我们感谢 Dr. Atsushi 宫和 Dr. 理查德 j 游乐共享 mt 马质粒和 mt 马联合 Hela 细胞。我们感谢 Raghavendra a. Shamanna 和 Dr. Croteau 对手稿的批判性阅读。这项研究得到了国立卫生研究院 (VAB)、国家老龄研究所的内部研究方案以及2014-2015 和2016-2017 的 intra-laboratory 赠款 (VAB) 的支持。HELSE 支持 RHF (项目编号: 2017056) 和挪威研究理事会 (项目编号: 262175)。
作者投稿:
设计了原稿, 起草了草稿;KP, NS, EMF, RDS, JSK, SAC, YH, 和 ED 写了不同的部分的文件;NT, JP, VAB 修改了手稿, 并提供了专门知识。

动物 (雄性和雌性小鼠) 是根据和批准 NIH 动物保育和使用委员会的认可而在动物设施中出生和繁殖的。安乐死的方法必须符合所有国家和机构的规定。
1. 人体细胞中 Mitophagy 的检测
<ol><li>用 mt-马质粒检测 mitophagy 注: 马蛋白, 融合到线粒体靶信号, 简化为 mt-马, 已证明是有用的体内mitophagy 检测。马是一种比率 pH 敏感的荧光蛋白, 在碱性或中性条件下呈绿色荧光 (激发 458 nm), 在酸性条件下具有红色荧光 (激发 561 nm)。健康的线粒体生长在碱性或中性条件下 (pH 值为 7-8), 在转染 mt 马时用绿色荧光表示。当线粒体经过 mitophagy 并与酸性溶酶体融合时, pH 下降到 4.5, 含有 mt-马的线粒体呈红色荧光6,13,14。重要的是, mt-马是对溶酶体蛋白酶的抗性, 能够在较长时期内对 mitophagy 进行评价。下面的协议是为6井板设计的。<ol>
<li>1天: 使用主要的人类细胞 (例如, 成纤维细胞) 或永生化的人类细胞 (如如, Hela 细胞或 U2OS 细胞)。种子大约 0.3-1 x 106细胞/井 (根据细胞生长速率, 使细胞在第二天达到 70% 70-100) 在一个6孔板。在完整的细胞培养基中维持细胞 (Dulbecco 氏改良的鹰培养基 (DMEM) 培养基辅以 10% FBS, 和1% 青霉素-链霉素混合溶液), 在细胞培养孵化器中孵育37° c (20% O2, 5% CO2).在6孔板中加入1毫升完整的细胞培养基/井。 注: 基因过度表达/击倒或药物治疗可在此阶段进行6。</li>
		<li>2天: 混合 mt-马质粒 dna6和 dna 转染试剂 (为6井板的一个井的混合物)。<ol>
<li>根据制造商的协议, 用150µL 的无血清培养基稀释15µL 的 DNA 转染试剂。</li>
			<li>用150µL 无血清培养基稀释2-5 µg 马质粒 DNA。</li>
			<li>添加稀释的 mt-马质粒到稀释的 DNA 转染试剂, 十年代的漩涡, 在室温下孵育10分钟。 注: DNA 转染条件的优化可能是必要的取决于板的大小 (见制造商的协议细节)。</li>
			<li>添加 DNA/转染试剂混合物滴到细胞, 然后轻轻摇动板 5-10 s, 直到解决方案完全混合。</li>
		</ol>
</li>
		<li>3天: 用新鲜完整的细胞培养基 (1 毫升/井) 取代 DNA 转染试剂培养基, 改变培养基。</li>
		<li>4天: 使用共聚焦显微镜成像细胞6。使用两个成像通道: 绿色通道 (激发 458 nm, 发射 570-695 nm) 来可视化正常的线粒体和红色通道 (激发 561 nm, 发射 570-695 nm) 来可视化正在 mitophagy 的线粒体。随机图像20面积在40x 放大率, 以覆盖100-200 细胞在每一个井。</li>
		<li>执行数据分析。计算 "mitophagy 指数" (mitophagy 线粒体的百分比), 利用图像分析软件对红色和绿色通道的荧光量进行量化: 以红色荧光与红 + 绿荧光的比值,乘以 1006。 注: 替代转染包括使用 mt-马稳定表达的 HeLa 细胞系14。由于 HeLa 细胞不表达帕金, 帕金必须稳定表达, 以研究帕金依赖性 mitophagy 在这个细胞线。</li>
	</ol></li>
	<li>细胞色素 C 氧化酶亚基 II (COXII) 与 LAMP2 定位的 mitophagy 检测 注: COXII 是线粒体基因组编码的内膜蛋白。"mitophagy 指数" 等于 COXII colocalized 与 LAMP2 的比值, 表示为 COXII 总金额的百分比。<ol>
