We would like to thank Dr Hamish Campbell and Prof Antony Braithwaite for their help in developing the transduced MCF-7-LeGO cell lines. We would like to acknowledge our funding support by the Bloomfield Group Foundation through the Hunter Medical Research Institute. B.C.M is supported by an APA scholarship through the University of Newcastle and the MM Sawyer Scholarship through the Hunter Medical Research Institute.

1.准备用于细胞增殖检测注:准备以相同的方式和种子中的两种细胞系在相同的格式对于要分析的每个方法。 <ol><li>使用生长的MCF-7-LEGO和MCF-7-Δ40p537细胞至75-80％汇合T中75cm 2的组织培养瓶无酚红的Dulbecco改良Eagle培养基（DMEM）中补充有10％胎牛血清（FBS）的，200 2mM L-谷氨酰胺，2微克/毫升的胰岛素和1μg/ ml的嘌呤霉素，在37℃，5％的CO 2。处理细胞II无菌生物安全柜类。 注意:需要的时间的细胞生长至汇合的量取决于播种稀释而变化。在用于电镀的细胞的增殖测定的准备，种子的细胞以1:3稀释在补充的DMEM，并在正常生长条件保持3天，直至75-80％汇合。 </li><li>从烧瓶中除去细胞，媒体倒出成一个废物容器。立即用2ml预热2×胰蛋白酶的洗细胞层。吸胰蛋白酶。在37℃的培养箱中添加进一步2毫升预温热胰蛋白酶的细胞层和地点的5分钟，用5％ 的 CO 2。 <ol><li>一旦细胞脱离，洗涤细胞，用10ml温热新鲜补充的DMEM。在RT细胞悬液转移到无菌的15毫升管和离心491 xg离心5分钟。小心吸取上清液处置。 </li></ol></li><li>重悬细胞沉淀在5毫升新鲜的DMEM加味的。进行细胞计数，以确定正确的密度播种的细胞在96孔板中。 <ol><li>稀释100微升在900微升1×Dulbecco氏磷酸盐缓冲盐水（DPBS）中的细胞悬浮液。将新的60微米传感器到自动细胞计数。按住柱塞和淹没在稀释的细胞悬浮液中的传感器。慢慢地松开细胞计数柱塞。从细胞凑取出传感器当完成NTER。 注:细胞计数，将显示在细胞/ ml的细胞计数器。 </li></ol></li><li>种子的细胞以在约20％汇合100微升（在DMEM）在一个96孔板的最终体积，以允许室超过3-5天（ 见表1）的增殖的细胞生长和测量。种子一式三份所有细胞系。 注意:在这个实验中，三个不同的细胞密度进行了评估，以确定最佳的细胞密度。所需的细胞数以较低的密度概括于表1中 。种子细胞，如果需要更长期的增长的测量。 <ol><li>对于血球和基于发光的测定，每准备时间点的一个板块（ 即每次检测4板）。对细胞成像测定法中，在实验的持续时间仅准备一个板。 </li></ol></li><li>维持细胞在37℃，5％CO 2的24小时。 </li></ol> 2.确定细胞计数我们ING血球<ol><li>在细胞培养培养箱预暖既介质和胰蛋白酶至37℃，烘箱或水浴中。从细胞中吸出培养基到废物容器中，用2×胰蛋白酶的30微升洗一次，并吸出胰蛋白酶。 </li><li>用2×胰蛋白酶的30微升再次洗涤细胞，并孵育在37℃下5分钟。轻轻敲击板的边缘与板打跑细胞。加入50μl补充的DMEM和直至细胞形成单细胞悬浮液，通过移液混合细胞。 </li><li>通过清洁表面和玻璃盖，用70％的乙醇制备血球。 </li><li>将玻璃盖玻片上计数室，并加盖直到"牛顿环折射"可以在盖玻片边缘和血球间可见。 注意:这些表明，盖玻片已正确地坚持的血球。 </li><li>轻轻吸取20微升细胞悬浮液的盖玻片下，通过毛细管运动填充计数室。将血球下放大10倍的显微镜和可视化的网格布局的计数室。 </li><li>算上在网格布局的4外广场的细胞。为了提高精确度，如果需要的附加 ​​外正方形重复计数，或者直到计数已经达到70 - 100细胞14,15。 <ol><li> （如果使用的话）每平方×稀释因子的平均细胞数×10 4:计算每毫升的细胞浓度如下</li></ol></li><li>重复步骤2.1-2.8，每24小时96小时后播种。 </li></ol> 3.确定细胞增殖使用基于发光的分析注意:这是一个终点测量。一旦所述试剂加入到细胞中，板只能使用一次定量。 <ol><li>解冻发光试剂在22℃水浴中30分钟。 </li><li>轻轻颠倒瓶子以获得均匀混合混合试剂。 </li><li>平衡接种细胞的一个板（来自步骤1.5）在室温30分钟。 </li><li>加入100μl发光试剂的每个孔中。混合内容上轨道摇床2分钟。允许板在室温下孵育10分钟。 </li><li>使用多模式平板阅读器软件设置发光实验。 <ol><li>开启多模板读数器和打开软件。在"任务管理器"，选择"实验"和"创建new&#39;.Select&#39;文件&#39;从侧面工具条，并根据"协议"选项卡中，选择"程序"。 </li><li>选择"格尼尔96平底&#39;板型，并选中"使用盖子"框。在选择"读法"盒子"读"的行动。选择"发光"的检测方法。点击"确定"。 </li><li>在阅读的步骤中，选择"全板"选项卡下要扫描的井，并选择井作为空白孔。 </li><li>设置过滤器设置为空过滤器（ 例如 ，插头，EM:孔镜:无）。将增益设置为135，每孔0.5秒的积分时间和6.5毫米的读高度。点击"确定"保存设置发光读。 </li></ol></li><li>执行发光读<ol><li>通过选择"仪器控制"选项卡弹出板夹，然后点击"板块out&#39;.Place 96孔板上板架，确保盖子上。通过点击&#39;板&#39;下&#39;的仪器控制"选项卡中关闭板固定器。点击&#39;现在读"从工具栏进行发光读。 </li><li>导出的每个的相对发光单位（RLU）测量很好地用于进一步分析电子表格。 </li></ol></li><li>重复步骤3.1-3.6种子细胞后，细胞增殖记录每24小时最多96个小时。 </li></ol> 4.确定细胞计数使用细胞成像仪<ol><li>使用多模式信元成像软件建立细胞成像实验<ol><li>打开多模式电池成像仪和开放日E软件。 </li><li>在"任务管理器"，选择"实验"标签和"创建new&#39;.On的"文件"工具栏上，单击"协议"选项卡，打开"程序"tab.Select"设定温度"的行动。孵化器设置为"开"和设定温度为37℃，请与接下来"框，然后再继续"预热"。点击"确定"保存设置。 </li><li>选择"读"的行动。点击"图像"检测方法，选择"端点/动力学"读类型和"过滤器"光学类型。上盘点击满盘&#39;标签&#39;的OK&#39;.Click&#39;，选择井成像。点击"确定"保存设置。 </li><li>从下拉"目标"选项中选择2.5X目标。在"频道"选项卡，选择两个渠道:GFP 469，525和明视野。检查两个通道"自动"曝光，并选择自动曝光好。 </li><li>要确定AUTØ聚焦设置，选择"自动对焦"，然后单击"选项"。对于自动对焦选项，选择"扫描，然后自动对焦"的方法。点击"确定"保存设置。 </li><li>设置的水平和垂直距离的井（微米）中心偏移到0.Select扫描每孔的多个图像在一个3×2的蒙太奇，以水平间距（微米）= 2881和垂直间距（微米）= 2127。点击"确定"保存设置程序。 注:请参见表2读取参数。 </li></ol></li><li>在板块选择执行读取实验<ol><li>点击"仪器控制"选项卡，选择"积垢"function.Place在板架96孔板，确保盖子上。根据"仪器控制"选项卡，选择功能"中板"。从板选项卡中选择"立即阅读。重复阅读每24小时最多96小时后播种。 </li></ol></li><li>细胞计数分析使用细胞成像软件。 <ol><li>为了分析图像，单击"数据"选项卡上的成像板，选择"图片[GFP 469，525 +明场]&#39;。双击一个很好成像。 </li><li>点击加载图像。选择"分析"，然后选择要分析蒙太奇的单一形象。点击"OK&#39;.Check了"绿色荧光蛋白"的唯一通道，并设置参数表3所列。点击"开始"，以参数适用于成像单元。 </li><li>观察细胞计数掩模放置在成像单元，它被用来确定每幅图像的细胞计数。点击整个实验成像每块板"应用更改"和维护这些设置。 </li><li>一旦回到成像板，单击"数据"下拉菜单，选择"细胞计数"。这将产生每孔的总细胞计数。 </li><li>通过点击进行​​进一步的分析Ø"导出"选项卡上导出所有的数据到电子表格˚F每孔的细胞计数。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

