Name: molecular probe optimization to determine cell mortality in a photosynthetic organism (microcystis aeruginosa) using flow cytometry
Uploaded: 2016-01-29T13:00:00.000+00:00
Duration: 10 min 16 s
Description: Watch this Scientific Journal Video about Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry at JoVE.com

作者要感谢博士生戴维哈特内尔和伯恩茅斯大学的支持和资助的研究和设施。

1.准备分子探针和流式细胞仪<ol><li>稀释储备溶液的核酸探针，它们被提供在二甲基亚砜（DMSO）5mM溶液至所需浓度的等分试样的超纯过滤的 H 2 O. </li><li>存储核酸探针在-5℃，与黑暗的条件- 25°C，直到使用。 </li><li>打开流式细胞仪和负载软件包（见表材料/设备的FCM规格）。 </li><li>放置在进样探针（SIP）的空溶血管（12×75毫米），点击疏通，然后再冲水启动FCM清洗过程。 注 :为SIP一些样品台可容纳多种类型的管子，包括离心管中。在FCM装置中使用的分子探针稀释剂和鞘流体是从分析级"1型"0.22μm滤膜过滤源。 </li><li>地点15分钟和流体速度快（或&gt; 66微升/分钟的流速） -一个新鲜溶血管与2ml超纯过滤H 2 O的的SIP上，为10设定时间限制。 </li><li>选择一个新的数据单元，把相关阈值，荧光和光散射渠道，降低背景噪音，然后单击"运行"。 </li><li>如果每秒的总事件不是低于制造商的建议，然后运行2ml样品的去污溶液对快2分钟，然后重复步骤1.4＆1.5。 注 :请检查制造商对FCM建议启动清洁的协议，因为他们可以型号而有所不同。测试对于特定阈值也需要在与FCM模型线作为一些设备允许用户申请的电压增益，以光电倍增管（PMT）增强或减少从光检测器记录的电信号。在这个experi使用的FCM模型换货有固定电压光电倍增管和使用80,000前向光散射（FSC-H）的阈值以排除颗粒小于2.0微米和电子噪音。 </li></ol> 2.准备工作文化与初始细胞计数<ol><li>高压釜98毫升超纯过滤的 H 2 O和2 ml的藻媒体（50倍 ​​浓度）在250ml烧杯中20分钟的在120℃。 </li><li>从微囊藻 （PCC 7806）的在一个高稳定状态密度地点2毫升样品的初始单一到SIP下的管。分解任何集落形成涡旋或超声处理20并通过光学显微镜确认均匀分散细胞。 注 :过度暴露于超声波可导致细胞裂解，应谨慎使用。因为超声波可以折叠气囊产出，如侧向光散射（如物种，如微囊藻中）（SSC-H）的强度可以变得更SEnsitive。 </li><li>内将FCM软件，选择直方图来记录从正向光散射（FSC-H）数据，并通过点击"登录"，以查看在轴线对数标尺配置情节规格。 </li><li>在一个单独的输出，选择另一个直方图（登录轴）来记录自然荧光，如附件光合色素藻蓝蛋白（FL4-H，675±12.5纳米），在微囊藻找到。 </li><li>使用能够激发藻蓝蛋白和一个检测器，它可以从所得荧光过滤排放的光源。 注 :光合色素如叶绿素可以兴奋与常用的488纳米的蓝色激光，而藻蓝蛋白是奋达600纳米，只会有一个红色的光源21进行检测。检查与流式细胞仪的制造商为激励光源和发射检测器的光谱，这里既一个488纳米，640纳米的激光被连同用于一个675±12.5 nm的光学滤波器。 </li><li>用于记录的最接近目标生物体（10微米）和相对慢的流速（14微升/分钟）的芯尺寸的最高分辨率选择设置。 注:最佳分辨率样品应按照制造商每秒事件的建议下运行。 </li><li>采集数据前使用门槛，门出光散射和/或荧光信号是由电子背景噪音或细胞样品碎片造成的。 </li><li>选择一个新的数据单元，创建具有FSC-H和SSC-H参数对数标度的密度的情节，然后点击运行。 </li><li>如果样品密度导致了过度的事件发生率，可以采取稀释步骤来增加准确度和精度。 </li><li>在密度图应用软件门从以前的直方图排除低电平散射信号，通过背景噪声或碎屑（FSC &lt;320000）生产的。反之门更高的相对荧光从细胞中藻蓝蛋白的信号，只包括这些事件（FL4，56000 - 1,950,000）。 </li><li>使用记录在门控区的细胞的数量和通过样品的总体积是通过将FCM已通过将其划分，制定出每毫升多少细胞。 注 :这里使用的FCM模式有一个微处理器控制的蠕动泵系统，允许被确定样本量。其他FCM设备可能需要校准珠暂停或计算的H 2 O的重量/体积的不同来验证总样本量。 </li><li>细胞的需要的体积添加到新鲜制备的介质，以便开始微囊藻的一个批次生长周期（250,000细胞/ ml）。 注 :种将不同生长率取决于它们的原位环境参数和养分有效性，所以一个批量周期应预先记录，以确定生命周期阶段。 </li></ol> 3.的Optim的分子探针细胞摄取化<ol><li>在微囊藻培养收获一半的指数相准备在步骤2中作为一个"活"的控制使用。 注 :样品由高密度培养稀释直奔指数期可以通过死细胞的营业额影响优化的结果，相比于文化从最初的滞后/诱导阶段接种的。 </li><li>制备的另一半通过使用方法如70％的乙醇，加热的样品在 60℃1小时，多聚甲醛或4％甲醛30分钟6,11,22一个"死"的控制。检查中的变化样品微环境（如，pH值）。 注 :正，热灭活，&#39;死&#39;在微囊藻的控制是通过其在藻蓝蛋白的信号下降一"活"样本区分。通过其它方法诱导的死亡率可能不会导致相同的输出和将VARŸ物种。 </li><li>设置为使用不同比例的混合样品"活"和"死"的样本（例如，0％，25％，50％，100％）。 </li><li>分列集落形成涡旋或超声检查pH值。 </li><li>选择488nm的激光沿着探测器可从绿色记录荧光（FL1，530±15纳米），橙色（FL2，585±20毫微米）的核酸探针和640纳米的激光通过其相应的检测器来记录藻蓝蛋白的信号。 注意:当与DNA结合，绿色核酸探针具有504 nm，发射523纳米的最大值近似的荧光激发波长，而橙色核酸探针具有547 nm和排放为570nm极大值的激发波长。甲的488nm氩离子的固态激光器可被用于激发两个分子探针，然而，一个绿色激光（高达547纳米）将产生一个较高的橙色荧光。 </li><li>作为一个起点，介绍分子探针与厂家推荐使用浓度为50％的"活"，50％"死"的文化和孵化在黑暗中。 </li><li>选择一个新的数据单元，将与阈值和触发器，这将降低背景噪音（FSC-H 80000）的SIP下的样本。 </li><li>创建具有FSC-H和SSC-H的参数和三个直方图密度的情节。一个直方图使用各自的分子探针光检测通道（FL1或2），一个用于检测排放藻蓝蛋白（FL4-H）和其它的FSC-H，所有对数尺度。 </li><li>孵育微囊藻与在黑暗的核酸探针的样品达60分钟，记录在单独的数据单元，在多个时间点（1，5，10，15，30和60分钟）。 注 :当调整参数，如pH检查与生产的某些化学品的潜在反应（用于测试核酸探针的缓冲不能含有磷酸盐或高单价或女主角的价阳离子，作为与DNA结合将减少）。 </li><li>应用软件门仅包括FSC-H直方图（320,000 - 1500000）为靶标生物细胞的大小，进入各自的荧光探针通道直方图。 注意:在这个协议中所用的浓度分别为0.05，0.1，0.5，1，5，10，50和100微米，这可以通过增加或减少微囊藻样品或核酸探针的初始储备液的体积可以改变。 </li><li>在荧光探针信道时，使用另一包容软件门到直方图中的最高峰（​​绿色FL1-H，240000 - 1650000，橙FL2-H，30000 - 165000），并随后栅极阳性探针荧光入密度图。 </li><li>运行步骤3.1 - 3.11 100％"活"，100％"死"和所有的混合培养样品，调整分子探针浓度（例如:X为0.1〜×10）和/或温度和pH水平如果必要的。 </li> &lt;利&gt;比较的阳性分子探针的荧光信号的数量为"死"细胞的原始细胞密度（从半总FSC-H或100％的"死"培养采取）找到细胞染色核酸的总百分比探头。 </li><li>每个浓度范围内做到这一点的每个时间段找到细胞核酸探针的吸收比例最高的最优协议不产生非特异性染色（使用方法在单因素方差分析和方差的秩和检验单因素分析，如果数据是非参数）。 </li></ol> 4.分子探针荧光歧视<ol><li>对于荧光干扰/重叠从固有的或不特定的细胞染色测试选择50％"现场"和50％的"死"混合培养数据和起飞所有闸门。 </li><li>应用软件门仅包括FSC-H直方图（320,000 - 1500000）为靶标生物细胞的大小，成藻蓝蛋白通道直方图。 </li><li>门最高的藻蓝蛋白峰和标签为"现场"，增加标记为&#39;死&#39;的最低峰。 </li><li>使用一次，在&#39;活&#39;，然后&#39;死&#39;藻蓝蛋白（FL4）信号，记录两者的平均波长执行另外的门一步进入各自的分子探针荧光直方图通道。 注 :该模式用于在这个实验中引起的死亡率是通过热处理，此协议下，显然降低了藻蓝蛋白信号完成。 &#39;死&#39;细胞产生群体的其它方法可能会产生不同的输出中获得的运行。 </li><li>比正分子探针荧光（"死"）和本征/非特异性信号（"现场"）的平均波长，以确定协议灵敏度。 注 :为提高"活"与"死"波波尔荧光歧视ations，增加碳源以活性防污摄取营养物耗尽的电池或乙二胺四乙酸（EDTA），以提高细胞壁的渗透性和产生的荧光信号6。在一些FCM模型光谱重叠有限的补偿可通过用户操纵成对校正期间样品采集（PMT电压的变化）或从分析后专门的软件来执行。 </li><li>选择优化的协议，其中已被染色的最高量的"死"细胞不发生非特异性染色。如果一个数测试的有类似的结果接受协议与最低浓度和温育时间，以及良好的荧光信号鉴别。按照制造商的流式细胞仪关机程序的指令。