<li>在1° c (37 O20%) 的细胞培养池中, 4 井室滑动 (1-5 x 104细胞在2毫升完全细胞培养培养基/井) 中的种子 HeLa 细胞 (或1.1.1 中提到的其他感兴趣的细胞) 5% CO2) 过夜。</li>
		<li>添加3.0 µL 10 毫米羰基氰化物-4-三氟甲氧基腙 (FCCP) 和孵育在细胞培养孵化器 (步骤 1.2.1) 为 3 h。 注: 其他影响 mitophagy 的药物可使用。</li>
		<li>使用1毫升吸管移除培养基, 用1毫升/冷 (0-4 ° c) 1x PBS 清洗两次电池。让解决方案坐在10-30 秒前的下一个洗涤。然后在 1x PBS 中加入0.5 毫升3.7% 甲醛 (粉煤灰), 并在冰上孵育10分钟以固定细胞。 注意: 粉煤灰是毒素。使用粉煤灰时, 在通风橱里工作。</li>
		<li>使用1毫升吸管丢弃粉煤灰溶液, 用冷 1X pbs (1 毫升/井) 冲洗两次电池; 让解决方案坐在十年代之前的下一个洗涤), 并 permeabilize 的细胞与1毫升/井0.25% 洗涤剂 (在 1x PBS) 为10分钟的冰。</li>
		<li>丢弃 permeabilizing 缓冲液, 用冷 1x PBS 轻轻冲洗两次, 丢弃 (让溶液坐在十年代之间洗)。在4° c 的晚上, 在 1x PBS 中, 用1毫升/井的 5% FBS 阻断细胞。盖住房间以防湿气流失。</li>
		<li>用 COXII 抗体 (小鼠, 1:250 稀释) 和 LAMP2 抗体 (兔, 1:20-1:50 稀释) 在 1x PBS 中准备0.5 毫升/好的混合抗体溶液, 5% FBS, 并在冰上储存 0.5-1 h。</li>
		<li>从冷室取出4井室滑动, 用1毫升吸管丢弃堵塞液。用1毫升/好的冷 1x PBS 和丢弃 (让溶液坐在十年代之间洗) 两次, 然后加入0.5 毫升/好混合抗体溶液。在37° c 的低转速下孵育1小时。</li>
		<li>丢弃第一个抗体溶液使用1毫升吸管和温和地洗涤细胞三次1毫升/井的冷 1x PBS 和丢弃。让解决方案坐在十年代之前的下一个洗涤。添加1毫升/好的荧光二次抗体 (例如, 山羊兔与波长 568 nm 的红色荧光蛋白 (RFP) 的 LAMP2; 和山羊 anti-mouse 与波长 488 nm 绿色荧光蛋白 (GFP) 为 COXII) 1:250 稀释与5%FBS 在 1x PBS。用铝箔覆盖4井室滑动, 在37° c 下孵育1小时, 在一个低转速的振动筛上。</li>
		<li>用1毫升吸管丢弃第二抗体溶液。用1毫升/好的冷 1x PBS 温和冲洗细胞六次。让溶液在下一次洗涤前坐1分钟。</li>
		<li>将50µL/antifade 安装介质的电池装入 DAPI, 并添加盖板。</li>
		<li>在共聚焦显微镜下, 用66x 放大倍数和浸入式油 (瓷砖扫描/马赛克) 对细胞进行成像, 可以增加软件的成像细胞数量。在所有图像处理过程中使用相同的设置, 如通道曝光时间、数字增益、数字偏移量、等(有关详细信息, 请参阅软件说明)15。</li>
		<li>使用定位分析软件对100单元的至少20随机图像进行定量分析15。使用 "闭合贝塞尔曲线" 函数选择整个单个单元格。比较皮尔逊的定位系数的 GFP, 以 RFP, 无论是加权或加权, 以确定 mitophagy 水平的分析细胞。在整个分析过程中维护定位分析软件的水平和垂直十字线的值。要精确设置这些十字线, 请准备两组额外的单元格, 并标记一个 COXII 和其他 LAMP2。然后, 在每个通道15的像素分布上方直接设置十字线 (有关详细信息, 请参阅软件说明)。</li>
	</ol></li>
	<li>高吞吐量成像测量 mitophagy 的筛选 注: 执行高吞吐量屏幕的 mitophagy 诱导或 mitophagy 抑制化合物从大型复合图书馆是技术要求。基于线粒体与体定位的成像技术是一种更简单, 但高度敏感的选择, mitophagy 筛选16。<ol>
<li>1天: 种子细胞 (例如, 5000 cells/100 mL 细胞培养基/井) 中的人原代成纤维细胞 (见步骤 1.1.1)。</li>
		<li>2天: 将第二天的细胞与 FCCP 或其他有兴趣的化合物用于指定的潜伏期 (见步骤 1.2.2)。</li>
		<li>按照步骤 1.2. 2-1. 2.9 使用 COXII 的主要抗体 (小鼠, 1:250 稀释与 5% FBS 在 1x pbs) 和 LC3B (兔子, 1:100-1: 200 稀释与 5% FBS 在 1x pbs)。此外, 还可使用 COXII 和 LAMP2 的主要抗体。</li>
		<li>使用荧光读取器16对单元格进行图像处理。收集图像在随机放置的领域和至少500细胞/井。</li>
