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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I., Rengefors, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Control+of+HAB+Species+using+Low+Biosurfactant+Concentrations.">On the Control of HAB Species using Low Biosurfactant Concentrations.</a> Harmful Algae. 8 (6), 857-863 (2009).</li><li>Bouchard, J. N., Purdie, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Elevated+Temperature,+Darkness,+and+Hydrogen+Peroxide+Treatment+on+Oxidative+Stress+and+Cell+Death+in+the+Bloom-Forming+Toxic+Cyanobacterium+Microcystis.">Effect of Elevated Temperature, Darkness, and Hydrogen Peroxide Treatment on Oxidative Stress and Cell Death in the Bloom-Forming Toxic Cyanobacterium Microcystis.</a> J. Phycol. 47 (6), 1316-1325 (2011).</li><li>Reynolds, C. S., Jaworski, G. H. 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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?...

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. ...

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

Research

JoVE Journal

Medicine

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

8.8K 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

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

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

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

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

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

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

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

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

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

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

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

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

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

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

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 ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

一种改进<em&gt;在体外</em&gt;入侵检测，以确定激素，在调节肿瘤细胞侵袭的细胞因子和/或生长因子的潜在作用

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

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

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

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

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

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

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D 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

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. Those tools are subjective or determine secondary physiological indicators which also have certain limitations particularly when exploring the effectiveness of analgesia. As cortical processing is essential for pain perception, brain activity measures may provide a useful approach to assess pain in infants. Here we present a method to assess nociception with electrophysiological brain activity recordings optimized for the use in newborn infants. To produce highly standardized and reproducible noxious stimuli we applied mechanical stimulation with a flat-tip probe, e.g., PinPrick, which is not skin-breaking and does not cause behavioral distress. The noxious-evoked potential allows the objective measurement of nociception in non-verbal patients. This method can be used in newborn infants as early as 34 weeks of gestational age. Moreover, it could be applied in different situations such as measuring the efficacy of analgesic or anesthetic drugs.

Measuring pain in non-verbal patients is a challenge. In this study we combine EEG recording with stimulation using a flat-tip probe to detect noxious-evoked brain activity in an objective manner.

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. ...