RK 和 RRG 得到了 UNM 生物和医学金属中心 （CMBM） 通过 NIH NIGMS 赠款 P20 GM130422 的资助。RRG 得到了 NM-INSPIRES P30 赠款 1P30ES032755 的试点奖励的支持。CX7 Cellomics 仪器的成像核心支持是通过 NIH 资助的 AIM 中心核心提供的P20GM121176。我们要感谢 Sharina Desai 博士和 Li Chen 博士在与使用 CX7 Cellomics 成像平台相关的技术问题上提供的宝贵帮助。

1. 细胞培养
<ol><li>将测试细胞（HepG2、HUH7 和 JHH4 肝细胞癌细胞）以 10,000 个细胞/孔的接种密度接种到 96 孔板中，最终接种体积为每孔 200 μL。<ol><li>在培养HepG 2细胞之前，在室温（RT）下用IV型胶原（50μg/ mL）涂覆96个板孔2小时。为避免原液凝固，将原液放入冰中，然后开始稀释过程至所需浓度。</li>
		<li>孵育期2小时后，吸出多余的胶原蛋白，并用1x PBS洗涤孔三次。</li>
	</ol></li>
	<li>在37°C和5%CO2 浓度下，在Dulbecco改良的Eagle培养基（DMEM）中培养细胞过夜。</li>
	<li>第二天，或达到80%-90%汇合度时，以较早者为准，分别用H2O2（未处理，250μM，500μM，750μM和1000μM）和甲萘醌（未处理，25μM，50μM，75μM，100μM）处理细胞30分钟以诱导代表性氧化应激。</li>
	<li>或者，选择使用能够在细胞内诱导氧化应激的所需测试物质来测试细胞。</li>
</ol>2. 细胞DHE染色的储备和稀释溶液制备
<ol><li>通过将 5 mg DHE 溶解到 1 mL DMSO 中来制备 DHE 储备溶液（5 mg/mL 或 15.9 mM）。</li>
	<li>用双蒸压水稀释DHE储备溶液至最终浓度为100μM。</li>
	<li>为了避免染料的冻融过程（随着时间的推移可能会破坏荧光特性），在浓度为100μM的1.5mL离心管中制备几种等分试样，并将其储存在-20°C。</li>
	<li>为了达到10μM的最终工作浓度，使用100μMDHE浓度的等分试样，并用预热的DMEM培养基稀释。 注意：工作溶液必须在实验当天新鲜制备。</li>
	<li>将工作荧光染料溶液涡旋5-10秒，以确保染料的适当混合。</li>
</ol>3.DHE染色工艺
<ol><li>从每个孔中取出含有药物或测试物质的培养基，并用 1x PBS 或 DMEM 轻轻洗涤一次。 注意：在实验中执行添加或洗涤步骤时，避免细胞与移液器之间的直接接触至关重要。这可以通过沿着孔的侧面轻轻移液 PBS 来实现，以尽量减少对细胞的任何潜在机械损伤。确保移液器不直接接触细胞将有助于在实验期间保持细胞的完整性和活力。</li>
	<li>向每个孔中加入100μL含有10μM DHE（工作溶液）的DMEM培养基，并在37°C孵育30分钟。</li>
	<li>孵育期30分钟后，除去含DHE的培养基，并用1x磷酸盐缓冲盐水（PBS）轻轻洗涤每个孔三次。这可以使用多通道移液器进行，以确保快速周转。</li>
	<li>在室温下向每个孔中加入 200 μL 1x PBS，用 Hoechst 33342 核染色染料 10 分钟。</li>
	<li>从孔中取出 Hoechst 33342 核染色溶液，并向每个孔中加入 200 μL 1x PBS。 注意：要获得固定细胞，请遵循与活细胞相同的方案，在处理和DHE染色后加入4%PFA5分钟。除去PFA溶液，用1x PBS洗涤细胞。然后，将细胞与核染色染料孵育10分钟。</li>
	<li>将板移至高内涵筛选平台、荧光显微镜或酶标仪，以尽快进行图像采集。</li>
</ol>4. 图像采集和强度测量
<ol><li>板加载<ol><li>打开 Cellomics CX7 高内涵筛选 （HCS） 软件进行数据采集。</li>
		<li>根据制造商的规格，使用特定的 96 孔板提前仔细校准成像平台，以避免数据中出现任何朦胧或模糊的图像。 注意：来自不同供应商的多孔板可能具有不同的材料特性，并可能影响获得的最终图像的质量。</li>
		<li>校准后，将特定于板品牌的系统参数保存在仪器数据库中，并在将来的数据采集中重复使用。</li>
		<li>小心地将装有制备样品的板放在HCS成像系统的加热台上。确保孔板位置牢固且方向正确，以便在微孔板支架上成像（即，在读板载物台上找到标有 A1 的标签，然后旋转微孔板，使孔 A1 的位置与贴纸的角相匹配）。</li>
		<li>轻轻按压微孔板，确保其平放在载物台上。HCS 系统中板的不均匀调平会导致图像采集错误。</li>
		<li>板就位后，按住 Ctrl 键，然后在软件的“板加载/卸载”对话框中单击 Ctrl-OK 。</li>
	</ol></li>