		<li>使用单元分析器自定义协议16分析数据。</li>
	</ol></li>
</ol>2. 在线虫中检测 Mitophagy
注意: 线虫 mitophagy) 提供了一个平台, 以分析个体水平的检测. 两个菌株可用于监测 mitophagy: (1) 线粒体靶向罗塞拉 (mtRosella) 或 (2) mitophagy 受体 DCT-1 与 autophagosomal 标记 LGG-1 融合与 Discosoma sp. 红色荧光蛋白 (DsRed)5,17。
<ol><li>基于罗塞拉生物传感器的 Mitophagy 监测 注: 罗塞拉是一种分子生物传感器, 它将 ph 不敏感的 DsRed 融合到 ph 敏感性的 GFP 变种。罗塞拉生物传感器利用酸性溶 (ph 值〜 4.5) 和其他细胞间的 ph 值的差异。该生物传感器已被用来成功地监测 mitophagy 在酿酒酵母和几个哺乳动物细胞系, 如 HeLa, HEK293, 和 HCT11618,19。我们改编了这一多才多艺的双荧光记者, 并产生了转基因的线虫线虫表达线粒体靶罗塞拉 (mtRosella) 在体壁肌细胞. Mitophagy 诱导是通过降低 GFP/DsRed 比 mtRosella 荧光表示。<ol>
<li>1天: 用解剖显微镜5, 在体壁肌肉细胞中选取表达线粒体靶向罗塞拉 (mtRosella) 的蠕虫幼虫, 将其 L4 到线虫生长培养基 (NGM) 上, 用大肠杆菌(OP50。安置10-20 蠕虫或板材在至少三 3.5 cm 板材。在20° c5的标准温度下孵化线虫。 注意: 有关蠕虫解剖的参考, 包括 L4 幼虫20的识别, 以及用于将蠕虫从一个位置传输到另一个21的选择的方法。</li>
		<li>5天: 通过选择 15-20 L4 转基因幼虫并将其转移到新鲜的 OP50 种子 NGM 板上, 使线虫同步。每个实验条件至少使用五板。</li>
		<li>7天: 准备汽车牌照, mitophagy 影响的药物或积极控制。<ol>
<li>杀死大肠杆菌(OP50) 细菌的种子, 暴露 NGM 板15分钟, 222 µW/cm2 (强度) 的 UV 光, 以确保 mitophagy 诱导化合物不会代谢细菌。必须用2000的 J/m2公开板。 注意: 需要曝光的秒数 = 2000/((强度在µW/cm2) * 100)。</li>
			<li>将感兴趣的化合物添加到种子 NGM 板的顶端。添加等量的复合车辆 (溶剂用于溶解化合物, 如二甲基亚砜), 以汽车牌照作为负控制。为积极控制 mitophagy 诱导, 线粒体毒物, 百草枯 (8 毫米最终浓度) 使用。添加一个解决方案, 含有10µL 的8米百草枯库存, 190 µL 的 ddH2O 在琼脂板的顶部为治疗组。对于车辆组, 添加一个解决方案包含10µL 的亚甲基亚砜与190µL 的 ddH2O 在琼脂板的顶部。 注: 百草枯工作液可在4° c 下保存1月。</li>
			<li>轻轻地旋转盘子直到药物或车辆覆盖整个表面。在转移蠕虫之前, 允许板在室温下关闭至少1小时以使其干燥。 注: 药物板也可以通过在液体 NGM 中加入药物来制备, 然后再固化。</li>
		</ol>
</li>
		<li>7天: 转移10-20 的2天的成年转基因动物在步骤 2.1.2, 以含有百草枯、化合物或汽车 (甲基亚砜) 的板材。在20° c 下孵育2天的转基因动物。</li>
		<li>9天: 准备2% 琼脂糖垫 (请参阅补充材料)。然后在每片 M9 中添加一滴 (10 µL) 20 毫米左旋咪唑。将转基因动物固定在 M9-levamisole 的下落中, 使其成象。轻轻地把片放在上面。</li>
		<li>使用附着在荧光显微镜上的照相机捕捉单个转基因动物。获取荧光图像的整个转基因线虫表达 mtRosella 体壁肌肉细胞在10X 倍放大倍数5。保存收集的图像。</li>
		<li>使用 ImageJ 软件处理获取的图像。分析位于蠕虫头部的体壁肌细胞, 以避免肠道组织中的自发荧光。测量每个荧光图像的平均像素强度值和总面积仅为每个动物的头部区域。分析感兴趣的具体领域:<ol>
<li>在 "图像" 和 "颜色" 下拉菜单中选择 "分割通道" 以转换图像。</li>
			<li>利用 "徒手选择" 工具捕捉感兴趣的荧光区域。</li>
			<li>在 "分析" 下拉菜单下选择 "测量" 命令, 并执行像素强度分析。</li>
			<li>通过对总面积的分析, 对像素强度进行归一化。正常化后, 计算 GFP 为 DsRed 比22。</li>
		</ol>
</li>
