1。样品制备<ol><li>与稀释剂ORFLO或适当的稀释剂稀释的细胞悬液，使细胞浓度的仪器（3000至50万细胞/ mL的M型磁带，或3000至2,500,000细胞/毫升为S型磁带）的经营范围内。 </li><li>一个1:5的稀释至1:20（M型磁带）或稀释1:5稀释（S型磁带）建议对于大多数哺乳动物细胞系，但适当的稀释，将取决于细胞类型和播种密度。一个准确的计数所需的体积约75微升。 </li></ol> 2。运行样品<ol><li>关闭末席Z在按电源按钮和主屏幕将显示。 </li><li>按托盘和磁带插入末席Z。 吸取75μL样品...屏幕将显示。 </li><li>吸取75μL样品到磁带填充口（蓝色椭圆标有1或2，根据录像带插入仪器）。这些都是一次性的录音带，它可以使用2测试。注：将直接进入负荷枪头〜45°角，使针尖被捕前唇下的井。配药时，重要的是，真空不“出人头地”的流体点胶，可能导致气泡进入录像带。据建议，在配药了良好的流体形式的小珠（条目以及大小）。 </li><li>对于大多数哺乳动物细胞计数，触摸屏幕上的任何地方开始。计数非常小的颗粒（4至8微米，平均直径为M型和S型的平均直径3-8微米），磨溪Z可以在小颗粒模式下运行。注：纸盒类型显示在屏幕上方的黑条。在这种模式下，没戏ž设置作为默认的2至10微米直径的规模和执行的计数ü唱小细胞检测参数优化。按这里的触摸小颗粒模式按钮（黑方）以启动测试，在这种模式下运行。 </li><li>磨溪Z将开始测试和细胞计数结果将在大约8秒（M型磁带）或15秒（S型磁带）的完整。 </li><li>曲线拟合和MVI计算自动开始，只需要一些额外秒。当找到一个合适的拟合，结果将自动显示在屏幕上。 </li><li>为了使浇注默认的采集模式，按曲线拟合计数按钮，切换到选通模式。 </li><li>在这一点上，磁带可以从单元中删除。 </li></ol> 3。管理数据<ol><li>如果自动模式缩放，结果初步显示为一个直径2-34微米尺度（M型磁带）或2-26μ曲线拟合计数M（S型磁带）如果自动模式缩放，然后的末席ž将自动调整水平轴（X轴）的范围，同时确保所有曲线拟合数据显示最接近的曲线拟合人口的宽度。 </li><li>如果自动模式缩放，以手动方式显示在一个更高的分辨率结果，触摸的2-26微米的图标（M型）的2-18微米的图标（S型）注：录像带类型显示在屏幕左上方的蓝色方块。数较小的细胞或颗粒改善曲线拟合功能以及用户的视觉区分数据的细微差别的能力时，建议</li><li>在提高水平分辨率，缩放按钮，将动态调整，以便循环之间现有的X轴的刻度范围：2-34，2-26，2-18，和2-10微米的M型盒式;或2 -26，2-18，2-10微米的S型盒式。此功能只availaBLE紧随细胞计数。 </li><li>根据直方图的左侧，显示计数信息曲线拟合或选通区域（细胞/ ml）在一个黑盒子。平均细胞大小的信息显示，在根据直方图右侧的黑盒子。 </li><li> MVI的值显示在直方图的右上角的蓝色框。如果小颗粒模式运行测试，MVI的不适用，将显示“MVI的SPM的”。此外，如果细胞的浓度是MVI的经营范围，“MVI的=超出范围”的消息之外，将显示。 </li><li>要保存当前的直方图，触摸完成的图标。这与“测试XXX”的，其中XXX是对应的测试号的名称保存数据。 </li><li>手动门直方图，触摸切换曲线拟合计数结果“按钮。这使得浇注默认的采集模式。然后将每个蓝色的门标记设置闸门或挖掘日é精细调整门的左，右箭头（这些出现在触摸蓝色的门标记）标记之间的细胞计数。基于曲线拟合的人口幅度直方图的垂直比例尺自动缩放。要调整这个垂直刻度，轻扫屏幕或直方图地区的。 </li><li>轻触门控计数结果“按钮返回显示模式曲线拟合和曲线拟合模式默认的收购模式。 </li><li>然后按“ 完成 ”图标，将结果保存并返回到主屏幕。要删除的结果，在任何时候按下“ 删除 ”图标，永久删除的测试结果。 </li></ol> 4。以前获得的数据分析<ol><li>打开一个保存的测试，按主屏幕上的直方图图标</li><li> 9保存直方图图标将显示在屏幕上。按相应的图标，利益的考验，或按向上或向下图标页面 ，以查看更多的测试结果。 </li><li>将打开每个直方图曲线拟合计数模式或门控计数模式，这取决于它是如何保存的。 在门控计数模式，浇注标记可以定位为独立滑动每个蓝色的门标记所需的自动门所期望的峰值接触。轻触门控计数和曲线拟合计数模式之间切换。 </li><li>按变焦和Unzoom的图标来调整直方图的垂直刻度。垂直刻度也可以改变垂直滑动手指上显示最多（增加）或向下（减少）。 </li><li>按“ 删除 ”图标永久删除的测试结果。 </li><li>按完成的图标关闭测试结果，并返回到主屏幕。 （注意：缩放视图将不会被保存。） </li></ol> 5。将数据传输到PC <ol><li>数据可以传输到PC使用Orflo Moxichart应用程序，或通过USB传输末席žUSB电缆插入PC或Mac。蓝牙传输（MoxiChart）按的末席z单位的主屏幕上的蓝牙图标，首先要确定单位的蓝牙ID。 </li><li>在计算机上，打开MoxiChart的应用程序，点击蓝牙图标在右上角。然后，选择对应到末席ž蓝牙ID的设备，并选择一个上传文件的目标文件夹。 </li><li> moxichart会自动转移通过蓝牙连接到指定的目录中的所有文件。 </li><li>通过蓝牙（MoxiChart）或USB传输的文件可以打开和分析直接与MoxiChart应用捷思锐，或，因为他们被格式化。csv文件，他们可以直接在Microsoft Excel或其他数据分析程序打开加/自定义分析。 </li></ol> 6。代表结果评估末席ž的精度和准确性，HEK-293细胞，HeLa细胞的CHO-K1，酵母的Jurkat E6-1细胞，PC12细胞，计数精度校准珠悬浮物浓度从0-500,000细胞/毫升（M型磁带）和0-2-2.5E 6细胞/ ml（S型磁带）计算和比较使用库尔特Z2的末席Z。所有哺乳动物细胞获得来自美国菌种保藏中心（ATCC）和培养如前所述7-11。简单地说，悬浮在75毫米2组织核心媒体的RPMI 1640（的Jurkat E6-1的PC12），MEM（的HEK-293，HeLa细胞）细胞，或F12K（的CHO-K1）细胞培养瓶细胞培养。所有的媒体，辅以10％胎牛血清和100的üpenicillin/100微克链霉素。瓶均保持在37℃，5％CO2培养箱中。细胞传代，每3天。酵母细胞（X5的， 白色念珠菌 ，VIN 13）的捐赠，并立即使用的规定。初始样品浓度〜50万细胞/毫升为M型盒式数据和1〜2 2.5E 6个细胞/毫升的S型磁带建立了使用库尔特Z2的。随后的理论浓度，准备通过系列稀释生理缓冲区（Isoton II或汉克的平衡盐溶液）。相同的样品，每个浓度测定两个系统，并绘制互相对抗。在图2可以看出，M型，有很强的线性相关（R 2&gt; 0.9886，S型R 2&gt; 0.9781）之间的所有样品的计数。浓度也比预期/理论没戏Z和“Z2的细胞浓度（ <strONG&gt;图3），再次表现出强大的线性关系（M型R 2&gt; 0.9758，S型R 2&gt; 0.9774）。 评估精度，HEK-293和HeLa细胞计数进行使用库尔特Z2的，没戏ž，1血细胞计数。