在这个协议中进行了检查三个不同测量在培养细胞的细胞增殖的方法。每一种方法是有能力的细胞增殖的可再现和精确的测量的超过96小时，并且结果是每个测试的方法之间比较的（ 图2和3）。同时基于发光的测定法和细胞成像方法所产生的最强大的结果，是表示之后96小时在细胞增殖的线性增加（ 图2b，c） 所示 。此外，随着时间的推移细胞成像描绘在转导的和非转导的细胞系（ 图4）之间的生长速率无显著差异。 有许多的优点和缺点对该协议检查的每个方法， 见表5为摘要。使用血球常规细胞计数法是一种低成本的方法，需要非常少的附加的试剂或effoRT准备和运行。此外，这种方法进行定量在细胞/ ml 14的绝对细胞数。但是，也有严重的缺点，其中包括消耗细胞计数，高错误率，其导致计数之间大的标准偏差的性质的时间，和一个事实，即高范围细胞数目的所必需的准确的细胞计数。这可以在图2a，其中，使用血球细胞计数表明在低细胞密度不同的结果，和大的标准偏差在稍后的时间点可以看到。这些缺点使得对小样本的细胞计数这种方法很有用，不够用于需要更小的板尺寸和接种密度较大的高通量测量。如果细胞密度增加，这样，计数细胞的最小数目开始以大于100个细胞的阈值这些限制可以得到缓解。越稀释的细胞悬浮液，或降低小区D密度，计数低于100个细胞，因此增加的可变性之间复制15的机会较大。但是，这种方法是不适合于96孔板，由于低细胞表面面积，因此，数量不足的细胞，可以在分析中使用。这凸显了缺乏这种方法和谁需要这种能力的用户明显的劣势的高吞吐能力。 基于发光的测定通过测定ATP的量，这是代谢活性细胞16的存在的度量确定细胞生存力。该测定是在96孔板形式设计用于多个样品的高通量筛选，以确定细胞增殖。这种简单的方法定量测定细胞增殖作为使用板读数器的相对发光单位（RLU），其正比于ATP的存在于代谢活性细胞的。然而，这种方法的主要缺点是叔他花费试剂，测定对细胞的代谢活性的依赖。各种细胞的培养条件，如温度或细胞周期的时间点，可以容易地影响由细胞，这是直接正比于由该测定17所产生的发光信号产生的ATP的量。因此，重要的是根据经验确定的实验条件下不与细胞的代谢活性干扰并可能妨碍由细胞产生的相对发光信号。 检测用于确定细胞增殖的第三种方法是一个活细胞成像和软件来执行的相对细胞数。细胞成像器具有高通量的能力，因为它可以自动捕捉多个图像的每个井，实时温度控制一起使用相同的细胞在测定法的持续时间。如在图4中所示，细胞增殖可以为monitored在同一板在一段时间过程中，它可以提供有关与细胞计数分析沿着细胞群的其他信息。这是因为它消除了交叉板变性和细胞接种错误，这是在96孔板格式司空见惯非常有利的。伴随细胞成像软件允许使用表2和3 中所概述的参数细胞计数的定量。因此，在高通量设置有用，可以很容易地被多路复用到其它测定法来测量细胞功能如细胞凋亡或细胞毒作用。该系统的主要缺点是软件，它是在其分裂触摸物体的能力受到限制。虽然它可以在较低的汇合细胞执行该功能，它是该测定的一个主要的限制，因此需要细胞类型特异性的优化。此外，虽然使用的成像器个别实验是在平板上逐片规模非常低的成本，这种方法将上如果在实验室仪器已经设置LY符合成本效益。因此，这提供了用于测定培养细胞的细胞增殖的新方法，并具有不需要另外的试剂的主要优势，并且能够96孔板自动计数，使得适合于高通量分析此方法。