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+method+for+the+assessment+of+the+minimal+inhibitory+concentrations+of+antibacterial+agents+to+Mycoplasma+agalactiae.">Flow cytometric method for the assessment of the minimal inhibitory concentrations of antibacterial agents to Mycoplasma agalactiae.</a> Cytom Part A. 69, 1071-1076 (2006).</li><li>Davey, H. M., Kell, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometry+and+Cell+Sorting+of+Heterogeneous+Microbial+Populations:+The+Importance+of+Single-Cell+Analyses.">Flow Cytometry and Cell Sorting of Heterogeneous Microbial Populations: The Importance of Single-Cell Analyses.</a> Microbiol. rev. 60 (4), 641-696 (1996).</li><li>Franklin, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explaining+the+Causes+of+Cell+Death+in+Cyanobacteria:+What+Role+for+Asymmetric+Division?.">Explaining the Causes of Cell Death in Cyanobacteria: What Role for Asymmetric Division?.</a> J. Plankton Res. 36 (1), 11-17 (2013).</li><li>Kaprelyants, A. S., Mukamolova, G. V., Davey, H. M., Kell, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+the+Physiological+Heterogeneity+within+Starved+Cultures+of+Micrococcus+luteus.+by+Flow+Cytometry+and+Cell+Sorting.">Quantitative Analysis of the Physiological Heterogeneity within Starved Cultures of Micrococcus luteus. by Flow Cytometry and Cell Sorting.</a> Appl. Environ. Microbiol. 62 (4), 1311-1316 (1996).</li><li>Waggoner, A., Melamed, M. R., Lindmo, T., Mendelsohn, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+probes+for+cytometry.">Fluorescent probes for cytometry.</a> Flow cytometry and sorting. , 209-225 (1990).</li><li>Gray, J. V., Petsko, G. A., Johnston, G. C., Ringe, D., Singer, R. A., Werner-washburne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#34;Sleeping+Beauty&#34;:+Quiescence+in+Saccharomyces+cerevisiae.">&#34;Sleeping Beauty&#34;: Quiescence in Saccharomyces cerevisiae.</a> Microbiol. Mol. Biol. Rev. 68 (2), 187-206 (2004).</li><li>Keer, J. T., Birch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Methods+for+the+Assessment+of+Bacterial+Viability.">Molecular Methods for the Assessment of Bacterial Viability.</a> J. Microbiol. Methods. 53 (2), 175-183 (2003).</li></ol>