平均变异系数（CV，N = 19）使用的末席z（M型磁带），库尔特Z2的1血球细胞计数，计算与CV的测量在200,000-300,000细胞/毫升范围从5单独计数。末席ž小的平均CV（ 图4）是可比的Z2的，是基于库尔特计数技术的精度只有达到较高水平的代表。 上浆的末席ž仪器的精度下，通过购买，精密校准的聚苯乙烯珠的测量评估。针对制造商报告的大小，平均粒径测量末席ž（N = 5）绘制。可以看出，我n 图5，末席ž测量一贯匹配的制造商值具有较高的线性相关（R 2 = 0.9989）。 大小和数量的信息之外，没戏Z提供试剂，哺乳动物细胞培养健康的总体评估报告末席活力指数（MVI）的价值。该指数是从一个细胞在利益方面的总颗粒计数的人口数的比例产生。为了证明MVI的测量能力，MVI的读数（M型磁带）标准手册和流式细胞仪现场/死测量技术的比较。通过隔夜孵化的活细胞死亡（&lt;10％可行）HEK-293和HeLa细胞的人口创造了营养，钠含稀释剂（Isoton二，贝克曼）氟在37°C。控制理论的可行性称为ratiometrically混合活的和死细胞的浓度水平。 RESulting解决方案进行了分析，一式两份，每份用血球计数和流式细胞仪测量的可行性水平。血球计数仪测量了使用传统的50/50 0.1％台盼蓝溶液和细胞的混合物。使用番石榴主成分分析仪（默克）使用Viacount试剂和制造商指定的协议（默克公司），流式细胞仪测量了。 如图6所示，Pearson相关系数的值（R 2）“MVI的数据类似血球活力百分比的线性拟合”的HEK细胞和Hela细胞的R 2 = 0.9937和r 2 = 0.9728，分别为。这些结果表明，这种方法提供了跨文化的细胞存活率媲美的活/死利用流式细胞仪或血球计数仪测量范围广泛的卫生信息。 表和图（从Dittami 等人 ，2011）15： 图1。薄膜库尔特计数：通过细胞薄膜盒式传感区的电流通过。作为细胞流经的CSZ的单个文件，测量电压瞬间增加末席Z。 <img alt="图2" src="/files/ftp_upload/3842/3842fig2.jpg" /> 图2末席的ž计数库尔特Z2的数量相媲美。细胞和珠悬浮相同罪名被同时使用库尔特Z2和末席Z.的计算平均计数值的末席ž计数的（N = 3-4）方面的绘制科尔特相应Z2的计数和R 2的线性拟合值进行了测定。一）M型卡式 - 浓度范围为0 - 50万细胞/毫升。乙）S型磁带-浓度范围为0 - 2 2.5E 6 / ml.Error酒吧表明±1个标准差。 图3。没戏的ž浓度测量亦或理论的细胞浓度。准备）M型卡式​​-理论浓度值从最初的50万细胞/毫升样品连续稀释）S型盒式磁带- 2.5E 6细胞/ ml样品-理论浓度值从最初的2系列稀释的准备。错误横道±1个标准差。 <img alt="图4" src="/files/ftp_upload/3842/3842fig4.jpg" /> 图4。库尔特Z2的末席z（M型磁带），和血球的变异系数。每种方法的HEK-293和HeLa细胞计数的平均变异系数（CV，N = 19）分别计算从5个独立的测量简历HEK-293和HeLa细胞在20万 - 30万细胞/ mL范围内的罪名。错误横道±1个标准差。 图5。使用的Z-末席ž 末席粒度测量精度测量粒径（M型卡式 ​​）与制造商报告珠大小。错误横道±1个标准差。 <img alt="图6" src="/files/ftp_upload/3842/3842fig6.jpg" /> 图6。文化使用MVI的健康评估- MVI的测量（M型卡式 ​​），与视觉，台盼蓝染色的可行性，使用血球计数番石榴PCA Viacount可行性比较。