		<li>使用统计分析软件进行学生t测试 (两个组之间的比较) 或方差分析 (多个组之间的比较) 以p < 0.05 作为重要的517.</li>
	</ol></li>
	<li>DCT-1 与 LGG-1 共存 mitophagy 的评价 注: DCT-1 是一种外线粒体膜蛋白, 它作为一种 mitophagy 受体反应于应激状态, 类似于其假定的 orthologues BNIP3 和哺乳动物的 NIX/BNIP3L (5。因此, mitophagy 的启动是评估共存之间的 DCT-1 mitophagy 受体和 autophagosomal 膜蛋白 LGG-1 (同源哺乳动物 LC3)。<ol>
<li>1天: 选取 L4 的转基因动物幼虫, 将 DCT-1::GFP 和 DsRed::LGG-1 在体壁肌细胞中的表达,5到 OP50 种子 NGM 板上。放置5-10 蠕虫/3.5 cm 板, 并使用至少三板。在20° c 孵育线虫。 注意: 转基因位于单独的质粒中, 它们没有被整合在基因组中。拾取带有6 (su1006)和pmyo-2 GFP contransformation 标记的线虫。只有两个 contransformation 标记的转基因蠕虫在体壁肌细胞中表达 DCT-1::GFP 和 DsRed::LGG-1。</li>
		<li>按照 2.1. 2-2. 1.5 的步骤执行。</li>
		<li>图像单体-壁肌细胞使用摄像头连接到共聚焦显微镜5,23。检测整个身体壁肌肉细胞的边界, 并采取 z 堆栈图像在63x 放大倍数。也可以使用更高的放大倍数。</li>
		<li>使用共焦软件获取的打开和处理图像。mitophagy 的启动是由 mitophagy 受体 (DCT-1::GFP) 的共存向 autophagosomal 标记 (DsRed::LGG-1) 指示的。在人体壁肌细胞的每个堆叠中手动测量共存事件5。必须保持所有的显微镜和相机设置 (镜头和放大镜, 过滤器, 曝光时间, 分辨率, 激光强度, 增益,等) 在整个实验中一致。应注意所有设置。</li>
		<li>使用统计分析软件进行学生t测试 (两个组之间的比较) 或方差分析 (多个组之间的比较) 以p < 0.05 作为重要的517.对于 two-way 比较使用学生的t测试 (p < 0.05 被认为是重要的)。对于多个比较, 使用单因素方差分析, 由post Bonferroni 测试更正。我们建议在每个实验条件下至少分析50-70 动物或30-50 体壁肌细胞。重复实验至少三次。</li>
	</ol></li>
</ol>3. 小鼠 Mitophagy 的检测
注意: 以前检测小鼠 mitophagy 的方法繁琐, 不敏感, 难以量化。一个表达了荧光报告马 (mt-马) 的线粒体靶向型的转基因小鼠模型现在可以用来评估 mitophagy 在各种生理和病理条件下的水平。
<ol><li>安乐使用由机构动物保育和使用委员会批准的协议14。</li>
	<li>使用四肢的针脚将鼠标 (腹侧向上) 固定到工作平台上。用显微外科剪刀和镊子在下腹切开切口, 使肝脏显露为24。用显微外科剪刀和镊子切一块肝脏 (例如, 1 x 1 x 1 厘米的右叶)。 注意: 检测小鼠的 mitophagy 是复杂的, 需要了解鼠标解剖和熟练的鼠标操作和小鼠手术。此协议提供了有关此方法的基本步骤, 并且可以使用更详细的方法24。</li>
	<li>将肝脏样品放在冰上的金属板上, 以迅速冷却组织。将组织置于冷金属板上, 并尽快处理。最理想的情况是, 在解剖1小时内使用。</li>
	<li>用弯曲钳提起肝脏样品, 用5毫升的 ice-cold 1x PBS 冲洗。</li>
	<li>将肝脏转移到一个培养皿 (Ø 100 mm) 上, 用铲刀的勺端将其转到冰上。</li>
	<li>用单边刀片将肝脏样品手工切割成 0.5-1 毫米厚的切片。</li>
	<li>用显微外科钳将组织切片小心地转移到玻璃底盘。</li>
	<li>小心地用钳子将肝切片放在底部。</li>
	<li>用3-5 滴冷 1x PBS 覆盖切片肝脏。 注意: 建议 DAPI 染色以确定某些组织 (如、大脑)24中的感兴趣区域。为了验证共存的红色 mt-马信号与溶, 溶酶体染料, 如葡聚糖级联蓝和 lysosensor, 可以使用24。</li>
	<li>通过两次连续激励24在共焦显微镜下成像组织切片。设置两个成像通道: "绿色" mt-马通道 (激发 458 nm, 发射 570-695 nm 范围) 和 "红色" mt-马 (激发 561 nm, 发射 570-695 nm 范围)。在实验条件之间保持成像设置。为了更有效地成像, 同时分析两只动物。</li>
</ol>