作者宣称，他们有没有竞争的财务权益。

使用分子探针的出版物的数目增加表明可靠和信息数据可以得到5,6,8 - 15,19,22,23。作为然而没有完美的染色细胞存活，可以是有效的在所有物种具有相同的浓度和温育时间6,10。即使是同一类型的探针具有改变的荧光发射的表示需要建立正确的浓度和温育时间（ 表1和3）。如使用此协议时看到的那样，最佳浓度和温育时间为橙色核酸探针是在1μM的10分钟，而绿色核酸探针将只需要一半的浓度，但不对三倍长（ 图5和6）。这两种细胞非透性花青单体与浓度大于1.0微米的开始，以产生在排放光谱与非膜损伤的细胞的重叠。 SI的这种重叠gnals提供的证据表明非特异性染色时，其中"活"细胞被占用的分子探针。在从非特异性染色所得超过荧光产生误报，强调必须仔细针对每个分子探针与靶生物确定，必须对协议的发展。在优化的分子探针的最重要的步骤是:1）如何理解分子探针进行交互与FCM仪器; 2）采用合适的活的和死的控制; 3）从发展FCM输出的门控参数的知识和4）通过现场环境的理解修改的协议。 的光源，滤光器，以改变流式细胞仪内的PMT电压和检测器的能力而变化，从一家生产厂商，所以下的发光光谱在于相对于荧光通道什么激发波长从激光和其中的认识是至关重要的。随着收购的数据，阈值可以征收增加的决议，否则将失去也许是背景噪音，碎片或死亡的细胞聚集。分子探针可以提供深入了解物种的生存力无培养，通过单一参数，例如，膜电位，DNA含量和酶活性。然而，环境样品中可以存在类似的细胞的大小和荧光的许多生物。此外uniagal培养内，细胞群将不可避免地具有一定程度的生理状态3，这可能影响分子探针摄取。一个FCM的主要优点是它的区分，并在实时的个体细胞水平，研究当原位微生物生存能力 ，因为它们通常经受快速和动态条件，这是不可或缺表征生理状态的能力。尽管它的频繁使用，细胞活力的概念仍然难以确定。通常用于细胞增殖，可行性是非常打进一个基础的增长方式。这托底假设可能功亏一篑，因为条件可能不适合单个细胞复制，但他们仍可能容忍的。在非增殖的生理状态的细胞已经进入一个静止或G（0）细胞周期阶段27可能无法使用分子探针提供DNA的循环或代谢基于探针漏报互动。 使用实验室生长或新鲜分离细胞，以优化分子探针将有助于生态研究中所用的数据的精炼。从分批培养在优化中使用的细胞通常采取指数或早期固定相并假定具有最高的存活率。然而，格外小心，必须使用高细胞密度，那些在后期稳定期或微生物产生的细胞外基质时，应考虑。在这个协议中对细胞的死人口控制暴露于60℃加热1小时，带确认的膜损伤和颜料降解通过显微镜看到的（ 图4）和 FCM荧光输出（ 图2和3）。模拟自然的细胞死亡的原因是极其困难的;摄取食草动物，病毒裂解，程序性细胞死亡或不对称分裂24的可能性并不总是可行或在实验室条件下观察到。道德的实验模式经常受到质疑，因为它们可能不等同于从环境中引起的应力（例如，热杀灭），并有可能是多相反应，其中某些个体死亡，其他细胞显示没有可观察的细胞退化或死亡3 25。为了避免这种矛盾由测量的实验，必须明确规定，以免引起混淆6,23，宽松的研究与标准化的操作定义。 各种替代方法甲肝Ë被用来分析细胞活力培养与分子探针结合。荧光显微镜是用传统的技术，尽管漂白或从附近的细胞中引入假象信号可能会给出一个低于或超过人口，如果没有记录足够快的生理状态的估计。这使得通过优化成像非常困难的，具有潜在的精度低。细胞活力的同时使用DNA和RNA扩增方法，如基因组的评估;聚合酶链反应（PCR），逆转录酶（RT-PCR）和核酸序列为基础的扩增（NASBA）也已经使用。然而，基因组的评估仅充当生存能力验证的间接方法，核酸的持久性在很大程度上取决于环境条件，与实际细胞活力的相关潜在变化非常28。通过自然的照片使用的光散射和/或荧光组合ynthetic颜料（ 图1）门的人群可以的:数据提高分辨率，找出稀有人群的反应，降低假阳性结果。 FCM沿分子探针突出从先前提到的技术在测试单细胞生理其速度，精度和记录的多个参数。 FCM是在水生微生物学，并与加成分子探针的一个强大的分析工具具有在多个领域，包括，单细胞和社区分析，以工业部门（例如，监测饮用水供应）宽电位。未来的发展可以看到，FCM将荧光原位杂交（FISH），它可以检测和定位在密集的异类样本中的个别细胞的特异性基因序列。 FCM和分子探针可移植性的用于原位录音的发展可以提供关于水源的重要数据来实现S早期trategies资源管理。下面这里开发的优化协议，FCM和分子探针可以潜在在提供有关微生物生理学在水生环境有价值的数据的关键作用。