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORFLO+Moxi+Z:+Revolutionizing+electronic+cell+count+accuracy+and+cell+viability+estimates+through+curve+fitting.">ORFLO Moxi Z: Revolutionizing electronic cell count accuracy and cell viability estimates through curve fitting.</a> Life Science Magazine, VWR International. , (2011).</li></ol>

员工，该公司设计，制造和销售的末席的Z.理查德·D·Rabbitt提供兼职，有偿咨询的专业知识，Orflo技术Orflo技术格雷戈里米Dittami何Ayliffe。 主办Orflo技术生产和自由地进入本文。

底层实现库尔特方法可以是高度确定性的整体测量精度。一个重要领域是“巧合事件”在两个或两个以上的细胞，同时通过光圈和电气作为一个单一的事件测量的原始细胞计数校正。 “巧合事件”作出贡献的错误而异，取决于许多因素，包括细胞的大小和集群程度（Davis等，1967）6。因此，对于任何给定的样本所需的巧合校正不能预测，广泛应用于细胞/颗粒类型之间，甚至相同的细胞类型的不同文化之间的不同。建立理论，扎根在最初的出版物由库尔特，涉及申请数调整的基础上，观察到的事件和经验确定的，粒子的特定的修正系数，Z的原始计数。虽然准确的计数，可以实现与T他的校正算法，它被限制在它的实用性，由于细胞类型和相应的困难，准确地预测其价值之间的z值大的变化。在这里，使用的“真实”的细胞计数没戏Z的巧合校正算法结合曲线拟合确定，没戏ž动态纠正巧合，以实现真正的浓度。当使用高端库尔特Z2的得到的结果相比，没戏ž可比计数跨越浓度范围为0 - 50万细胞/毫升（M型磁带）和3000 - 2 2.5E 6细胞/ ml（S型盒式） 。此外，没戏ž类似计数的变异系数低，是金标准，库尔特细胞计数技术的特点和颗粒尺寸精度达到这个计数的准确性。 此外，这里给出的数据表明，在末席Z可以提供有价值的信息关于细胞培养的整体健康。末席ž利用曲线拟合的方法，与专有软件算法，以确定这种文化的健康评估。与细胞死亡相关的形态学变化，如出泡，分裂，体积扭曲（冈和Tsuruo的1996，Liegler等1995年，谢里登等人，1981年）12-14，有可能使末席-Z的之 ​​间区分impedimetric差异居住人口和死细胞/碎片群。 MVI的是所产生的文化/粒度分布分析，以确定粒子碎片的相对贡献，坏死细胞萎缩，曲线拟合感兴趣的细胞群。 MVI的价值，从而反映了人口指数的整体粒子的人口方面的单分散的人口数或比例的措施。然后这个值是基于算法调整人口统计数字和经验性观测。 becaus电子MVI的参数比传统的染色方法，它不预期，以反映这些技术，而是提供了一个健康的细胞培养成有价值的另一种观点，特别是碎片和微生物污染方面，。 总之，没戏-Z的细胞计数器是一个超小型台式仪器，提供准确，重复性检测细胞计数，细胞大小，细胞的健康使用拥有专利的薄膜传感器技术相结合的库尔特原理和曲线拟合软件算法。随着M型盒，它是能够计数的粒子，包括从4 - 8秒 - 25微米（直径34微米的动态范围，2）。与S型纸盒计数从3不等的颗粒的能力 - 在15秒 - 20微米（直径26微米的动态范围2）。此外，没戏-Z的健康评估，没有使用的试剂。这少得多的劳动集成nsive和主观比手工计数。在标准的库尔特计数相比，磨溪速度较快，明显较小，提供更多的信息，需要相当少的维护，更容易使用，是一小部分的成本。

<table border="1" ><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> ORFLO稀释剂</td><td> ORFLO </td><td> MXA006 </td><td></td></tr><tr><td>卡式饮水机</td><td> ORFLO </td><td></td><td>最多可存储25卡带，为方便配药</td></tr><tr><td> USB电缆</td><td> ORFLO </td><td></td><td>仪器连接到PC / Mac或电源适配器</td></tr><tr><td>电源适配器（仅限于美国和欧盟的机型） </td><td> ORFLO </td><td></td><td> USB电缆连接到交流电源插座</td></tr><tr><td> USB闪存驱动器</td><td> ORFLO </td><td></td><td>商店莫克斯我Z软件和用户手册。 </td></tr><tr><td>校准检查珠</td><td> ORFLO </td><td></td><td></td></tr><tr><td>电子校准盒式</td><td> ORFLO </td><td></td><td>电子核查系统的正常运行和校准录像带</td></tr></tbody></table>

determination of mammalian cell counts, cell size and cell health using the moxi z mini automated cell counter