<ol>
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S., Horvath, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+ROS+signaling+in+organismal+homeostasis.">Mitochondrial ROS signaling in organismal homeostasis.</a> Cell. 163 (3), 560-569 (2015).</li><li>Palikaras, K., Lionaki, E., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+mitophagy+and+mitochondrial+biogenesis+during+ageing+in+C.+elegans.">Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans.</a> Nature. 521 (7553), 525-528 (2015).</li><li>Lazarou, 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=The+ubiquitin+kinase+PINK1+recruits+autophagy+receptors+to+induce+mitophagy.">The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.</a> Nature. 524 (7565), 309-314 (2015).</li><li>Kerr, J. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+autophagic+activity+in+mammalian+cells.">Measurement of autophagic activity in mammalian cells.</a> Curr Protoc Cell Biol. , 16 (2012).</li><li>Katayama, H., Kogure, T., Mizushima, N., Yoshimori, T., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+quantitative+technique+for+detecting+autophagic+events+based+on+lysosomal+delivery.">A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery.</a> Chem Biol. 18 (8), 1042-1052 (2011).</li><li>Sun, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+In+Vivo+Mitophagy.">Measuring In Vivo Mitophagy.</a> Mol Cell. 60 (4), 685-696 (2015).</li><li>Fang, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+mitophagy+in+XPA+via+PARP-1+hyperactivation+and+NAD(+)/SIRT1+reduction.">Defective mitophagy in XPA via PARP-1 hyperactivation and NAD(+)/SIRT1 reduction.</a> Cell. 157 (4), 882-896 (2014).</li><li>Diot, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+quantitative+assay+of+mitophagy:+Combining+high+content+fluorescence+microscopy+and+mitochondrial+DNA+load+to+quantify+mitophagy+and+identify+novel+pharmacological+tools+against+pathogenic+heteroplasmic+mtDNA.">A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA.</a> Pharmacol Res. 100, 24-35 (2015).</li><li>Schiavi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron-Starvation-Induced+Mitophagy+Mediates+Lifespan+Extension+upon+Mitochondrial+Stress+in+C.+elegans.">Iron-Starvation-Induced Mitophagy Mediates Lifespan Extension upon Mitochondrial Stress in C. elegans.</a> Curr Biol. 25 (14), 1810-1822 (2015).</li><li>Rosado, C. J., Mijaljica, D., Hatzinisiriou, I., Prescott, M., Devenish, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosella:+a+fluorescent+pH-biosensor+for+reporting+vacuolar+turnover+of+cytosol+and+organelles+in+yeast.">Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast.</a> Autophagy. 4 (2), 205-213 (2008).</li><li>Sargsyan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+parallel+measurements+of+macroautophagy+and+mitophagy+in+mammalian+cells+using+a+single+fluorescent+biosensor.">Rapid parallel measurements of macroautophagy and mitophagy in mammalian cells using a single fluorescent biosensor.</a> Sci Rep. 5, 12397 (2015).</li><li>Altun, Z. F., Hall, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+C.+elegans+anatomy.">Introduction to C. elegans anatomy.</a> WormAtlas. , (2009).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+worm+lab:+from+culturing+worms+to+mutagenesis.">An introduction to worm lab: from culturing worms to mutagenesis.</a> J Vis Exp. (47), e2293 (2011).</li><li>Palikaras, K., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Mitophagy+Monitoring+in+Caenorhabditis+elegans+to+Determine+Mitochondrial+Homeostasis.">In vivo Mitophagy Monitoring in Caenorhabditis elegans to Determine Mitochondrial Homeostasis.</a> Bio Protoc. 7 (7), e2215 (2017).</li><li>Palikaras, K., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+Mitochondrial+Selective+Autophagy+in+the+Nematode+Caenorhabditis+elegans.">Assessing Mitochondrial Selective Autophagy in the Nematode Caenorhabditis elegans.</a> Methods Mol Biol. 1567, 349-361 (2017).</li><li>Sun, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence-based+imaging+method+to+measure+in+vitro+and+in+vivo+mitophagy+using+mt-Keima.">A fluorescence-based imaging method to measure in vitro and in vivo mitophagy using mt-Keima.</a> Nat Protoc. 12 (8), 1576-1587 (2017).</li><li>Eiyama, A., Okamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+for+Mitophagy+in+Yeast.">Assays for Mitophagy in Yeast.</a> Methods Mol Biol. 1567, 337-347 (2017).</li><li>McWilliams, T. G., 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=mito-QC+illuminates+mitophagy+and+mitochondrial+architecture+in+vivo.">mito-QC illuminates mitophagy and mitochondrial architecture in vivo.</a> J Cell Biol. 214 (3), 333-345 (2016).</li></ol>