细胞是一个复杂的系统，它不断地通过修改的生理参数和改变其功能响应环境。无论是在自然和实验室等基因微生物种群的种群动态是由亚群的发展受到影响，即使在相对 ​​恒定的环境条件发生1 - 3。天然微生物群落的变异性的出现是由于环境条件的高度可变的性质。这些有时随机过程随后产生亚群是非常不同的人群的平均水平。最近的证据表明，这些亚群生理反应不同的环境条件，并且能够产生信号的化合物或抑制剂显着影响，并影响整体人口3,4。 建立以群体内定义的异质性的方法，关键是未derstanding在各种环境中的微生物的生态和建设时滋扰蓝藻知识，如有毒的微囊 ，从而影响严重人类水的安全性。物种如鱼腥响应于环境波动显示形态异质性，开发专门细胞如异形是必不可少和akinetes 2。与此相反， 微囊细胞不期间应激反应显示明显的形态的异质性。可行的和非存活细胞之间的区别是生理分化的最重要的方面，并且允许更好地理解微生物种群动态的。然而，细菌存活本身的概念性的问题仍然是困难和特征1,5,6很差。 流式细胞术（FCM）是分析单个细胞的可靠和快速的方法。为了提高单细胞生理的理解通过FCM迟缓，分子探针已被用于区分一个号码代谢和生化过程7。这导致了物种对细胞和群体水平增加的知识和反过来帮助水资源管理8,9。然而，在生物分子探针摄取和排出方面由于孔隙和泵在蜂窝壁和细胞膜，这都导致了许多分子探针设计和协议实现6,10,11不同。可用于商业和研究用途分子探针通常与一个通用的协议，该协议可以是适用于非常不同的细胞类型提供。我们必须在传输的一种细胞类型开发协议， 另外 6非常谨慎，因此它是一个重要的任务，使用前有效地优化分子探针。 绿色和橙色核酸探针结合于两个双和单链核酸与最小化的基本选择ivity和用于评估细胞质膜完整性。绿色核酸探针具有显着改善的细胞标记的荧光信号相对于其它分子探针，如碘化丙啶-基化合物12，它也可以作为细胞活力的指标。术语"细胞活力"这里假定DNA降解的细胞膜完整性的丧失后出现。所述核酸探针是不对称花青染料具有三个正电荷和不能进入细胞胎膜完整特征的浓度下，在这两种真核11,13和14,15原核生物有机体。的核酸探针与核酸的结合可导致多达从内源性信号荧光发射中的细胞，具有其膜完整性破坏一个&gt; 500倍增加。虽然分子探针如绿色核酸探针可以是单细胞生理学的良好指标，有必要吨Ø优化每个探头的预定目标生物体，如孵化时间从7分钟变化-在微囊藻实验中0.5微米单独15 - - 30分钟，浓度为0.1μM19。 在这里，我们提出了一个协议，以优化绿色的流式细胞检测和比较新的橙核酸探针（其至今尚未测试的蓝藻种类微囊藻 ）。下面发达方法然后可以转移到其他物种和用作其它分子探针优化协议，从而增加微生物的理解及其生态行为的平台。

