种具有成本效益的替代方案，用于使用靶向线粒体和溶酶体的市售荧光染料测量线粒体自噬。该方法有效地测量线虫秀 丽隐杆 线虫和人肝细胞中的线粒体自噬，这表明其在其他模型系统中的潜在效率。

Mitochondria are essential for all aerobic animals, including humans. They convert the chemical energy of biomolecules to adenosine triphosphate (ATP) via oxidative phosphorylation1, synthesize heme2, degrade fatty acids through &#946; oxidation3, regulate calcium4 and iron5 homeostasis, control cell death by apoptosis6, and generate reactive oxygen species (ROS), which play a vital role in redox homeostasis7. Two complementary and opposite processes maintain the integrity and proper function of the mitochondria: the synthesis of new mitochondrial components (biogenesis) and the selective removal of damaged ones through mitochondrial autophagy (i.e., mitophagy)8.
Several mitophagy pathways are mediated by enzymes, such as PINK1/Parkin, and receptors, including FUNDC1, FKBP8, and BNIP/NIX9,10. Notably, the selective degradation of mitochondrial components can occur independently of the autophagosome machinery (i.e., through mitochondrial-derived vesicles)11. However, the endpoints of the different selective mitophagy pathways are similar&#160;(i.e.,mitochondrial degradation by lysosomal enzymes)12,13. For this reason, various methods for identifying and measuring mitophagy rely on the colocalization of mitochondrial and lysosomal markers14,15,16,17 and decreased levels of mitochondrial proteins/mitochondrial DNA18.
Below is a concise description of the existing experimental methodologies for measuring mitophagy in animal cells using fluorescence microscopy, emphasizing the mitophagy endpoint phase.
Mitophagy biosensors 
Mitochondrial degradation occurs within the acidic environment of the lysosome19. Therefore, mitochondrial components, including proteins, experience a shift from a neutral to an acidic pH at the endpoint of the mitophagy process. This pattern underpins the mechanism of action of several mitophagy biosensors, including mito-Rosella18 and tandem mCherry-GFP-FIS114. These sensors contain a pH-sensitive green fluorescence protein (GFP) and a pH-insensitive red fluorescence protein (RFP). Therefore, at the endpoint of mitophagy, the green-to-red fluorescence ratio drops significantly due to the quenching of the GFP fluorophore. The major limitations of these sensors are (1) possible F&#246;rster resonance energy transfer (FRET) between the fluorophores; (2) the differential maturation rate of GFP and RFP; (3) dissociation between the GFP and RFP due to proteolytic cleavage of the polypeptide that connects them; (4) fluorescence-emission overlap; and (5) differential fluorophore brightness and quenching15,16.
A sensor that overcomes some of these limitations is the Keima mitochondrial sensor17. The mt-Keima sensor (derived from the coral protein Keima) displays a single emission peak (620 nm). However, its excitation peaks are pH-sensitive. As a result, there is a transition from a green excitation (440 nm) to a red one (586 nm) when shifting from a high pH to an acidic pH16,17. A more recent mitophagy sensor, Mito-SRAI, has advanced the field by allowing for measurements in fixed biological samples20. However, despite the many advantages of genetic sensors, such as the ability to express them in specific tissues/cells and target them to distinct mitochondrial compartments, they also have limitations. One limitation is that the genetic sensors need to be expressed in cells or animals, which can be time-consuming and resource-intensive.
Additionally, the expression of the sensors within mitochondria themselves may influence the mitochondrial function. For example, expressing mitochondrial GFP (mtGFP) in the C. elegans worm body wall muscles expands the mitochondrial network21. This phenotype depends on the function of the stress-activated transcription factor ATFS-1, which plays an essential role in the activation of unfolded protein response in mitochondria (UPRmt)21. Therefore, although genetically encoded mitochondria/mitophagy biosensors are extremely useful for monitoring mitochondria homeostasis in vivo, they may affect the very process they are designed to measure.
Mitochondria/lysosome-specific antibodies and dyes 
Another strategy for testing mitochondrial/lysosome colocalization is to use antibodies against mitochondrial/lysosomal proteins, such as the mitochondrial outer membrane protein TOM20 and lysosomal-associated membrane protein 1 (LAMP1)22. In most cases, secondary antibodies that are conjugated to a fluorophore are used to detect the fluorescence signal via microscopy. Another strategy is to combine genetic constructs with mitochondrial/lysosomal dyes, such as expressing a LAMP1::GFP fusion construct in cells while staining them with a red mitochondrial dye (e.g., Mitotracker Red)16. These methodologies, while effective, require specific antibodies and often involve working with fixed specimens or generating cells/transgenic animals expressing fluorescently labeled mitochondria/lysosomes.
Here, we outline the utilization of a commercial lysosome/mitochondria/nuclear staining kit for assessing the mitophagy-activating properties of synthetic diamine O,O (octane-1,8-diyl)bis(hydroxylamine), hereafter referred to as VL-85023, in C. elegans worms and the human cancer cell line Hep-3B (Figure 1). The staining kit contains a mixture of lysosomal/mitochondrial/nuclear-targeted dyes that specifically stain these organelles23. We previously used this kit to demonstrate the mitophagy activity of 1,8 diaminooctane (hereafter referred to as VL-004) in C. elegans23. Importantly, we validated the staining kit results with the mito-Rosella biosensor and qPCR measurements of the mitochondrial:nuclear DNA content23. This staining kit offers the following advantages. First, there is no need to generate transgenic animals or cells expressing a mitochondrial biosensor. Therefore, we can study unmodified wild-type animals or cells and, thus, save much time, money, and labor. Moreover, as mentioned, expressing mitochondrial biosensors can change the mitochondrial function. Second, the kit is cost-effective, easy to use, and fast. Third, although we demonstrate the method in C. elegans and human cells, it could be modified for other cell types and organisms.
With that said, like any method, the staining kit protocol has drawbacks. For example, the incubation of the worms with the reagent is carried out in the absence of food (we have seen that even dead bacteria significantly decrease the staining efficiency). Although the incubation time is relatively short, it is possible that even in this time frame, homeostatic responses may be altered, including mitophagy. In addition, the binding of the dyes to the ER/mitochondrial/nuclear proteins and other biomolecules may affect the activities of these organelles. Moreover, unlike mitophagy measurement with genetic sensors, we work with worms and cells that have undergone chemical fixation. Therefore, it is impossible to continue monitoring the same worms/cells at different times. Hence, we recommend combining different methodologies to validate the function of mitophagy in a particular physiological process. Below, we present new data demonstrating that VL-850 induces robust mitophagy in C. elegans worms and Hep-3B cells. Therefore, these data further support the hypothesis that VL-850 extends the lifespan of C. elegans and protects C. elegans from oxidative damage through the induction of healthy mitophagy. We have used the proton ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), which is a potent mitophagy inducer24, as a positive control.