	<li>选择成像参数<ol><li>在HCS成像系统中，在Cellomics生物应用界面中打开 靶标激活 模式，并使用与（a）核染色和（b）DHE荧光探针数据采集兼容的双通道设置方案创建新方案。在当前方案中，过滤器 386_BGSRS_BGSRS、480_BGS_RS 和 386_BGS_RS 分别用于 （a） Hoechst 33342 核染色、（b） 总 ROS 产生和 （c） 超氧自由基检测。这些过滤器的选择是由作为该协议一部分的每种染料（DAPI和DHE）的发射特性的特定重叠驱动的。 注：滤光片的命名约定，例如386-23_BGRFRN_BGRFRN，是指用带宽为23nm的386 nm光激发样品，然后是二向色滤光片，该滤光片在特定波长范围内通过蓝色（B），绿色（G），红色（R），远红外（FR）和近红外（N）光，386_BGS_RS是指在二向色性之后通过BGS和RS发射波长光的发射滤光片。</li>
		<li>根据需要，在软件协议界面中指定图像采集过程的目标，例如 10 倍或 20 倍放大倍率。</li>
		<li>根据所需的成像细节水平和实验所需的分辨率选择合适的物镜放大倍率。但是，每个目标的分辨率都是固定的。更高分辨率的细节将需要改变平台上的目标。在当前协议中，使用20x，0.45 NA物镜，这是该协议中使用的高内涵筛选平台的一部分。 注意：20倍物镜代表了成像分辨率细节和捕获ROS随时间变化所需的采集速度之间的良好权衡。可以选择更高放大倍率的物镜（例如，40倍），但是，这将导致采集时间增加。</li>
		<li>根据协议的具体要求，指定图像采集相机的曝光设置，如曝光时间、相机像素合并、z-stack间隔等。 注意：曝光时间（通常以毫秒为单位）根据所用特定荧光团的信号强度和灵敏度而变化。根据染料浓度和染色质量，这也可能因实验而异。在当前协议中，采集参数固定如下 - 目标百分比 - 35%，图像采集模式 - 1104 x 1104（2x2 像素合并），物镜 20x，0.45 NA。这些参数可以根据具体的实验条件进行设置。</li>
	</ol></li>
	<li>映像配置参数 注意： 设置成像参数后，可以使用软件界面启动图像采集过程。<ol><li>图像采集参数<ol><li>使用仪器中可用的不同算法（光学 - 等值数据、固定和三角形）识别感兴趣的主要跟踪对象（细胞核）。 注意：本协议中使用用于识别和标记主要物体的等值数据方法。</li>
			<li>按形状或强度参数对对象（如果有）进行分割。</li>
			<li>根据每种细胞类型特有的所需大小、形状和强度参数验证主对象。</li>
			<li>然后，定义此主要对象周围的“感兴趣区域”（ROI）。 注意：ROI是数据采集的一个关键参数，它定义了荧光染料强度被鉴定为在不同细胞类型中一致且可重复的位置。ROI 定义了关键参数（例如染料的强度），这些参数是针对仪器不同通道中的每个主要跟踪对象（即细胞）测量的。ROI可以以围绕每个细胞核的圆圈或环的形式定义。HCS 软件在通道 2 中自动获取 DHE 荧光染料标记物的强度，该强度在以自动方式分割和分析的每个细胞的 ROI 范围内。</li>
			<li>在主要物体（原子核）周围设置一个 20 μm 的圆圈。本研究根据本方案中使用的各种细胞系（HepG2、JHH-4 和 HUH-7）的近似大小选择了 20 μm 的距离。细胞周围的 20 μm 环 ROI 以一致且可重复的方式获得荧光强度。 注意 选择 ROI 的关键参数是充分覆盖单元区域的能力;圆形 ROI 的大小取决于单元格大小。较大的单元可能需要更大的投资回报率，反之亦然，投资回报率较低。这些参数必须提前确定每种细胞类型和感兴趣的荧光染料。</li>
		</ol></li>
	</ol></li>
	<li>种群特征和选择要存储的要素<ol><li>在 HCS 成像协议中定义各种采集参数后，将 HCS 系统设置为数据采集模式。</li>
		<li>该协议的图像采集参数设置如下：扫描限制为 1000 个细胞/孔，每个区域至少要求 10 个物体（细胞）。 注意：每孔评估的字段数是根据达到 1000 个的最终细胞计数确定的。HCS系统继续搜索孔内的各个位置，直到获得1000个细胞/孔的目标群体。因此，采集速度取决于 96 孔板的每个孔中的汇合度和总细胞数。在目前的方案中，从每个孔中收集通道2的总强度和平均强度（代表超氧自由基和总ROS产量），用于进一步的下游分析。</li>
	</ol></li>
	<li>图像捕获、处理和分析<ol><li>调整并最终确定图像采集参数后，启动每个 96 孔板的扫描过程。 注：Cellomics CX7 成像系统能够对各种参数的样品进行高内涵筛选和自动分析，包括以高通量方式在细胞内产生 ROS。除了 ROS 生产外，HCS 系统还能够提供有关各种参数的额外有价值的信息，例如细胞形态、亚细胞定位和许多其他感兴趣的细胞特征。一旦针对采集进行了优化，HCS 成像平台就可以以稳健的方式对细胞参数进行全面、定性和定量表征。</li>
		<li>扫描过程完成后，启动 视图分析 软件并以.csv格式导出各种定量数据参数以进行其他分析。</li>
		<li>使用第三方软件，复制并粘贴各种感兴趣的定量值，以便进行其他下游分析。 注意：在当前协议中，GraphPad Prism软件用于分析DHE强度值，以响应由于H2O2 和甲萘醌暴露引起的氧化应激。</li>
	</ol></li>
	<li>数据和统计分析<ol><li>计算通道 2 的平均平均强度（代表总 ROS 值和超氧自由基）。 注意：所有计算强度值的实验重复三次，每个条件在每个剂量水平重复 6 次。</li>
		<li>执行单因素方差分析或 t 检验，以测量各种测试条件之间强度值的差异。</li>
	</ol></li>
</ol>

