这项工作得到了韩国国家研究基金会 (2016R1D1A1B03931949) (j.u.) 和韩国政府 (MSIT) (2016R1A2B2008887, 2016R1A5A2007009) 资助的韩国国家研究基金会 (NRF) 赠款的支持。.)

1. 表达 Keima (mt-Keima) 的 HeLa 细胞的生成
<ol><li>
Keima 慢病毒北疆的制备
	<ol>
<li>用2毫升0.001% 聚 l-赖氨酸/磷酸盐缓冲盐水 (PBS) 涂上100毫米的培养基, 在室温下可以站立5分钟。</li>
		<li>使用连接到真空的玻璃吸管去除聚 l-赖氨酸溶液, 并通过添加2毫升 1x PBS 来洗涤培养皿。</li>
		<li>板 1.5 x 106 HEK293T 细胞在涂层培养皿与10毫升的 DMEM 含有10% 的血清和1% 青霉素/链霉素, 并培养细胞在37°c 在一个 CO2组织培养孵化器一天。</li>
		<li>染2µg 的 mt-Keima 慢病毒载体质粒29 DNA 连同包装脱氧核糖核酸 (psPAX 2 µg 和 pVSVG 0.25 µg) 使用的转染试剂 8 h 根据制造商的指示。</li>
		<li>去除转染介质, 加入8毫升的新鲜介质。</li>
		<li>收集含有病毒粒子的介质48小时后。通过离心 500 x g 5 分钟, 从收集的介质中去除细胞碎片, 并用0.45 µm 注射器过滤器进行过滤。 注:为长期使用, 使整除数的慢病毒载体颗粒和储存样品在-80 摄氏度。避免将病毒样本置于冰冻解冻循环中, 以有效地感染病毒。</li>
	</ol>
</li>
	<li>
Keima 慢病毒北疆感染 HeLa 细胞
	<ol>
<li>5 x 104 hela 细胞, 在60毫米的培养皿中表达, 其中4毫升的 DMEM 含有10% 个血清和1% 个青霉素/链霉素, 在37摄氏度前一天 Keima 慢病毒北疆。 注:由于在 hela 细胞中缺乏内源性的 CCCP表达, 在这里使用的 hela 细胞更清楚地显示了 mitophagy。此过程也可应用于其他主要或永生化的细胞系。</li>
		<li>第二天去除生长介质, 加入1毫升的 Keima 慢病毒载体介质。添加额外的2毫升的生长培养基, 含有3µL 的凝聚胺溶液 (8 毫克/毫升蒸馏水)。在2组织培养孵化器中孵化一天。</li>
		<li>第二天去掉 Keima 慢病毒载体混合物, 用 1x PBS 清洗两次细胞。添加4毫升的生长培养基, 并治疗细胞与2µg/毫升嘌呤霉素两天。</li>
		<li>经过两天的嘌呤霉素选择, 去除生长培养基, 并清洗细胞两次与 1x PBS。添加生长培养基, 并培养细胞在 CO2孵化器, 直到使用。 注:该方法可用于生成稳定表达 Keima 的细胞, 包括其他细胞系和主细胞。</li>
	</ol>
</li>
</ol>2. 诱导 mitophagy 使用 CCCP 处理和制备外地药物管制样品
<ol style="margin-left: 40px;"><li>板 5 x 104 HeLa 细胞表达 mt-Keima 在60毫米培养皿与4毫升的 DMEM 含有10% 个血清和1% 青霉素/链霉素和培养他们一天在37°c 在 CO2组织培养孵化器。</li>
	<li>第二天去除生长培养基, 加入4毫升新鲜 DMEM, 含10µM 羰基氰化腙 (CCCP)。在 CO2组织孵化器中孵育 HeLa-Keima 细胞, 6 小时。</li>
	<li>去除生长培养基6小时后, 用2毫升 1x PBS 冲洗细胞。用胰蛋白酶/EDTA 溶液将细胞分离, 加入含有10% 血清的生长培养基, 使胰蛋白酶钝化。将细胞转移到15毫升锥形管中。</li>
	<li>离心细胞在 500 x g 5 分钟。</li>