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  

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

Degradation of damaged mitochondria by mitophagy is essential for the function of the mitochondrial network. Unfortunately, this process declines with age. We develop and use drugs that activate mitophagy, thereby promoting healthy aging to understand the function of mitophagy in aging and age-related diseases, including Alzheimer's.One of the biggest challenges is to measure the mitophagy process without affecting it. Recent studies have shown that GFP expression in the mitochondria of muscle cells induces stress that is MT UPR. Therefore, using dyes in non-transgenic animals or cells is essential and complements genetic technologies.Our protocol has three main advantages. First, there is no need to generate transgenic animals or cells expressing mitophagy biosensors. Second, no need for expensive infrastructure and expertise such as in case of electron microscopy.And third, the organelle dye cocktail is commercially available and the protocol is simple and fast. The new mitophagy-activating compound VL-850 induces robust mitophagy, and in this way protects from oxidative stress and promote lifespan. Now the big question is to determine the underlying mechanism of action.We explored the mechanism at multiple levels, from the cellular to the organismal using C.elegans and mammalian cells.

用VL-850诱导 秀丽隐杆线虫 和Hep-3B细胞中强大的线粒体自噬反应 VL-850 保护 秀丽隐杆线虫 和人角质形成细胞（HaCaT 细胞）免受氧化应激23。为了进一步探索其作用机制，我们研究了VL-850是否诱导 秀丽隐杆线虫 和其他人类细胞的线粒体自噬。为了测试这一点，我们将 秀丽隐杆线虫 （年轻人，L1后3天）暴露于62.5μM VL-850，5μM FCCP（阳性对照...