在肝癌细胞中使用二氢乙锭进行新型 ROS 定量

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作者声明他们没有竞争利益。

在这项研究中，在高内涵筛选系统上建立了使用二氢乙锭 （DHE） 荧光染料评估超氧化物驱动的细胞内活性氧 （ROS） 产生的方案。文献中现有的大多数当前方案都使用DCFH-DA作为荧光成像探针来量化ROS物种。然而，多项研究表明，DCFH-DA并不是测量细胞内ROS的理想探针。DCFH-DA不适合作为探针的各种原因包括：（i）DCFH-DA不与H2O2发生直接反应，这使其成为评估细胞内H2O2</s...

活性氧 （ROS） 是一组天然存在的、高反应性的、时间不稳定的化学自由基，是细胞正常细胞代谢的一部分。ROS 在调节细胞中发生的正常生理和生化过程中起着关键和必不可少的作用 1,2。细胞中ROS产生的主要来源是线粒体电子传递链（ETC）途径，作为正常生物能量循环的一部分。ROS产生的重要其他来源包括酶促反应，例如细胞中的细胞NADPH氧化酶。食物分子（例如葡萄糖）的代谢通过线粒体基质中的氧化磷酸化途径发生。ROS产生的基线水平对于调节正常的生理细胞信号传导过程至关重要。已知许多属于葡萄糖代谢信号通路的关键蛋白质分子（例如 Akt 和 PTEN）对细胞内 ROS 水平有反应。此外，ROS 由各种细胞内酶产生，例如黄嘌呤氧化酶、一氧化氮合酶和过氧化物酶体成分，作为细胞酶途径的一部分 1,2。与活性氧的天然来源相比，某些环境因素，如外源性物质、传染性病原体、紫外线、污染、吸烟和辐射，也会导致活性氧的过量产生，而活性氧是细胞内氧化应激的关键驱动因素1,3。细胞氧化应激升高可对细胞内的天然生物分子（如脂质、蛋白质和 DNA）造成损害，导致各种疾病，如癌症、糖尿病、心血管疾病、慢性炎症和神经退行性疾病 1,3,4。因此，ROS的准确测量对于了解氧化应激诱导的疾病病理生理学所涉及的细胞机制至关重要。
由于细胞内 ROS 产生和消除的时间很短，因此对各种 ROS 自由基的定量测量仍然是一个挑战。电子顺磁共振 （EPR）5、高压液相色谱 （HPLC） 和基于荧光探针的成像等方法用于测量各种细胞 ROS6。虽然 EPR 和 HPLC 等方法可产生定量准确的估计，但这些方法涉及细胞空间形态的破坏，并且通常采用样品的全局和批量测量形式。相比之下，基于成像的方法（例如基于荧光探针的方法）保留了ROS生成的细胞形态和空间背景。然而，各种荧光探针对不同类型 ROS 自由基的特异性尚未得到充分证实 7,8。几种荧光探针，如二氢乙铵 （DHE）、二氯二氢荧光素二乙酸酯 （DCFH-DA）、二氢罗丹明 （DHR）、二甲基蒽 （DMA）、2,7 二氯二氢荧光素 （DCFH）、1,3-二苯基苯并呋喃 （DPBF） 和 MitoSox 可用于商业 ROS 检测。在过去的几十年中，DHE、MitoSox 和 DCFH-DA 是测量细胞和组织中 ROS 的常用荧光染料 8,9。DCFH-DA 是一种广泛使用的染料，用于检测细胞内 H、2、O2 和氧化应激。尽管 DCFH-DA 很受欢迎，但之前的多项研究表明，它不能可靠地用于测量细胞内 H2O2 和其他 ROS 水平8、9、10、11、12、13、14。
相反，荧光探针二氢乙锾 （DHE） 显示出对细胞内超氧自由基 （O2-） 的特异性反应。虽然超氧自由基是在细胞中观察到的许多 ROS 物质之一，但它是一种重要的自由基，参与过渡金属的还原、转化为过氧硝酸盐和氢过氧化物的形成，以及其他细胞内效应。DHE迅速被细胞吸收，并在红色波长范围15内具有荧光发射。在与超氧自由基发生特异性反应时，DHE形成红色荧光产物2-羟基乙锭（2-OH-E+）。因此，DHE可以被认为是用于超氧化物检测的特异性探针。然而，DHE也可以与ONOO-或OH.、H2O 2和细胞色素c进行非特异性氧化，形成第二种荧光产物乙锭E+，其可以干扰测量的2-OH-E+水平。然而，当用 DHE 染色时，这些 2-OH E+ 和 E+ 产物组合在一起，代表了细胞内观察到的总细胞 ROS 物质的主要部分。E+ 插入 DNA，大大增强其荧光8、9、10、11、13、14、15、16。由于乙锭和2-羟基乙锭的荧光光谱仅略有不同，因此可以使用DHE荧光产物检测和测量继发于超氧化物产生的细胞中观察到的大多数ROS水平。使用 480 nm 波长激发和 610 nm 波长发射 15,16,17 鉴定这些 ROS 物质。