	<li>通过吸入和并用重悬1毫升冷 1x PBS 的细胞, 小心地去除生长培养基。</li>
	<li>将细胞转换成适当的流式管, 放在冰上。 注:准备未感染的 HeLa 细胞作为阴性对照。 注:细胞在冰上存活数小时, Keima 荧光信号也保持稳定。</li>
</ol>3. mitophagy 的资产管制分析
注: 在本研究中, 用 405 nm 和 561 nm 激光的流式细胞仪对单元进行分析。细胞兴奋与紫激光 (405 nm) 与发射检测到 610, 10 毫微米与一个 BV605 探测器, 并与黄绿色激光 (561 nm) 与发射检测 610 @ 10 nm 由一个 PE-CF594 探测器同时 (图 1)。
<ol><li>
浇细胞
	<ol>
<li>打开流式细胞仪和计算机, 登录分析软件。</li>
		<li>生成新的实验或复制现有的实验。要激发细胞与紫罗兰 (405 毫微米) 和黄绿色 (561 nm) 激光器和检测发射在 610 @ 10 nm 探测器, 选择 BV605 和 PE-CF594, 除了在参数窗口中的正散射 (FSC) 和侧散射 (SSC), 根据需要显影。 注:应在线性模式中添加所有显影。</li>
		<li>涡旋每个细胞样品以分散细胞团聚体。</li>
		<li>将包含控制的 Keima 细胞样本的管放置在取样端口上, 不感染 mt-慢病毒北疆, 并按下 "运行" 按钮。</li>
		<li>在 fsc 与 ssc 的点图中, 调整 fsc 和 ssc 电压, 将单元格放在点图的中心。</li>
		<li>使用围绕活细胞的多边形图标绘制一个 P1 门, 并消除死细胞和细胞碎片 (图 2A)。</li>
	</ol>
</li>
	<li>
调节紫、绿激光器的电压
	<ol>
<li>将含有 Keima 慢病毒北疆感染的 HeLa 细胞的导管放置在样品注射口, 然后按下 "运行" 按钮。</li>
		<li>在 BV605 (405 nm) 与 SSC 的点图中, 调整 BV605 电压, 使表达 mt-Keima 的 HeLa 细胞明显有别于不表达 mt-Keima 的控制单元 (图 2B)。</li>
		<li>在 PE-CF594 (561 nm) 与 SSC 的点图中, 调整 PE-CF594 电压, 使表达 mt-Keima 的 HeLa 细胞明显有别于控制细胞 (图 2B)。</li>
	</ol>
</li>
	<li>
评估 mitophagy 中细胞的百分比
	<ol>
<li>利用线性尺度获取 BV605 与 PE-CF594 点图。用多边形图标在表达 mt-Keima 的细胞周围画一个门, 消除没有表达 mt-Keima 的细胞 (图 3A)。Keima 荧光阳性细胞群称为 "mt-Keima" 门。</li>
		<li>创建另一个点图 BV605 vs PE-CF594 只显示 "mt-Keima"-门控细胞。在这个点图中, 在未经处理的 mt-Keima 阳性细胞周围画一扇门。控制 HeLa 细胞的基底 mitophagy 活性低, PE-CF594/BV605 的发射率小于1。因此, 这个门被称为 "低" 门。</li>
		<li>绘制另一个包含 "低" 门区域的门。这个门被称为 "高" 门, 因为它包含高 mitophagy 活动的细胞, 即在 PE-CF594/BV605 中发射率高的细胞 (图 3B)。</li>
		<li>要计算 "高" 门中单元格的百分比, 请选择 "显示人口层次结构" 菜单。"父百分比" (%Parent) 值表示 mt-Keima 阳性人群中 "高" 门中细胞的百分比。</li>
		<li>设置至少1万事件记录在 "mt-Keima" 停止门和运行每个样本。</li>
	</ol>
</li>
</ol>