这是以往的血球细胞计数方法的显著的改进，并且是一种更具成本效益的选择到基于发光的测定。 使用细胞成像器时，关键的步骤是所捕获的图像和每幅图像的随后的细胞计数的分析过程中。这些图像可以使用多种细胞成像平台来处理。因此，最重要的，以确定所研究的细胞类型最为合适的软件。这将是对验证从通过使用不同的成像软件的细胞成像器获取的图像的细胞计数是有用的。该协议可以被应用到一个ý培养细胞，但是分析参数将必须对每个细胞系进行定义。这可以是一次在第一耗时，但一旦参数被设定，该方法可以在同一时间内自动向图像整体板。 要注意的是，这些试验是实际上增殖的间接测量，因为它们测量细胞数目（血球计数器，细胞成像器），或代谢活性（基于发光的测定法），过了一段时间，它是重要的。这些方法可以很容易地修改，以测量可以影响扩散等的重要因素。例如，台盼蓝排除法可以很容易地掺入到任何的血球或细胞成像的方法来排除死细胞从而确定细胞生存力13。的基于发光的测定法可与细胞毒性测定被多路复用，以提供有关细胞的健康的其他信息，并且该测定期间所测量的代谢活性也是细胞viabi的测量lity 12。因此，这些试验的灵活性，以与其它测定法进行复用，以提供额外的蜂窝信息是这些方法的一个很强的优势。 此协议中使用的人乳腺癌细胞系MCF-7已经用所LEGO-IG2-普罗+载体含有GFP基因已经稳定转导。这允许使用GFP的光学滤波器来图像的细胞，而不需要添加任何染料试剂到培养基中。这种方法可以很容易地，或者通过用增殖标志物标记细胞，无论是通过成像使用亮场光学类型的细胞修饰以图像GFP阴性细胞或甚至原代细胞系。在这个协议比较的方法是足够在多种不同的细胞系的情况下使用健壮的，但是，对于每个单独的细胞系的最佳协议设置应首先确定。使用明场通道细胞成像代表主要CE的最佳选择LLS或那些生长缓慢，由于这种方法的长期性。终点测定法，如基于发光的测定中，没有很好地适合于长期的细胞生长的分析。 这个协议描述了三种不同的方法用于体外测定细胞增殖。每种方法可以在实验室中进行例行检测细胞生长，大多数实验室将拥有必要的技能来执行这些方法之一。然而，也有可用于测量分裂细胞其它测定法的范围内。这些可包括测定DNA合成，代谢活性的方法，增殖或ATP浓度9的相关抗原。例如，已经开发了许多试验中通过用放射性物质标记的细胞，如3H-胸苷来测量细胞DNA合成的速率。这种方法的缺点是明显使用放射性物质，以及它们的处置。其他它们经常用于测量细胞增殖的方法包括使用新形成的DNA的BrdU标记，可使用流式细胞仪18进行测量的。流式细胞仪可用于分析细胞数以及细胞周期的信息。这可以是用于特定实验的有利的结果，但是这种方法的缺点包括额外的时间和步骤，昂贵的试剂和增加用户训练技能，以流式细胞仪操作的流动，并分析所得到的数据18。因此，存在可用于科学家以评估细胞的生长许多不同的测定，以及所选择的方法在很大程度上取决于所使用的细胞类型，用户和他们的技能水平，并在实验的终点所需的数据的类型。 总之，三种不同的测量细胞增殖的方法使用乳腺癌细胞进行了比较。每种方法具有许多优点和disadvantagES，并且因此依赖于用户的，并根据他们的每个实验的要求，确定最合适的测定来进行细胞计数的条款。