除了选择特定的荧光ROS检测探针外，选择一种灵敏的检测方法来测量细胞内ROS也很重要。因此，准确评估细胞内 ROS 水平是识别患病细胞或暴露于各种环境应激源（如辐射、毒理化合物和遗传毒性物质）的细胞中发生的氧化还原平衡状态紊乱的关键18。由于 ROS 是细胞中常见的现象，负责调节各种细胞信号传导活动，因此可靠的 ROS 检测方法至关重要。为了能够对细胞内的ROS产生进行这种高通量评估，该协议使用高内涵筛选（HCS）平台来测量ROS种类。目前的方案允许对细胞内ROS的产生进行高通量分析，这在许多毒理学研究中至关重要19。该协议旨在提供一种简单且通用的解决方案来检测和测量贴壁性肝细胞癌细胞中的细胞内ROS。H2O2 和甲萘醌的化学试剂用作有效的 ROS 刺激剂，以测量受控和高通量环境中 ROS 产生的相对水平。根据需要，可以对该方案进行微调，以在适当条件下测量贴壁和非贴壁细胞中ROS的产生。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>1.5 mL centrifuge tubes&#160;</td>
			<td>VWR&#160;</td>
			<td>20170-038&#160;</td>
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			<td>96- well plate&#160;</td>
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			<td>07-200-90&#160;</td>
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			<td>Collagen&#160;</td>
			<td>Advanced Biomatrix&#160;&#160;</td>
			<td>5056&#160;</td>
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			<td>DHE (Dihydroethidium)&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>D1168&#160;</td>
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			<td>DMEM&#160;</td>
			<td>Sigma&#160;&#160;</td>
			<td>6046&#160;</td>
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			<td>FBS&#160;</td>
			<td>VWR&#160;</td>
			<td>97068-085&#160;</td>
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			<td>GraphPad Prism</td>
			<td>GraphPad</td>
			<td>Version 6.0</td>
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			<td>HepG2 cell line</td>
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			<td>Hoechst&#160;</td>
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			<td>33342&#160;</td>
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			<td>HUH7 cell line</td>
			<td>ATCC</td>
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		<tr>
			<td>Hydrogen Peroxide&#160;</td>
			<td>Sigma&#160;</td>
			<td>88597&#160;</td>
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			<td>JHH4 cell line</td>
			<td>ATCC</td>
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			<td>Menadione&#160;</td>
			<td>Sigma&#160;</td>
			<td>M5625&#160;</td>
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</table>