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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=In+vitro+and+in+vivo+detection+of+mitophagy+in+human+cells,+C.+Elegans,+and+mice.">In vitro and in vivo detection of mitophagy in human cells, C. Elegans, and mice.</a> Journal of Visualized Experiments. 129, e56301 (2017).</li></ol>

作者没有什么可透露的。

在这里, 我们提出了一个快速和灵敏的方法, 使用流式细胞仪来测量细胞 mitophagy 活动在表达 mt Keima。接受高水平 mitophagy 的细胞表现为 PE-CF594 (561 nm)/BV605 (405 nm) 激发的比例增加。因此, mitophagy 活动可以表示为高561/405 比例的细胞百分比。我们计算了 PE-CF594 (561 nm) 与 BV605 (405 nm) 的斑点图中 "高" 门区域内细胞的百分比, 结果表明 CCCP 的治疗导致了 HeLa 细胞 mitophagy 的增加。我们以前证明, 慢病毒北疆介导的 mt Keima 的过度表达并不影响线粒体功能或细胞生理学31。虽然我们的手稿显示, 流式细胞术分析可以成功地检测 CCCP 诱导的 mitophagy 在 HeLa 细胞表达, 这一技术可以应用于任何类型的细胞后引入 mt-Keima。我们以前表明, 缺氧诱发 mitophagy 可以发现在 HeLa 细胞没有异位的29。此外, 以往的研究, 包括我们的和其他组, 表明, mt-Keima 荧光分析可以检测 mitophagy 的主要细胞, 如小鼠胚胎成纤维细胞 (MEFs) 和永生化细胞, 如 HeLa, HEK293 和 SH-SY5Y 细胞28,29,32。
与其他基于显微或免疫印迹法的 mitophagy 检测方法相比, 流式细胞仪检测具有一定的优势, 可以快速、重现性地分析大量细胞31。因此, 在显微镜下的分析中, 研究者的偏倚会扭曲结果, 这种偏倚在使用流式细胞仪检测时不是问题。
流式细胞术分析需要少于 1 x 106细胞。另外, 由于样品的分析只需要一到两分钟, 最多可以在一到两个小时内进行分析。此外, 流式细胞仪检测 mitophagic 细胞的增加具有良好的灵敏度。在我们的流式细胞术分析中, 我们发现在 CCCP 治疗6小时后, mitophagic 细胞增加了10倍。
用流式细胞仪分析 mitophagy 的关键步骤是正确设置 "高" 和 "低" 门, 以区分处于高水平 mitophagy 的细胞。由于基底 mitophagy 水平可以不同, 取决于细胞类型, 设置 "高" 和 "低" 门可能是困难的一些细胞系。在这种情况下, 它有助于包括一个低基底 mitophagy 活动, 如 HeLa 细胞的控制细胞线。此外, 对于用溶酶抑制剂 (如氯喹) 治疗的细胞, bafilomycin a 可以作为低 mitophagy 控制来设置正确的门控。虽然慢病毒通常用于异位表达的有趣的蛋白质, 一些细胞系可能容易感染病毒。如果慢病毒北疆感染本身导致细胞生长或线粒体功能的变化, 应考虑另一种方法 Keima 表达。Keima 的表达水平也会因细胞类型而异。如果 mt-Keima 荧光信号太弱或太不均匀, 只有具有强 mt Keima 荧光信号的细胞可以使用细胞分拣器或 mt-Keima 细胞再用更高浓度的嘌呤霉素抗生素处理数天.Keima 信号的分离细胞是稳定的, 只要细胞是可行的, 但我们建议通过流式细胞术尽快分析样品。
流式细胞仪检测是一种稳健、方便的 mitophagy 分析方法, 但存在一定的局限性。首先, 由于细胞在流式细胞术中应分离为单细胞水平, 因此很难在不改变生理变化的情况下对组织或器官的 mitophagy 进行分析。此外, Keima 荧光在固定后失去了 pH 依赖性的特性, 因此应将所有样品作为活细胞进行分析。这就导致了上面提到的样本存储的限制, 以及在应用额外的免疫荧光染色时的另一个限制。这种技术可以定量地测量 mitophagy 活动, 但它无法监测线粒体形态学或动力学的变化, 这在 mitophagy 过程9中起着重要的作用。研究人员应该牢记 mitophagy 的复杂性和不确定的方面。因此, 为了避免可能的误判, mitophagy 过程应进一步证实, 其他共同的 mitophagy 分析方法的综合结果, 包括电子显微学, 线粒体蛋白周转, 减少线粒体 DNA 和荧光显微镜测量线粒体定位的溶酶体或自噬体, 如前所述33,34,35。任何观察到的 mitophagy 的增加, 也可以通过共聚焦显微镜的细胞表达 mt-Keima 进一步检查。在 mitophagic 细胞中, mt-Keima 表达在一个明亮的点状模式, 高586/440 的比例, 可以观察到共焦显微镜28,29。
由于它能够快速、灵敏地检测 mitophagy, 这种流式细胞仪检测提供了一步逼近, 对各种研究都有价值。通过简单地用 mt-Keima 慢病毒北疆感染细胞, mitophagy 活动的变化可以很容易地在任何期望的细胞类型中测量。结合细胞分选功能, 具有特定水平的 mitophagy 活动细胞可以被具体隔离, 并用于研究 mitophagy 的作用和调控机制。