Watch this Scientific Journal Video about Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes at JoVE.com

通过电子显微镜、基因传感器和免疫荧光探索线粒体自噬需要昂贵的设备、熟练的人员和大量的时间投资。在这里，我们证明了商业荧光染料试剂盒在量化 秀丽隐杆线虫 和肝癌细胞系的线粒体自噬过程方面的功效。

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

线粒体自噬对受损线粒体的降解对于线粒体网络的功能至关重要。不幸的是，这个过程会随着年龄的增长而下降。我们开发和使用激活线粒体自噬的药物，从而促进健康老龄化，以了解线粒体自噬在衰老和年龄相关疾病（包括阿尔茨海默氏症）中的功能。最大的挑战之一是在不影响线粒体自噬过程的情况下测量线粒体自噬过程。最近的研究表明，肌肉细胞线粒体中的GFP表达会引起MT UPR的压力。因此，在非转基因动物或细胞中使用染料是必不可少的，并且是对遗传技术的补充。我们的协议有三个主要优点。首先，不需要产生表达线粒体自噬生物传感器的转基因动物或细胞。其次，不需要昂贵的基础设施和专业知识，例如电子显微镜。第三，细胞器染料混合物是市售的，协议简单快捷。新的线粒体自噬激活化合物VL-850诱导强大的线粒体自噬，并以这种方式防止氧化应激并延长寿命。现在最大的问题是确定潜在的作用机制。我们使用秀丽隐杆线虫和哺乳动物细胞在多个层面上探索了从细胞到生物体的机制。

detection-mitophagy-caenorhabditis-elegans-mammalian-cells-using

Research

JoVE Journal

Biology

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

1.5K 次观看.  The Hebrew University of Jerusalem. 线粒体自噬对受损线粒体的降解对于线粒体网络的功能至关重要。不幸的是，这个过程会随着年龄的增长而下降。我们开发和使用激活线粒体自噬的药物，从而促进健康老龄化，以了解线粒体自噬在衰老和年龄相关疾病（包括阿尔茨海默氏症）中的功能。最大的挑战之一是在不影响线粒体自噬过程的情况下测量线粒体自噬过程。最近的研究表明，肌肉细胞线粒体中的GFP?...

视频: 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  

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.

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.

科研

生物学

Protocol for Measuring Mitophagy in Caenorhabditis elegans

Protocol for Measuring Mitophagy in Caenorhabditis elegans  

To begin, take the synchronized young adult C.Elegans hermaphrodites in a micro centrifuge tube. Add 200 microliters of M9 buffer, containing Poloxamer 188, Pluronic F127, and two microliters of the staining kit reagent to the worm pellet. Cover the tubes with aluminum foil to protect the dyes from light.Let the mixture rotate at 20 RPM for one hour at room temperature. Then spin the worms at 500 G for one minute. After that, remove the staining solution without disturbing the worm pellet.Wash the worms with M9 buffer thrice and transfer them into a seeded NGM agar plate containing the appropriate treatment. Wash the worms off the plates into fresh micro centrifuge tubes with one milliliter of M9 buffer. Afterward, fix the worms with 1%formaldehyde on ice for 30 minutes.And wash the worms with one milliliter of M9 buffer thrice to remove any residual formaldehyde. After pelleting the worms, aspirate the maximum amount of supernatant, keeping the pellet intact in 10 microliters of M9.Next, to prepare 2.5%agarose, weigh 0.125 grams of agarose into a 10 milliliter borosilicate glass test tube. Add five milliliters of M9 buffer and dissolve the agarose by gently heating the tube with a bunsen burner.Transfer the melted agarose to a dry bath set at 75 degrees Celsius. Add 100 microliters of the melted agarose onto a deck glosser microscope cover glass using a one milliliter tip. Immediately, put another slide perpendicularly on the agarose drop forming a cross shape.After two minutes, gently separate the slides by pushing the upper cover glass leaving the agarose pad on the bottom cover slide. Transfer the worms onto the agarose pad with a Pasteur glass pipette. Use a wick made of a laboratory wipe to remove excess liquid.Then cover the worms with a smaller cover slip and apply transparent nail polish to the periphery to prevent evaporation. Place the slide in a dark box to protect it from light. Use a confocal microscope to image the worms within 24 hours at the appropriate wavelengths using a 60 times magnification lens.Next, open the imaging software and right click on the gray area. From the popup that appears, select acquisition. Ti2 full pad.ND acquisition, and lookup tables. Under Ti2 full pad, select 60 times and adjust the microscope fine focus knob to bring the worms into focus. Then select spinning disc and choose the 16 bit-no binning option.For each fluorescence filter, set the exposure time to 500 milliseconds and 20 milliseconds for bright field. Once these parameters are set, select run now and wait for the output image to be generated as an ND2 file.