玻尔实验室与 ChromaDex 和葛兰素史克 CRADA 安排。

精确测量 mitophagy 是技术上的要求。在这里, 我们提出了一些强有力的技术, 允许定性检测 mitophagy 和量化的 mitophagy 水平在最常见的实验室实验模型。
为了获得可复制的数据, 必须进行至少三生物重复的实验设计。所有参与试验和分析的研究人员都必须对实验组的特性视而不见。此外, 成像场必须在图像采集过程中随机选择。对于细胞培养和线虫研究, 至少应该进行三生?...

Mitophagy 对线粒体的维护至关重要。线粒体与多个细胞信号通路相交, 是细胞能量产生、细胞新陈代谢和钙稳态的通用细胞器,1,2,3,4. 线粒体经常面临来自内源性和外部来源的挑战, 如活性氧种 (ROS) 和线粒体毒物, 这些都导致了 "衰老" 和功能失调的线粒体的产生。受损线粒体的积累降低了 ATP 生产的效率, 同时增加了有害活性氧的数量, 并与年龄有关的疾病, 如代谢性疾病, AD 和 PD1,5,6.为了防止线粒体诱导的细胞功能障碍, 细胞需要特别识别受损的线粒体, 并通过细胞过程中的线粒体自噬 (mitophagy) 有效地去除它们。这表明 mitophagy 在健康和疾病中的重要性说明了需要准确和有效的方法来检测 mitophagy 的体外和体内。
Mitophagy 是一个多步过程涉及许多蛋白质和蛋白质复合物5,7,8。简言之, 受损的线粒体首先被 double-membraned phagophore, 它可以来源于细胞膜、内质网、高尔基体、细胞核或线粒体本身9,10。球形 phagophore 拉长, 并最终封闭线粒体内, 构成线粒体 autophagosome (mitophagosome)。mitophagosome 然后与溶的降解, 形成一个 autolysosome, 其中受损的线粒体退化和回收利用7,8。主要的噬蛋白也涉及 mitophagy 包括: 自噬相关的 7 (ATG7) 和 Beclin1 (启动), 微管相关蛋白 1 a/1 b 光链 3 (LC3-II) (LGG-1 在C. elegan) 和 p62 (phagophore 的组成部分), 并溶酶体相关膜糖蛋白 2 (LAMP2)6,7。此外, 有几种基本的蛋白质 mitophagy, 包括 PTEN 诱导的假定激酶 1 (PINK-1), Parkin1, 核点蛋白 52 kDa (NDP52), optineurin, BCL2 交互蛋白3像 (NIX/BNIP3L) (DCT-1 在C. 线虫),在其他5、6、11中。
一种检测自噬水平变化的常用方法是 LC3-II/LC3-I 或 LC3-II/actin 的比值。然而, 这种方法是非特异性的, 因为增加这一比例可能反映了增加启动或受损融合的 mitophagosome 到溶12。另一种方法是评估自噬标记 (例如, LC3) 和线粒体蛋白 (如如, Translocase 外线粒体膜 20 (TOMM20, 可被蛋白酶降解)) 之间的定位。但是, 这只能指示总 mitophagy 级别的变化, 并且不能区分发生阻塞的步骤。这可以通过使用溶酶体抑制剂 (例如, E64d+Pepstatin A, 称为 EP) 来澄清, 从而导致 mitophagosomes 的积累。mitophagosomes 在基线上的数目与 EP mitophagosomes 后的数量之间的差异可以表明 mitophagy。这些限制促使了新的 mitophagy 检测技术的发展。鉴于 mitophagy 在广泛的人类疾病中的相关性日益增强, 我们提出了几种健壮的 mitophagy 检测技术, 这可能对研究人员有用。我们包括体外和体内技术, 并建议结合多种技术来验证 mitophagy 的变化。

