我们承认Juliann Jaumotte为节省在ATP检测试剂的量的概念。对此，我们深表感谢玛丽卡鲁索，德布威尔森和成龙花拉和以药剂的Mylan公司为学校提供这些研究的财政支持高超的行政支持。还要感谢的Hunkele可怕的疾病基金会和帕金森氏症和运动障碍基金会主神经元研究的资金支持。

的协议的原理图如图1所示。 1。细胞电镀板的细胞在96孔板中以不同的接种密度（ 图2）。有关的N2a成神经细胞瘤细胞系，板2.5K，5K，10K，和每孔15000细胞在3或6孔/组的线性度检查。在大鼠原代皮质神经元，板25k，50K，100K，每200K良好的细胞在3个或6孔/组的线性度检查。如果细胞系或感兴趣的原代细胞看起来很健康在不同的接种密度，板在和周围的最佳细胞密度的细胞类型。 注意：在本研究中，的N2a细胞铺板在100μl介质和原代皮质神经元在200μl介质上而设计的低蒸发板。有关电池的处理，培养基，血清，抗生素和毒素治疗的详细信息，请参阅Unnithan 等 。为的N2a CELLS 8和Posimo 等 。原发性皮质文化22。 <ol><li><ol><li>重复该镀上的第二的96孔板。一个板将被测定ATP（ 图2A），而另一个与红外分析（ 图2B）。人们可以不使用相同的板的ATP和红外线检测由于细胞必须对细胞内ATP的测量被裂解开。 </li><li>请务必板块在红外检测为背景减除至少3个额外的井在最佳接种密度（第2列; 图2B）。 </li></ol></li><li>新增无介质或细胞，更廉价，无菌水外井排A和H和列1和12。避免依赖于从沿边缘井数据，存活率通常是低级这里从媒体蒸发和温度梯度的影响。这些问题与微孔板通常被称为边缘效应23,24。不要让这些井空因为再行B和G和列2和11遭受的边缘效应。 边缘效应可以通过在环境条件下孵育新鲜接种板在室温下转移到CO 2培养箱中24之前被降低。此外，最近的一项研究Carralot介绍了使用一个单一的控制列23酶标板的数学校正方法。奥利弗和他的同事使用了一种特别设计的强制空气微量滴定板孵化器，以减少热梯度25。如果边缘效应是显著的关注，微孔板稳定性试验箱（ 如：BT＆C股份有限公司）就可以买到创建一个均匀的微环境湿度高，温度均匀梯度。 </li><li>如果需要比图1所示更多的井，因为额外的试剂的稀释或多个接种密度将被测试，用于减去背景边缘的孔中。 </li><li>等待通宵附件细胞和测定法，如下所述第二天早晨的。 </li></ol> 2。发光的ATP含量<ol><li>按照细胞滴度格洛制造商的建议与缓冲基材的重建和孵育时间。 <ol><li>从100或200微升电镀媒体，删除50或150微升媒体分别。略少于50微升将在井中留在后面。加入25微升的试剂（衬底加缓冲液）中，以1:2稀释的列2-6（ 图2A）。 注：媒体变卷后留下去除可能的差异跨井稀释或浓缩的ATP检测试剂。小心，以确保液体中的所有多通道移液器吸头的水平是等价的。立即加入ATP检测试剂，以确保准确性之前测量在整个板中选择孔​​中的剩余液体用吸管。如果存在从媒体的蒸发罐差别税率高变异横跨板的表面振动性，除去所有旧的介质，并立即加入ATP检测试剂之前的新鲜培养基或磷酸盐缓冲盐水（PBS）中相同的体积加至所有孔中。如果从板块蒸发变量的水平受到影响，尝试切换到从Costar公司康宁的低蒸发板。在后者的板，在图2所示的60内部的井只遭受从平均0.995％±0.41介质蒸发过夜。还有从而在测定时在媒体音量变化可以忽略不计。 </li><li>在列7到11中，行B至D，除去足够的媒体，留下50微升的后面，如上面详述的，并以1:1的稀释度加入50微升的试剂。 </li><li>列7至11，行E到G，离开细胞在100μl的媒体和1:1稀释加入100μl试剂，再次。公司建议，将100μl试剂应该在100μl的媒体进行1:1稀释。 注：为了为了节省成本，也能够在数量和在稀释切断这些建议。