肿瘤抑制基因，p53基因，是一个数字的细胞过程，包括细胞周期停滞，细胞凋亡和衰老1的一个重要调节器。它是负责维持基因组稳定性，因此对于维持细胞死亡和细胞生长的平衡是至关重要的。 p53基因的突变是在癌症常见并且是p53的失活的主要原因，导致不受控制的细 ​​胞增殖2。有趣的是，在p53基因突变只占乳腺癌3的大约25％，这表明其他机制负责p53功能的丧失。最近发现的p53同种型已被证明在许多人类癌症中过表达，并能调节p53功能4,5。我们以前曾表明，p53的同种型，Δ40p53，是在乳腺癌中最高度表达的同种型，并且在乳腺癌细胞中显著上调，相比正常相邻做卷烟时UE 6。在此之后，我们稳定转导的人乳腺癌细胞系MCF-7中使用LEGO-IG2-普罗+载体（GFP +）7以过表达Δ40p53。这些细胞被用来研究是否高Δ40p53表达增加乳腺癌细胞的细胞增殖率。 有体外测定8,9-培养细胞的细胞增殖的许多直接和间接的方法。这些都可以无论是作为连续测量随着时间的推移，或作为端点检测10进行。传统的方法仍然是有用的，诸如使用血球细胞计数。该测定法是一种成本低，细胞数的直接测量，但它确实依赖于大细胞计数和高技能训练，以尽量减少计数误差和大的标准偏差。以执行与高通量格式兼容的测量的需要已经导致多孔板测定法的发展。这些基于发光的测定measu再根据正比于电池11,12的代谢活性的发光信号的细胞数。最近，引进高含量的成像平台已经允许其监控细胞增殖，同时提供定量和定性的表型数据采集新的工具，包括各种系统13。所有的这些方法提供途径来测量细胞生长，或者通过连续测量或端点测定法，和每个具有与问候灵敏度，可以通过样品的数字，和小单元信息的范围内的优点和缺点，它们都可以相应地称量取决于所研究的问题。 这个协议描述了测量细胞增殖在体外 ，具有利用灵敏度，重复性和多井板格式的不同范围的每个方法三种不同的方法。该协议的目的是比较使用一个血球计数的茶MBER，一个基于发光的细胞生存力测定法，和细胞成像仪，在细胞增殖超过96小时时间过程的测定。要做到这一点，载体转导的细胞（MCF-7-LEGO）的生长相比转导至过表达Δ40p53（MCF-7-Δ40p53）细胞，使用三种不同的细胞密度。细胞增殖测定每24小时的多达96小时。发现每一种方法有它自己的优点和缺点，并且根据实验的目的，每一个仍然是对增殖的速率提供信息的有价值的方法。