high throughput screening assessment of reactive oxygen species (ros) generation using dihydroethidium (dhe) fluorescence dye

活性氧（ROS）在生理和病理过程中调节细胞代谢中起着关键作用。生理 ROS 的产生在正常细胞功能（如增殖、信号传导、细胞凋亡和衰老）的空间和时间调节中起着核心作用。相比之下，慢性 ROS 过量生产会导致多种疾病，例如癌症、心血管疾病和糖尿病等。因此，以准确和可重复的方式量化ROS水平对于了解正常的细胞功能至关重要。基于荧光成像的方法来表征细胞内 ROS 物种是一种常用的方法。文献中的许多成像 ROS 方案使用 2'-7'-二氯二氢荧光素二乙酸酯 （DCFH-DA） 染料。然而，这种染料在使用和可解释性方面存在重大局限性。目前的实验方案演示了使用二氢乙铓（DHE）荧光探针作为在高通量环境中量化ROS总产量的替代方法。高通量成像平台CX7 Cellomics用于测量和量化ROS的产生。这项研究是在三种肝细胞癌细胞系 HepG2、JHH4 和 HUH-7 中进行的。该协议深入描述了评估细胞内ROS所涉及的各种程序，包括- DHE溶液的制备，用DHE溶液孵育细胞以及表征ROS生产所需的DHE强度的测量。该方案表明，DHE荧光染料是一种稳健且可重复的选择，可以以高通量方式表征细胞内ROS的产生。用于测量 ROS 产生的高通量方法可能有助于各种研究，例如毒理学、药物筛选和癌症生物学。

二氢乙铵 （DHE） 是一种超氧化物响应荧光染料，可提供有关细胞内 ROS 状态的特定信息。DHE染料本质上在细胞质中发出蓝色荧光。然而，在与超氧自由基相互作用时，它被转化为 2-羟基乙鎓，其发出红色波长 （>550 nm） 的荧光（图 1）。DHE染料很容易转运到细胞和细胞核中。发射的荧光可以通过许多实验室中常见的荧光显微镜装置进行可视化。在本研究中，在高内涵筛选平?...