线粒体是细胞增殖和生理的基本器官。线粒体负责产生超过80% 的 ATP 供应通过氧化磷酸化, 他们也提供了各种代谢中间体的生物合成和新陈代谢1,2。除了它们在能源供应和新陈代谢中的作用, 线粒体在许多其他重要过程中扮演中心角色, 包括活性氧 (ROS) 生成、细胞死亡调节和胞内 Ca2 +动力学3.线粒体功能的改变与人类疾病有关, 包括癌症、糖尿病和各种神经退行性疾病4、5。增加的线粒体功能障碍和线粒体 DNA 突变也被认为有助于正常衰老过程6,7,8。因此, 质量控制是线粒体中的一个关键问题。线粒体是高度动态的细胞器, 可以在拉长的网络和短而零碎的形态之间不断改变它们的形状。线粒体动力学在维持线粒体功能以及通过 mitophagy9的降解过程中起着重要的作用。
Mitophagy 是一种机制, 它涉及利用自噬机械对整个线粒体进行选择性降解。Mitophagy 是线粒体更替和去除受损或功能失调的线粒体的原理机制。在这个过程中, 线粒体首先被膜包围, 导致自噬体的形成, 然后与溶酶体融合水解降解9。以往果蝇的基因研究表明, 两种帕金森病相关基因, PTEN 诱导的激酶 1 (PINK1) (PARK6) 和 PARK2, 功能在同一途径10,11。子序列研究表明, PINK1-Parkin 通路是负责消除受损的线粒体和缺陷的这一途径导致功能失调的 mitophagy, 可能导致人类疾病12,13。最近在各种人类疾病中发现了 mitophagy 过程的缺陷, 包括癌症、心脏病、肝病和神经退行性疾病14。此外, 最近的研究也表明, mitophagy 是关键的许多生理过程, 如分化, 发展和免疫反应15,16,17,18 ,19, 表明 mitophagy 可能在控制细胞功能方面发挥更积极的作用。
尽管最近证实 mitophagy 在线粒体和人类疾病的质量控制中起着重要作用, 但 mitophagy 的分子机制仍然不太清楚。虽然在酵母中系统地研究了 mitophagy 相关的机制, 但旨在探索哺乳动物细胞 mitophagy 相关机制的研究主要集中在 PINK1-Parkin 途径上。以前的研究已经证实, PINK1-Parkin 途径主要负责选择性地去除受损的线粒体通过 mitophagy12,20,21。然而, 最近的研究报告说, 在某些模型中, 即使在没有功能 PINK122、23、24的情况下, mitophagy 也可以被激活。这些结果表明, 除了 PINK1-Parkin 通路, 有一个额外的不明途径, 可以激活 mitophagy。
缺乏一种方便的评估 mitophagy 活动的方法是探索调节 mitophagy 及其在病理生理事件中作用的途径的主要障碍。电子显微术是直接可视化 autophagosome 吞噬的线粒体的有力工具。然而, 电子显微镜在量化 mitophagy 活动方面有局限性。虽然使用微管相关的 protein-1 光链 3 (LC3)-共轭荧光探针 (如 GFP-LC3) 的策略是目前应用最广泛的方法25, LC3-based 信号的瞬态性及其高速率假阳性信号限制了其检测26细胞 mitophagy 的灵敏度。结合西方印迹测量线粒体蛋白水平, 线粒体 DNA 的量化, 和荧光显微分析 colocalize 线粒体与自噬体或溶酶体将是一个很好的方法来评估mitophagy。然而, 量化限制和现有方法的缺乏方便要求采取新的办法。引入一种新的 pH 依赖性荧光蛋白, 线粒体靶 Keima (mt-Keima), 大大提高了检测 mitophagy27的能力。将细胞色素 c 氧化酶亚基 viii (COX viii) 的线粒体靶向序列融合成 Keima, Keima 被定向到线粒体基质。Keima 的励磁峰的大位移从440毫微米到 586 nm (分别对应于 ph 7 和 ph 4) 可以用来评估 mitophagy在体外和体内实验28,29.更重要的是, Keima 对溶酶体降解的抗性导致了 Keima 信号在溶体蛋白中的整合, 使得 mitophagy 活性的定量测量变得容易。在 Keima 中发生的荧光变化可以用共焦显微镜或流式细胞术28,29进行分析。然而, 目前还没有详细提供基于流式细胞仪的测量 mitophagy 活动的方法--Keima。
在这里, 我们描述了一个详细的协议, 以流式细胞术为基础的技术, 以测量细胞 mitophagy 活动, 使用 mt-Keima。虽然我们在这里表明, 流式细胞术分析成功地检测到 CCCP 诱导 mitophagy 在 HeLa 细胞表达, 这一技术可以应用于多种细胞类型。