<table border="0" cellpadding="0" cellspacing="0" width="1031">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium, no phenol-red</td>
			<td style="width:99px;">ThermoFisher Scientific</td>
			<td style="width:119px;">21063-045</td>
			<td style="width:597px;">Supplemented with 10% FBS, 200mM L-glutamine, 2&#181;g/ml insulin and 1&#181;g/ml puromycin</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">L-glutamine solution (100x)</td>
			<td style="width:99px;">ThermoFisher Scientific</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insulin solution human</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">I9278-5ML</td>
			<td style="width:597px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fetal bovine serum (FBS)</td>
			<td style="width:99px;">Bovogen Biologicals</td>
			<td style="width:119px;">SFBS-F-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puromycin dihydrochloride</td>
			<td style="width:99px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9620-10ML</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">0.5% trypsin-EDTA solution (10x)</td>
			<td style="width:99px;">ThermoFisher Scientific</td>
			<td style="width:119px;">15400-054</td>
			<td>Dilute to 2x in DPBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS) (1x)</td>
			<td style="width:99px;">ThermoFisher Scientific</td>
			<td style="width:119px;">30028-02</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:216px;">Tissue culture flask, 75cm2 growth area</td>
			<td style="width:99px;">Greiner Bio-One</td>
			<td align="right" style="width:119px;">658175</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scepter 2.0 Cell Counter</td>
			<td style="width:99px;">Merck Millipore</td>
			<td style="width:119px;"></td>
			<td>Automated cell counter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96 well multiwell plate, flat bottom</td>
			<td style="width:99px;">Nunc</td>
			<td align="right" style="width:119px;">167008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Improved Neubauer Hemocytometer</td>
			<td style="width:99px;">BOECO Germany</td>
			<td style="width:119px;">BOE 01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus IX51 inverted microscope</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:119px;">IX51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CellTiter-Glo 2.0 Assay</td>
			<td style="width:99px;">Promega</td>
			<td style="width:119px;">G9242</td>
			<td>Luminescence-based assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytation 3 Cell Imaging Multi-Mode Reader</td>
			<td style="width:99px;">BioTek</td>
			<td style="width:119px;"></td>
			<td>Plate reader for luminescence, fluorescence and brightfield cell imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gen5 Data Analysis Software</td>
			<td style="width:99px;">BioTek</td>
			<td style="width:119px;">GEN5</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparison of three different methods for determining cell proliferation in breast cancer cell lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