Watch this Scientific Journal Video about High Throughput Screening Assessment of Reactive Oxygen Species ROS Generation using Dihydroethidium DHE Fluorescence Dye at JoVE.com

作者聚焦：肝细胞系中细胞内 ROS 水平的高通量测量

该方案描述了一种使用二氢乙铵（DHE）作为荧光染料探针，使用高通量筛选方法定量细胞内活性氧（ROS）的新方法。该方案描述了三种不同肝细胞癌细胞系中细胞内活性氧（ROS）的定量评估方法。

使用二氢乙锓 （DHE） 荧光染料生成活性氧 （ROS） 的高通量筛选评估

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS ...

我们的实验室研究环境污染物（如重金属）对肝功能和与活性氧生产相关的疾病的影响。该协议旨在使用二氢乙锓荧光探针定量测量细胞中的ROS水平。该协议使用高内涵筛选平台定量测量肝细胞系中的细胞内ROS物种水平。在高通量毒理学研究中，准确测量细胞间ROS水平至关重要，这是我们实验室关注的一个关键领域。DHE荧光是一种高度特异性的测量总ROS水平的方法，特别是针对超氧自由基的方法，与其他常用的ROS荧光探针相比，具有显着优势。此外，该协议还提供了在高通量平台中测量总细胞ROS水平的独特能力。我们的方案是表征细胞内总ROS产生的稳健且可重复的选择。测量 ROS 水平的高通量方法有助于各种研究，例如毒理学、药物筛选和癌症生物学，这将是我们未来的重点。

high-throughput-screening-assessment-reactive-oxygen-species-ros

Research

JoVE Journal

Bioengineering

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

3.7K 次观看.  University of New Mexico. 我们的实验室研究环境污染物（如重金属）对肝功能和与活性氧生产相关的疾病的影响。该协议旨在使用二氢乙锓荧光探针定量测量细胞中的ROS水平。该协议使用高内涵筛选平台定量测量肝细胞系中的细胞内ROS物种水平。在高通量毒理学研究中，准确测量细胞间ROS水平至关重要，这是我们实验室关注的一个关键领域。DHE荧光是一种高度特异性的测量总ROS水平的方法，特?...

视频: 作者聚焦：肝细胞系中细胞内 ROS 水平的高通量测量

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

基于细胞中钙含量TRP通道功能的高通量筛选，使用FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

科研

生物工程

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

白色念珠菌生物膜芯片（CA BChip）为高通量的抗真菌药物筛选

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat and indistinguishable with white light imaging alone. As a result, a modality that highlights glandular architecture would be useful to discriminate neoplasia from benign epithelium in the distal esophagus. VFI is a novel technique that uses an exogenous topical fluorescent contrast agent to delineate high grade dysplasia and cancer from benign epithelium. Specifically, the fluorescent images provide spatial resolution of 50 to 100 &#956;m&#160;and a field of view up to 2.5 cm,&#160;allowing endoscopists to visualize glandular morphology. Upon excitation, classic Barrett&#8217;s metaplasia appears as continuous, evenly-spaced glands and an overall homogenous morphology; in contrast, neoplastic tissue appears crowded with complete obliteration of the glandular framework. Here we provide an overview of the instrumentation and enumerate the protocol of this new technique. While VFI affords a gastroenterologist with the glandular architecture of suspicious tissue, cellular dysplasia cannot be resolved with this modality. As such, one cannot morphologically distinguish Barrett&#8217;s metaplasia from BE with Low-Grade Dysplasia via this imaging modality. By trading off a decrease in resolution with a greater field of view, this imaging system can be used at the very least as a red-flag imaging device to target and biopsy suspicious lesions; yet, if the accuracy measures are promising, VFI may become the standard imaging technique for the diagnosis of neoplasia (defined as either high grade dysplasia or cancer) in the distal esophagus.

Vital-dye enhanced fluorescence imaging (VFI) is a novel in vivo technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal esophagus.

用生命染增强荧光成像瘤在Barrett食管诊断

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

阴离子氧化铈纳米粒子催化清除体内植物活性氧的研究

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

使用微图案弹性体对单细胞收缩力进行简化的高通量分析

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...

使用二氢乙锓 （DHE） 荧光染料生成活性氧 （ROS） 的高通量筛选评估

使用二氢乙锓（DHE）荧光染料生成活性氧（ROS）的高通量筛选评估