Name: molecular probe optimization to determine cell mortality in a photosynthetic organism (microcystis aeruginosa) using flow cytometry
Uploaded: 2016-01-29T13:00:00
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。...

<ol>
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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?...

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 border="0" cellpadding="0" cellspacing="0" width="926">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:168px;">Cyanobacteria Media</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:123px;">C3061-500ML</td>
			<td style="width:501px;">BG-11 Freshwater concentrated solution (x50 dilution)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Decontamination Fluid</td>
			<td>BD Biosciences</td>
			<td>653155</td>
			<td>Run for 2 mins when outputs are more than 12 events per second on fast or a flow rate of 66 &#181;L/min.&#160; Followed by 2mins of sheath H2O.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>N/A (by request)</td>
			<td>BD Accuri C6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYTOX Green</td>
			<td>Life Technologies</td>
			<td>S7020</td>
			<td>Nucleic acid stain &#8211; 5mM solution in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYTOX Orange</td>
			<td>Life Technologies</td>
			<td>S11368</td>
			<td>Nucleic acid stain &#8211; 5mM 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。

正向光散射（FSC）和侧向光散射（SSC）的输出从指数生长期的微囊藻分批培养提供了分别对细胞尺寸（直径）和内部粒度信息（图1A）。 FSC可以区分细胞太大和/或小到可以微囊藻 。这个判别或选通可以由FSC输出（图1C）的某些点之间精制数据来完成。藻蓝蛋白， 微囊藻光合装置的主要结构产生强信号时由红色光源询问（例如?...

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. ...

Research

JoVE Journal

Medicine

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

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

制备髓源性抑制细胞从幼稚和胰腺癌荷瘤小鼠，用流式细胞仪和自动磁性细胞分选（AutoMACS）（MDSC）

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

科研

医学

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

目标移植的肌肉祖细胞的分子成像

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

半自动适应循环肿瘤细胞（CTC）分析临床和临床前研究中的应用

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

注重形成：一个基于Cell的检测，以确定一个基因的致癌性

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

单元位置的Imaging-和流式细胞仪为基础的分析和3D球体黑色素瘤细胞周期

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

Electrophysiological Measurement of Noxious-evoked Brain Activity in Neonates Using a Flat-tip Probe Coupled to Electroencephalography

应用平板探针结合脑电图对新生儿有害诱发脑活动的电生理测量

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. ...