<table><tbody><tr><td>Cyanobacteria Media</td><td>Sigma-Aldrich</td><td>C3061-500ML</td><td>BG-11 Freshwater concentrated solution (x50 dilution)</td></tr><tr><td>Decontamination Fluid</td><td>BD Biosciences</td><td>653155</td><td>Run for 2 min when outputs are more than 12 events per second on fast or a flow rate of 66 &amp;micro;l/min.&amp;nbsp; Followed by 2 min of sheath H&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Flow Cytometer</td><td>BD Biosciences</td><td>by request</td><td>BD Accuri C6</td></tr><tr><td>SYTOX Green</td><td>Life Technologies</td><td>S7020</td><td>Nucleic acid stain &amp;ndash; 5 mM solution in DMSO</td></tr><tr><td>SYTOX Orange</td><td>Life Technologies</td><td>S11368</td><td>Nucleic acid stain &amp;ndash; 5 mM solution in DMSO</td></tr></tbody></table>

molecular probe optimization to determine cell mortality in a photosynthetic organism (microcystis aeruginosa) using flow cytometry

在野外和实验室研究微生物亚群已经示出，以显示高的异质性的形态和生理参数。确定微生物细胞的实时状态超出活的或死的类型，如微生物可以处于休眠状态，由此细胞的分裂和代谢活性被降低存在。鉴于需要检测和微生物的定量，流式细胞术（FCM）用分子探针提供了一种快速，准确的方法，以帮助确定总体人口生存能力。通过使用SYTOX绿色和橙色SYTOX模型中的蓝藻铜绿微囊藻检测细胞膜的完整性，我们开发了单细胞死亡率迅速指示的转移方法。在该刊物中使用的分子探针将被分别称为绿色或橙色核酸探针（虽然也有其他的产品与具有共同行动的一个可比较的方式类似激发和发射波长N，我们专门请参考前面提到的探针）。利用分子探针协议物种之间有所不同，在浓度和温育时间不同的主要。根据这一协议，载于微囊藻绿色核酸探针孵育30分钟，并在1μM橙色核酸探针10分钟后，经过优化的0.5μM的浓度。在这两个探测器的浓度低于规定的优化导致了在报告细胞的细胞膜破坏。相反地​​，5μM的浓度和较高的两种探针表现出型非特异性染色的，由此"活的"细胞产生的目标的荧光，导致"非活"的细胞数目过表示。阳性对照（热灭活）提供的可检验的死亡生物量，虽然在控制一代的适当性还有待讨论。通过展示步骤的逻辑顺序进行优化绿色和橙色核酸探针，我们去monstrate如何创建，可以用来有效地分析蓝藻生理状态的协议。