研究测定培养细胞的增殖，-Δ40p53MCF-7转导的细胞的细胞增殖的不同的方法进行比较的非转导的MCF-7-LEGO乳腺癌细胞系。进行了比较，三种方法-分析-在本示意图中概括的常规血球方法，细胞活力发光测定和细胞成像（ 图1）。每种方法都有优点和缺点，以精确地测量随着时间的推移细胞计数，而最有效的方法取决于实验的终点的要求和可以为一个细胞群获取的信息。 MCF-7-LEGO和MCF-7-Δ40p53细胞的生长进行后4天24小时进行测定。正如图1所示，两种细胞系在三种不同的细胞密度（1.5×10 3细胞接种</s达&gt;; 3×10 3; 5×10 3细胞/孔）和细胞计数被播种后进行24，48，72和96小时。每个方法证实增加的细胞增殖，使用血细胞计数器，一个基于发光的测定法和细胞成像器（ 图2a，b和c，分别）超过96小时。在细胞增殖的最大增加被认为在最高细胞密度（5×10 3个细胞），并且也显示在每个时间点的最可重复的结果，这表明这是为在这些细胞中测量增殖的最佳细胞密度。有一个在细胞系之间的细胞增殖没有显著差异。 不同的方法之间的细胞增殖的测量是通过线性回归分析6（ 表4图3）相比较。有每个测试的不同方法之间的显著的相关性，比较当c在所有的细胞密度在两种细胞系ELL增殖（ 图3a和b）。的基于发光的测定法的比较，以及细胞成像器之间观察到最强的相关性（R 2 = 0.8899，P≤0.0001; R 2 = 0.9805，P≤0.0001，分别图3a和b）。 使用细胞成像的细胞增殖的可视表示中示出（ 图4）。由于该方法使用单一板的连续测量，细胞可以直到细胞达到接近100％汇合进行成像，每24小时。 如图2中所示，MCF-7-LEGO和MCF-7-Δ40p53细胞的生长率分别为在所有的时间点相当。这种方法提供了有用的细胞的信息，包括能够直观地监控多日的细胞生长，并比较细胞的大小和形态的细胞不同细胞系之间的迟缓。 <img alt="图1" src="/files/ftp_upload/54350/54350fig1.jpg" /> 图 1: 三种不同的方法测量细胞增殖的示意图细胞在三个不同的细胞密度接种到多个96孔平板并培养在37℃，5％CO 2的多达96小时。每隔24小时，细胞增殖通过或者使用一个血球，一个基于发光的分析或细胞成像测量。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54350/54350fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54350/54350fig2.jpg" /> 图 2: 细胞增殖的测量后96小时细胞计数，测定每24小时的96小时的MCF-7-腿O和MCF-7-Δ40p53细胞接种在三个不同的细胞密度。细胞计数在细胞/ ml用（ 一 ）血球，（b）使用在相对 ​​的发光单元的基于发光的测定（RLU），或（c）在细胞/图像用细胞成像的连续细胞计数测量。对于细胞成像条件见表2。所有实验代表三个独立实验的平均值，±在SD <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54350/54350fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54350/54350fig3.jpg" /> 图 3: 三种不同方法对乳腺癌细胞株测量细胞增殖的比较进行线性回归分析，比较不同的M之间的相关关系<html>编制方法检查（a）中的MCF-7-LEGO细胞和（b）MCF-7-Δ40p53细胞测定细胞增殖。皮尔森相关系数计算和显着性的不同方法测量细胞增殖之间的决定（P &lt;0.05）。所有的结果代表平均值±三个独立的实验的SD。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54350/54350fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54350/54350fig4.jpg" /> 图4:GFP阳性的MCF-7-LEGO和MCF-7-Δ40p53乳腺癌细胞图像的细胞的代表性图像从5×10 3个接种的细胞用细胞成像器每24小时拍摄的。比例尺条为1000微米。对细胞成像参数总结在表2中。<html> HREF ="htt​​ps://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54350/54350fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td colspan="3">细胞密度（细胞/孔​​） </td></tr><tr><td>电池类型</td><td> 1.5×10 3 </td><td> 3.0×10 3 </td><td> 5×10 3 </td></tr><tr><td> MCF-7，乐高</td><td> 76微升的8.4毫升媒体</td><td> 152μl，以8.3毫升媒体</td><td> 254μl，以8.2毫升媒体</td></tr><tr><td> MCF-7-Δ40p53 </td><td> 93微升的8.4毫升媒体</td><td> 185μl，以8.3毫升媒体</td><td> 308μl，以8.2毫升媒体</td></tr></tbody></table> 表1: 细胞接种卷96孔板中。 "FO:保持与 - next.within页="总是"&gt; <tbody><tr><td colspan="2">阅读参数</td></tr><tr><td>板型</td><td> 96 </td></tr><tr><td>模式</td><td>图片</td></tr><tr><td>目的</td><td> 2.5X </td></tr><tr><td>颜色</td><td> GFP（469，525），明场</td></tr><tr><td>曝光</td><td>汽车</td></tr><tr><td>焦点</td><td>自动（扫描然后自动聚焦） </td></tr></tbody></table> 表2:读取参数细胞成像利用细胞成像。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2">成像参数</td></tr><tr><td>阈</td><td> 5000 </td></tr><tr><td>最小对象大小（微米） </td><td>三十</td></tr><tr><td>最大对象大小（微米） </td><td> 300 </td></tr></tbody></t能够&gt; 表3:成像参数细胞计数分析。