用于各种应用，包括常规的细胞培养，血液分析，工业控制1-5粒子和细胞计数。细胞/颗粒计数技术的一个重要突破是的科尔特技术的发展华莱士科尔特超过50年前。该技术涉及电场跨微米大小的孔径的应用和流体力学重点是通过光圈的单粒子。颗粒闭塞的光圈导致产生可测量的变化中可以直接，准确地与细胞尺寸/体积的电阻抗。识别的方法在细胞/颗粒计数基准源于非凡的精度，其颗粒大小和计数的准确性，特别是相比，手册和成像技术（98％与75库尔特计数器精度 - 手册和基于视觉系统的80％）。这可以归结为事实，库尔特技术使成像为基础的细胞计数的方法不同，计算精确的容量信息，一个真正的立体（三维）细胞/颗粒测量极大地减少了计数的干扰，从碎片和聚类有关的细胞/颗粒。总体而言，这提供了一个列举和大小细胞在更准确，更繁琐，耗时少，少主观手段比其他计算技术6。 尽管库尔特细胞计数技术突出，在例行的生物学研究已经广泛使用的成本和规模的传统乐器由于望而却步。虽然更便宜的的库尔特基于仪器已制作完成，它具有比其更昂贵的同行在校正“巧合事件”在两个或两个以上的细胞通过光圈和同时测量的局限性。 AnotheR与现有的库尔特技术的限制是缺乏对整体健康的细胞样本的指标。因此，必须经常被用于其他技术结合库尔特计数来评估细胞活力。这延伸自可行性评估的传统方法需要细胞染色和/或使用昂贵而笨重的设备，如流式细胞仪实验的设置时间和成本。 末席ž迷你自动细胞计数器，这里描述的，是一种超小型台式仪器，用薄膜传感器技术相结合的库尔特原理的准确性，使精确的范围从3-25微米的颗粒大小和计数，这取决于用细胞计数卡带。 M型盒式可以用来计数颗粒平均直径4 - 25微米（动态范围2 - 34微米），S型的卡带可以用来计算与粒子和平均直径为3 - 20微米（动态响E 2 - 26微米）。由于该系统采用了体积的测量方法，在4-25微米，相当于34个细胞体积范围 - 8,180 FL和3 - 20微米，相当于14细胞体积范围 - 4200 FL，这是有关非球面粒子被测量。使用末席ž执行哺乳动物细胞计数，计算细胞稀释与ORFLO或类似的稀释剂。一个细胞计数磁带插入仪器，样品装载到磁带端口。成千上万的细胞被拉到一个“细胞传感区”（财税字），单文件，在8-15秒薄膜膜。运行之后，仪器的使用与专有软件算法的专有随着整体文化的健康评估，确定利益的人口数量的细胞总数的比例提供巧合事件校正曲线拟合粒子。含总粒子计数é萎缩，打破死细胞，以及其他碎片和污染物。结果是在直方图的格式，可以根据需要手动调整的大门，自动曲线拟合。 最终，没戏ž使计数到库尔特Z2的，目前的黄金标准相比具有精度和准确性，同时提供更多的文化健康信息。此外，它在更短的时间内实现这些结果，与更小的体积，显着更容易操作和维护，并在一小部分的成本可比技术。

Watch this Scientific Journal Video about Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter at JoVE.com

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

末席ž微型自动细胞计数器是一种新型的仪器，结合专利的薄膜传感器技术和专有软件算法进行上浆和一个广泛的尺寸范围的颗粒计数以及确定的单分散的整体健康库尔特原理哺乳动物细胞培养。该协议，介绍了该仪器的使用计数和评估健康的细胞培养。

测定哺乳动物细胞计数，细胞的大小和使用末席ž迷你自动细胞计数细胞的健康

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

determination-mammalian-cell-counts-cell-size-cell-health-using-moxi

Research

JoVE Journal

Biology

24.5K 次观看.  Orflo Technologies. 末席ž微型自动细胞计数器是一种新型的仪器，结合专利的薄膜传感器技术和专有软件算法进行上浆和一个广泛的尺寸范围的颗粒计数以及确定的单分散的整体健康库尔特原理哺乳动物细胞培养。该协议，介绍了该仪器的使用计数和评估健康的细胞培养。

视频: Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

N -连接糖链的糖蛋白在哺乳动物细胞中的脉冲大通分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

确定在细胞培养的细胞数量，使用权杖细胞计数

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

钙调蛋白结合蛋白的下拉

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&amp;#945; Promoter Using Gibson Assembly

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

质粒载体产生表达FLAG标记的蛋白质在人延伸因子1α启动子的使用吉布森大会规则

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt;锌指域

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...