Watch this Scientific Journal Video about Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry at JoVE.com

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

微生物群含有大量细胞的异质性，它可以决定整体行为。通过流式细胞分子探针分析可以决定细胞的生理状态，然而它的应用物种之间变化。本研究提供了一个协议，以精确地确定一个蓝藻群体内的细胞死亡，而不低估或记录假阳性结果。

分子探针的优化，以确定细胞死亡的光合生物（<em&gt;铜绿微囊藻</em&gt;）使用流式细胞仪

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

molecular-probe-optimization-to-determine-cell-mortality

Universidad San Sebastian

Research

JoVE Journal

Medicine

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

8.9K 次观看.  Bournemouth University. 微生物群含有大量细胞的异质性，它可以决定整体行为。通过流式细胞分子探针分析可以决定细胞的生理状态，然而它的应用物种之间变化。本研究提供了一个协议，以精确地确定一个蓝藻群体内的细胞死亡，而不低估或记录假阳性结果。 

视频: Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

科研

免疫与感染

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

流式细胞仪对水生生物膜的表征

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

环境科学

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

微生物动力学的特征-基于流式细胞仪的工作流从纯文化到自然群落

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-&#946;-Gal). 
Typically, the SA-&#946;-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-&#946;-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-&#946;-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.
Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

远红荧光衰老相关β-半乳糖苷酶探针，用于通过流式细胞术鉴定和富集衰老肿瘤细胞

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...

癌症研究

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing need for the development of methodologies that improve the detection of antigens immobilized on the surface of spores together with their quantification. Flow cytometry-based analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of B. subtilis. Herein, we propose the use of flow cytometry to evaluate the display efficiency of a fluorescent antibody (FA) on the surface of the spore and quantify the number of spores using counting beads. 
For this, we used ethidium bromide as a DNA marker and an allophycocyanin (APC)-labeled antibody, which was coupled to the spores, as a surface marker. The quantification of spores was performed using counting beads since this technique demonstrates high accuracy in the detection of cells. The labeled spores were analyzed using a flow cytometer, which confirmed the coupling. As a result, it was demonstrated that DNA labeling improved the accuracy of quantification by flow cytometry, for the detection of germinated spores. It was observed that ethidium bromide was not able to label dormant spores; however, this technique provides a more precise determination of the number of spores with fluorescent protein coupled to their surface, thus helping in the development of studies that focus on the use of spores as a biotechnological platform in different applications.

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry

This protocol focuses on the use of flow cytometry and counting beads to quantify bacterial spores labeled with ethidium bromide. The method is also efficient for analyzing the covalent coupling of proteins on the surface of intact spores.

枯草芽孢杆菌孢子计数和标记分析在流式细胞术中的改进

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing ...

生物化学

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代谢标记和流式细胞仪测定细胞周期进程的动力学

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

生物学

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

使用荧光抗生素探针对细菌耐药性的可视化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

使用2'，7'-二氯荧光素双乙酸探针和流式细胞术在穆勒神经胶质细胞中定量活性氧

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

神经科学

Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis

Source: McBride, J. E., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54779">Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood.</a> J. Vis. Exp. (2017).
This video demonstrates the lytic activity of natural killer cells against fluorescently-stained target cells. The method enables NK cell-induced cytotoxicity measurement by staining dead cells and using a flow cytometer to quantify live and dead target cells.

分子探针的优化，以确定细胞死亡的光合生物（

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