<table>
	<tbody>
		<tr>
			<td>mt-Keima mouse</td>
			<td>Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 DNA transfection reagent</td>
			<td>Thermofisher</td>
			<td>#11668027</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM medium (Gibco)</td>
			<td>Thermofisher</td>
			<td>#31985062</td>
			<td>serum-free medium</td>
		</tr>
		<tr>
			<td>mtKemia plasmid: pCHAC-mt-mKeima</td>
			<td>addgene</td>
			<td>#72342</td>
			<td></td>
		</tr>
		<tr>
			<td>COXII antibody (mouse)</td>
			<td>abcam</td>
			<td>#ab110258</td>
			<td></td>
		</tr>
		<tr>
			<td>LAMP2 antibody (rabbit)</td>
			<td>NOVUS</td>
			<td>#CD107b</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-anti-rabbit with wavelength 568 nm of red fluorescent protein (RFP)</td>
			<td>Thermofisher</td>
			<td>#Z25306</td>
			<td>Alexa Fluor 568 dye</td>
		</tr>
		<tr>
			<td>goat-anti-mouse with wavelength 488 nm of green fluorescent protein (GFP)</td>
			<td>Thermofisher</td>
			<td>#Z25002</td>
			<td>Alexa Fluor 488 dye</td>
		</tr>
		<tr>
			<td>prolong gold antifade with DAPI</td>
			<td>Invitrogen</td>
			<td>#P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>SIGMA 
			Corning Costar</td>
			<td>#CLS3516</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well chamber slide</td>
			<td>THermofisher, Nunc Lab-Tek</td>
			<td>#171080</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc F 96-well plate</td>
			<td>Thermofisher</td>
			<td>#152038</td>
			<td></td>
		</tr>
		<tr>
			<td>LC3B antibody rabbit</td>
			<td>NOVUS</td>
			<td>#NB100-2220</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA antibody</td>
			<td>Progen Biotechnik</td>
			<td>#anti-DNA mouse monoclonal, AC-30-10</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermofisher</td>
			<td>#D1306</td>
			<td>antifade mounting medium with DAPI</td>
		</tr>
		<tr>
			<td>IN Cell analyzer (fluorescent reader )</td>
			<td>GE Healthcare Life Sciences</td>
			<td>#IN Cell analyzer 2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse TE-2000e confocal microscope</td>
			<td>Nikon</td>
			<td>#TE-2000e</td>
			<td></td>
		</tr>
		<tr>
			<td>Colocalization software</td>
			<td>Volocity</td>
			<td>#Volocity 6.3</td>
			<td>alternative Zeiss ZEN 2012 software</td>
		</tr>
		<tr>
			<td>IN Cell Investigator Software</td>
			<td>GE Healthcare Life Sciences</td>
			<td>#28408974</td>
			<td></td>
		</tr>
		<tr>
			<td>cell culture medium</td>
			<td>Thermofisher</td>
			<td>#DMEM&#8211;Dulbecco&#39;s Modified Eagle Medium</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermofisher</td>
			<td>#15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>#12003C-1000ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture Incubator</td>
			<td>Thermofisher</td>
			<td>#Thermo Forma 3110 CO2 Water Jacketed Incubator</td>
			<td></td>
		</tr>
		<tr>
			<td>epifluorescence microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Zeiss Axio Imager Z2</td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Olympus</td>
			<td></td>
			<td>Olympus DP71</td>
		</tr>
		<tr>
			<td>confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Zeiss Axio Observer Z1</td>
		</tr>
		<tr>
			<td>confocal software</td>
			<td>Zeiss</td>
			<td></td>
			<td>ZEN 2012</td>
		</tr>
		<tr>
			<td>image analysis software</td>
			<td>Image J</td>
			<td>colocalization analysis, etc</td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>statistical analysis software</td>
			<td>GraphPad Software Inc., San Diego, USA</td>
			<td></td>
			<td>GraphPad Prism software package</td>
		</tr>
		<tr>
			<td>material to make a worm pick</td>
			<td>Surepure Chemetals</td>
			<td>#4655</td>
			<td>The pick is made of 30 gauge 90% platinum 10% iridium wire</td>
		</tr>
		<tr>
			<td>IR: N2;Ex[pmyo-3 TOMM-20::Rosella]</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td>Maintain transgenic animals at 20 &#176;C</td>
		</tr>
		<tr>
			<td>IR: N2; Ex[pdct-1 DCT-1::GFP; pmyo-3 DsRed::LGG-1]</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pmyo-3 TOMM-20::Rosella</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pdct-1 DCT-1::GFP</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pmyo-3 DsRed::LGG-1</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraquat solution</td>
			<td>see supplementary data for preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M9 buffer</td>
			<td>see supplementary data for preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M9-levamisole buffer</td>
			<td>see supplementary data for preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Microscope Slides and Coverslips</td>
			<td>Fisher Scientific</td>
			<td>#B9992000</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td>STERIS Animal Health</td>
			<td>19 Piece Canine Spay Pack Economy</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>STERIS Animal Health</td>
			<td>19 Piece Canine Spay Pack Economy</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Thermofisher</td>
			<td>#AM9625</td>
			<td>10x PBS needs to be diluted to 1x PBS by using ddH2O</td>
		</tr>
		<tr>
			<td>shaker</td>
			<td>Fisher Scientific</td>
			<td>#11-676-178</td>
			<td>Thermo Scientific MaxQ HP Tabletop Orbital Small Open Air Platform Shaker Package A</td>
		</tr>
		<tr>
			<td>2% agarose pad</td>
			<td>see supplementary data for preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vibroslice blades</td>
			<td>World precision instruments</td>
			<td>#BLADES-2</td>
			<td>single-edge blade</td>
		</tr>
		<tr>
			<td>metal plate</td>
			<td>MSC</td>
			<td>#78803988</td>
			<td>0.012 in thick x 6 in wide x 12 in long, 430 Stainless Steel Sheet</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td></td>
			<td></td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>Methyl viologen dichloride hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>#856177</td>
			<td>paraquat</td>
		</tr>
		<tr>
			<td>Incubator for nematodes</td>
			<td>AQUALYTIC</td>
			<td></td>
			<td>Incubator to maintain 20 &#176;C</td>
		</tr>
		<tr>
			<td>Dissecting stereomicroscope</td>
			<td>Olympus</td>
			<td>SMZ645</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>AxioObserver Z1</td>
			<td>For nematodes (step 2)</td>
		</tr>
		<tr>
			<td>epifluorescence microscope</td>
			<td>Zeiss</td>
			<td>AxioImager Z2</td>
			<td>For nematodes (step 2)</td>
		</tr>
		<tr>
			<td>UV crosslinker</td>
			<td>Vilber Lourmat</td>
			<td>BIO-LINK &#8211; BLX-E365</td>
			<td>UV light source; 356 nm</td>
		</tr>
	</tbody>
</table>