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> MCF-7，乐高 </td><td></td><td></td></tr><tr><td></td><td> 与正方 </td><td> p值 </td></tr><tr><td>血球与基于发光的分析</td><td> 0.6301 </td><td> 0.0021 </td></tr><tr><td>细胞成像与血球</td><td> 0.7524 </td><td> 0.0003 </td></tr><tr><td>基于发光的分析与细胞成像</td><td> 0.8899 </td><td> &lt;0.0001 </td></tr><tr><td> MCF-7-Δ40p53 </td><td></td><td></td></tr><tr><td></td><td> 与正方 </td><td> p值 </td></tr><tr><td>血球与鲁基于minescence-分析</td><td> 0.8983 </td><td> &lt;0.0001 </td></tr><tr><td>细胞成像与血球</td><td> 0.9303 </td><td> &lt;0.0001 </td></tr><tr><td>基于发光的分析与细胞成像</td><td> 0.9805 </td><td> &lt;0.0001 </td></tr></tbody></table> 表4:测试了三种不同增殖的方法的线性回归分析。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 方法 </td><td> 优点 </td><td> 缺点 </td><td> 技术说明 </td><td> 最终输出 </td></tr><tr><td rowspan="4"> 血球 </td><td>低成本</td><td>高人为错误</td><td>吸取多次以制备单细胞悬浮液</td><td rowspan="4">细胞/ ml </td></tr><tr><td>需要最少的设备</td><td>需要单细胞悬液</td><td>执行多个计数达到精度</td></tr><tr><td>直接细胞计数</td><td>所需的细胞数的准确的评估细胞的高数</td><td></td></tr><tr><td></td><td>端点</td><td></td></tr><tr><td rowspan="6"> 基于发光的测定 </td><td>与多孔板格式使用</td><td>昂贵的试剂</td><td>避光</td><td rowspan="6">相对发光单位（RLU）/孔</td></tr><tr><td>容易执行</td><td>需要发光酶标仪</td><td>包括对照孔，以确定背景发光</td></tr><tr><td>快速检测</td><td>温度敏感</td><td></td></tr><tr><td>提供细胞活力的信息</td><td>可变取决于细胞的代谢活性</td><td></td></tr><tr><td></td><td>间接测量</td><td></td></tr><tr><td></td><td>端点</td><td></td></tr><tr><td rowspan="6"> 细胞成像 </td><td>连续测量</td><td>贵成像仪</td><td>确保细胞成像器被设置为37℃ </td><td rowspan="6">细胞/图片</td></tr><tr><td>温度控制</td><td>技能强化</td><td>避免电池不必要的晃动或中断</td></tr><tr><td>提供移动信息</td><td>可变取决于细胞汇合</td><td></td></tr><tr><td>成本效益（如果你有像仪） </td><td>相对计数</td><td></td></tr><tr><td>直接测量</td><td></td><td></td></tr><tr><td>多孔板格式的自动成像</td><td></td><td></td></tr></tbody></table><stroNG&gt;表5:优势和不同的细胞计数方法的缺点比较。

Watch this Scientific Journal Video about Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines at JoVE.com

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

的三种不同方法对乳腺癌细胞株确定细胞增殖的比较

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

comparison-three-different-methods-for-determining-cell-proliferation

Universidad San Sebastian

Research

JoVE Journal

Biology

19.4K 次观看.  Hunter Medical Research Institute. This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

视频: Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

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 ...

科研

生物学

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

（TET-ON）与四环素诱导shRNA或cDNA表达生成稳定的人类细胞系

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

基因缺失在通过CRISPR / Cas9哺乳动物细胞系生成

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

测量去泛素酶的活性细胞系和组织样品的方法

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

通过热烫完整植株或提取物热处理烟草宿主细胞蛋白去除方法的比较

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

诱导LAP标记的稳定细胞系用于研究蛋白质功能，时空定位与蛋白质相互作用网络

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

哺乳动物细胞系的厌氧生长与维持

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...