测量秀丽隐杆线虫线粒体自噬的协议

Analyzing the Images of Caenorhabditis elegans after Drug Treatment

Analyzing the Images of Caenorhabditis elegans after Drug Treatment  

After imaging the drug-treated C.elegane, open the ND file in the image J server with the colocalization plugin and select the split images option in the dialogue box. Each ND file contains image planes taken at three wavelengths and visible light. Work with brightfield, green, and red images.Generate duplicates of these images to keep the original image untouched by clicking on image and selecting duplicate or using the keyboard shortcut shift plus D.Then reduce the background by generating another image duplicate as demonstrated earlier. Subtract the background with a rolling radius of 100 and select the create background option to generate an image with the given image's Background. Now go to process, click image calculator and subtract the first duplicated image from the second duplicated image.Use the resulting images for the colocalization analysis. To use the colocalization plugin, convert the green and red channel images to eight bit by clicking image, selecting type, followed by eight bit. Next, click on plugins and select colocalization.To measure the colocalization of mitochondria and lysosome signals, set the ratio to 75%the threshold red channel to 80.0, and the threshold green channel to 50.0. An eight bit binary image containing colocalized puncta and combining the three eight bit images in an RGB image is generated as output. Next, focus on the puncta in the head body wall muscle of the worms by manually selecting the area and creating a mask by clicking edit, selection, and create mask to select the region of interest.To select the particles in the region of interest, use the image calculator to select the colocalized eight bit and masked images. Utilize the operation and to select the puncta in the region of interest generating an image with puncta in the region of interest. Finally, to analyze the area of the colocalized mitochondria and lysosomes, select analyze, followed by analyze particles and measure the summation of the puncta between 0.1625 square micrometers and four square micrometers.The confocal images of the head body wall muscles of worms showed colocalization of mitochondria and lysosomes. VL-850 induced robust mitophagy in the worm's head body wall muscles, indicating a potent mitophagy inducer. The mitophagy potency of VL-850 was similar to that of FCCP.

药物治疗后秀 丽隐杆线虫 图像分析

Protocol for Measuring Mitophagy in Hep-3B Cancer Cells 17

To begin, take 70 to 80%confluent Hep-3B cells. Remove 250 microliters of medium from each of the wells and add 50 microliters of medium with the appropriate treatment or vehicle. Add 50 microliters of medium to each well containing the staining reagent, and incubate the cells with the dye.After removing the medium and washing the cells with pre-warmed PBS, fix the cells with 200 microliters of fixing solution for 15 minutes at room temperature. Afterward, decant the fixative solution and wash briefly with 200 microliters of PBS. Finally, add 200 microliters of PBS.Keep the cells covered and protected from light at four degrees Celsius, and image within 24 hours.

测量Hep-3B癌细胞线粒体自噬的协议 17

Image Analysis of  Hep-3B Cells after Drug Treatment

After imaging the drug treated Hep-3B cells, open the confocal images in image J with the colocalization plugin. Open the ND file in the image J server and select the split channels option in the dialogue box. Work with green and red images.Generate duplicates of these images to keep the original image untouched by clicking on image and selecting duplicate or using the keyboard shortcut shift plus D.To reduce the background, generate another image duplicate, subtract the background with a rolling radius of 100, and select the create background option to generate an image with the given images background. Then, go to process. Click image calculator and subtract the first duplicated image from the second duplicated image.Utilize the resulting images for the colocalization analysis. To use the colocalization plugin, convert the green and red channel images to 8 bit by clicking image, selecting type, followed by 8 bit. Next, click on plugins and select colocalization.To measure the colocalization of mitochondria and lysosome signals, set the ratio to 75%the threshold red channel to 80.0, and the threshold green channel to 50.0. An 8 bit binary image containing colocalized puncta and combining the three 8 bit images in an RGB image will be generated. Convert these images to 8 bit images.To obtain the region of interest of the cells, generate an image that highlights the area of cells by selecting a colocalized point's image. To select the entire cell area, select the resultant colocalized points image and click on image, adjust threshold, and set threshold to ensure all the cell area is highlighted. To analyze the area of the cell, select analyze, click analyze particles, and measure all the particles in the image from zero to infinity, the default setting for analyze particles.To analyze the area of the colocalized mitochondria and lysosomes, select the colocalized 8 bit image. Then, select analyze. Click analyze particles and measure the summation of puncta between 0.1625 square micrometers and four square micrometers.Divide the area of the colocalized mitochondria and lysosomes by the total cell area to measure the colocalized puncta. The confocal images of Hep-3B cells displayed colocalization of mitochondria and lysosomes. VL 850 and FCCP induced significant mitophagy in the Hep-3B cells.