in vitro and in vivo detection of mitophagy in human cells, c. elegans, and mice

线粒体是细胞的动力, 以 ATP 的形式产生细胞能量。线粒体功能障碍有助于生物老化和多种疾病, 包括代谢疾病, 过早衰老综合征, 和神经退行性疾病, 如阿尔茨海默氏病 (AD) 和帕金森氏症 (PD)。线粒体健康的维持取决于线粒体生物和有效清除功能性线粒体通过 mitophagy。实验方法, 以准确检测自噬/mitophagy, 特别是在动物模型, 一直是挑战, 以发展。最近在了解 mitophagy 分子机制方面取得的进展, 使新的 mitophagy 检测技术得以发展。在这里, 我们介绍了几种通用的技术来监测人类细胞中的 mitophagy,线虫线虫(如, 罗塞拉和 DCT-1/LGG-1 菌株), 和小鼠 (mt-马)。这些 mitophagy 检测技术的结合, 包括物种评估, 将提高 mitophagy 测量的准确度, 并有助于更好地了解 mitophagy 在健康和疾病中的作用。

人体细胞中 Mitophagy 的检测:
采用本方法, 对人 HeLa 细胞进行转染 mt-马质粒。健康细胞展示了一个组织良好的线粒体网络 (GFP, 488 nm), 很少发生 mitophagy (RFP, 561 nm)。然而, 用线粒体剂 FCCP (30 µM 3 h) 进行预处理的细胞表现出 mitophagy 发生率的严重增加 (图 1A)。还使用 LAMP2 和 COXII (图 1B) 之间...

Watch this Scientific Journal Video about In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice at JoVE.com

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

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitophagy, 清除受损线粒体的过程, 是线粒体稳态和健康维护所必需的。本文介绍了人类细胞、线虫线虫和小鼠的一些最新的 mitophagy 检测方法。

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

in-vitro-vivo-detection-mitophagy-human-cells-c-elegans

Research

JoVE Journal

Medicine

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

17.4K 次观看.  National Institutes of Health. Mitophagy, 清除受损线粒体的过程, 是线粒体稳态和健康维护所必需的。本文介绍了人类细胞、线虫线虫和小鼠的一些最新的 mitophagy 检测方法。

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

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

评估癌症药物过敏的有效性<em&gt;在体外</em&gt;和<em&gt;在体内</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

体内结肠细胞 Photolabeling 对新生小鼠造血细胞迁移潜能的评价

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...