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sensitive measurement of mitophagy by flow cytometry using the ph-dependent fluorescent reporter mt-keima

Mitophagy 是一种选择性去除受损或不必要的线粒体使用自噬机械的过程。有缺陷的 mitophagy 和各种人类疾病 (包括神经退行性疾病、癌症和代谢性疾病) 之间存在着密切的联系。此外, 最近的研究表明, mitophagy 参与正常的细胞过程, 如分化和发展。然而, mitophagy 的确切作用和分子机制需要进一步研究。因此, 建立一种稳健、方便的测量 mitophagy 活动变化的方法至关重要。在这里, 我们描述了一个详细的协议, 以定量评估 mitophagy 活动, 通过流式细胞术使用线粒体靶向荧光蛋白 Keima (mt-Keima)。这种流式细胞仪可以比常规显微术或免疫印迹法方法更快速、灵敏地分析 mitophagy 活动。该协议可应用于分析各种细胞类型中的 mitophagy 活动。

如图 4所示, CCCP 诱导的 mitophagy 细胞的流式细胞术分析。应用上述流式细胞仪分析方法, 可以检测出 "高" 门 mitophagic 细胞的急剧增加。与未经处理的控制细胞相比, "高" 门细胞的百分比增加了10倍以上 (图 4A)。mitophagy 活动的增加完全被取消了与 bafilomycin A (论坛) 的合作 (图 4A和4B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58099/58099fig1.jpg"> 图 1.用流式细胞仪测量 mitophagy 的方法的原理图表示法.该方案概述了用流式细胞仪量化 mitophagy 细胞的主要步骤。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58099/58099fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58099/58099fig2.jpg"> 图 2.设置流式细胞仪参数。(A)对活细胞进行门控。在 FSC 和 SSC 图中确定了一个活细胞区域, 以排除死细胞和细胞碎片。(B)使用控制 hela 细胞和 Keima 表达的 hela 细胞, 调整 405 nm 和 531 nm 激光器。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58099/58099fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58099/58099fig3.jpg"> 图 3.用流式细胞仪计算 mitophagic 细胞的比例。(A) Keima 表达的 HeLa 细胞。(B)使用统计工具对 mitophagic 细胞进行浇口和计算比例。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58099/58099fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58099/58099fig4.jpg"> 图 4.流式细胞仪定量测定 HeLa 细胞中 CCCP 诱导的 mitophagy。(A)表达 mt-Keima 的 HeLa 细胞以10µM CCCP 为6小时, 有或不 100 nM bafilomycin A (博鳌)。用 BV605 和 PE-CF594 检测器对细胞进行了分析。(B)选择了高 PE-CF594/BV605 比的细胞 ("高"), 计算了它们的比例。用标准偏差绘制了三次重复实验结果。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58099/58099fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima at JoVE.com

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy, 线粒体的选择性降解, 与线粒体稳态有关, 在各种人类疾病中被解除管制。然而, 方便的实验方法测量 mitophagy 活动是非常有限的。在这里, 我们提供了一个敏感的检测 mitophagy 活动使用流式细胞仪。

pH 依赖性荧光 Keima 对流式细胞术 Mitophagy 的敏感性测定

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

sensitive-measurement-mitophagy-flow-cytometry-using-ph-dependent

Nanyang Technological University

Research

JoVE Journal

Biology

14.7K 次观看.  Dong-A University. Mitophagy, 线粒体的选择性降解, 与线粒体稳态有关, 在各种人类疾病中被解除管制。然而, 方便的实验方法测量 mitophagy 活动是非常有限的。在这里, 我们提供了一个敏感的检测 mitophagy 活动使用流式细胞仪。

视频: Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM:免疫沉淀，流式细胞仪检测

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

科研

生物学

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

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

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

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

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

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

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

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

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

对于外小泡的使用流式细胞仪分析技术

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

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

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

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

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

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

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

流式细胞仪检测盘基柄柄Macropinocytosis 的高通量

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