然而，在此之前，确保它不降低测定的线性度和灵敏度。 </li><li>一旦一个特定的量和所用试剂的稀释因子被发现是令人满意的，坚持使用他们的所有后续实验。该标准令人满意的数据在结果部分说明。 </li><li>未使用的试剂配制可再冷冻并使用在稍后的日期，以节省成本。据生产商，再造试剂可以储存在-20℃下进行21周，活动〜3％的损失。 </li></ol></li><li>将板在定轨摇床上，持续2分钟或章动振荡机中10分钟。如果该板的一部分被检测为不同的测量，并且是不可动摇的，搅拌的介质用简单的向上和向下的移液仅在感兴趣的孔中。然而，在此步骤中产生过多的气泡将与LUMI干扰nescent输出。 <ol><li>加入试剂后10分钟，转移60微升的孔内容到白色96孔板中。发光值较高的白酒板块较透明或黑色的板，因为它们反射光线向上直到检测。 注：根据我们的经验，细胞生存的低蒸发更好，清晰Costar平板比其他板块。这些具体的板不与白色的墙壁出售。其次，不透明壁和水清见底板是不是完全清楚的板更贵。如果一个发光液体转移到白色板，白色面板可洗涤并重复使用，从而节省了使用新的白色板为每一个实验的成本。液体转移因此，从长远来看更便宜的选择。 </li><li>弹出气泡之前读取该板用针，或者最好是用由塑料移液管灯泡的强制空气。读板，上有1秒的积分时间（该公司recomme光度计NDS 0.25-1秒为足够的积分时间）。读加入ATP检测试剂后板10-12分钟。定时是用于在不同的板比较关键的，因为该发光信号是瞬时与快速衰变速率，如由吉尔伯特和同事26。 </li></ol></li><li>从3个或6个孔中各基团和情节中，作为细胞数在散点图的函数的平均发光值（参见图3）。从每个相应的数据点首先减去相应的空井的平均本底荧光值（11G井2B - 2G，井11B - - 11D和11E井）。会出现在这个初始实验（ 图2A）三个不同的群体对每个电镀密度。 ATP水平可以被线性地与细胞密度在1这些基团的部分或全部。继续进行的最高稀释度的试剂仍然给人满意的结果，以节省成本。令人满意的结果的标准描述我n个结款。 注意：对于在本研究中使用的媒体，在该测定法的背景值不高，因此能够跳过空白孔。然而，制造商Promega公司的报告不同的培养基中发光的差异。本研究采用Hyclone公司胎儿克隆三，胎牛血清为的N2a细胞和初级皮质培养小牛血清合成版本。根据制造商，小牛血清减小发光值，但不降低该测定的灵敏度。然而，如果这是显著的关注，稀释在PBS代替媒体ATP的测定试剂在测定的时间。 <ol><li>如果细胞出现生长不均匀跨列过夜，尽量在6小时镀层的测定它们。但是，记住，在检测的正常时间（例如，24治疗后HR）的密度是指线性度必须达到的密度。 </li></ol></li></ol> 3。 Infrareð测定<ol><li>电镀后的早上，固定细胞在所述第二板在室温下在通风橱中。一定要戴手套，这一步和处置固定液为化学废物的，因为甲醛是一种致癌物质。在0.1M磷酸盐缓冲液中添加4％的甲醛和4％蔗糖，以1:1稀释的现有介质。媒体还可以用PBS冲洗掉固定在全强度的固定剂前。无论哪种方法效果很好。 <ol><li>孵育固定液中20分钟，然后取出固定液，并在200微升PBS洗涤3次。 </li></ol></li><li>用0.2％的叠氮化钠的储存板在PBS中于4℃下，如果测定将不会发生在同一天。否则，继续进行试验，然后将细胞阻断的解决方案，以尽量减少抗体的非特异性结合。 <ol><li>淡化鱼血清奥德赛块1:1的PBS，并添加0.3％的Triton X-100作为细胞渗透剂。创建足够的封闭液以及小学和中学antiboDY解决方案，使35微升可以很好地吸取到每个。然而，如果大量的气泡移液过程中的观察，使&gt; 50微升每个反应孔，否则气泡到达成从结合的细胞和块的抗体。过度的肥皂泡沫将显示为未染色圆形光斑在阱的中间。尝试，如果发生这种情况，调整移液。 </li><li>孵育在室温下30-60分钟，这封闭溶液。 注：来自相同物种作为第二抗体5％牛血清白蛋白或正​​常血清也可以用来作为封闭液保存在鱼血清块的成本。阻塞的解决方案可以影响抗体的性能。因此，对于每个抗体的最佳块是最好的经验确定。 注：一些研究者把内嵌式西片上呼风唤雨孵化期间。然而，这是没有必要的。 </li></ol></li><li>使第一抗体：1:10,000稀释的抗-α-微管蛋白（小鼠单克隆抗体， 表1）和1:2000稀释的抗-MAP2（小鼠单克隆抗体， 表1）。这些抗体是针对在这些细胞模型感兴趣的蛋白质，特异性为任何免疫细胞化学染色必不可少的。 注意：MAP2是一个标记为神经元和适合混合性神经元/神经胶质培养物。这里显示的产后的文化也含有一些胶质细胞（〜25％），因为星形胶质细胞首先出现在胚胎第18天，与他们的人数在高峰新生儿早期27,28。我们不能星形胶质细胞和神经元ATP和DRAQ5 +蓝宝石检测区分。为了分别测量神经元和星形胶质细胞，同时用MAP2和GFAP染色在800和700 nm的渠道，所描述的Mullett和同事4,29，留下了DRAQ5 +蓝宝石。但是，如果所有的细胞在培养要进行评估，使用一个泛细胞标记，如α-微管蛋白，β-肌动蛋白，或GAPDH。 注：这些稀释已优化的的N2a和初级皮层细胞，可能无法推广到每一个模型。因此，尝试在至少两个初级抗体稀释液，分别在板的左半部分和一个在右边一半（ 见图2B）。或者，尝试在每个3口井3-4稀释一抗。例如，在抗体的插入片的建议稀释可侧接有两个倍的变化。换句话说，如果对于免疫细胞化学的推荐稀释度为1:500，也尝试1:250和1:1000的稀释因子。 <ol><li>抗体稀释1:1奥德赛块：PBS中，加入0.3％的Triton-X。为了节省成本，尽量使抗体在被施加到所述单元在步骤3.2.1通过在孵育结束时除去该溶液中的封闭溶液。保持细胞在PBS，而这样做，他们一定不能干。 </li><li>孵育在一抗或者1-2小时，在室温或4℃过夜°C。对于弱binding抗体和蛋白质，是不丰富，孵育过夜可以帮助提高信号。 注：至少保留3口井堵塞的背景减法的解决方案在每个二级抗体浓度（井2B-2D和2E水井-2G 图2B）。不要这些井承受任何一抗任何责任。他们揭示了由次级抗体的非特异性结合的程度，并为信号与噪声的比值计算是有用的。如果存在这样的担忧，该第二抗体将导致高水平的非特异性结合的，还包括对照孔中未暴露于初级或次级抗体，但收到后洗涤的数目相同。这些井和“二次唯一的”井之间的差值将揭示的非特异性结合引起的二次抗体单独的程度。在我们对小鼠的N2a细胞测试，信号抗鼠二抗强独显0.557±0.032，与抗兔信号单独二抗为0.533±0.041，无二次抗体信号是0.357±0.003，与第一和第二抗体信号为11.867±0.911。我们在小鼠细胞使用抗鼠二抗，即使这样也没有观察到高浓度的非特异性结合的。有几个制造商的红外线的第二抗体;我们建议只买高交吸附的。注意，这取决于源次级IgG抗体的浓度可以变化。 </li></ol></li><li>以每孔，每10分钟洗涤3次的200微升PBS洗去一抗。初级抗体可在0.2％叠氮钠保存于4℃几周和重用，直至碎片的微小斑点变得明显时，溶液被保持到光。这只是做，以节省成本。如果成本不是问题，作出新的抗体，每次使用。 </li><li>由1:1,000或1:2,000 1:1奥德赛块稀释第二抗体：PBS 0.3％三吨-X（ 图2B）。加入1:1000稀释的板和1:2,000的上半部分的底部板的一半。这些浓度可进一步减少，以节省成本，但承诺此之前检查线性度。 注意：一定要添加相应的二级抗体溶液的背景减除井（ 图2B 2列）。 <ol><li>如果第二种蛋白将被检测到位DRAQ5 +蓝宝石，标记细胞中的α-微管蛋白或MAP2与700nm的山羊抗小鼠IgG和第二蛋白质与来自一个物种比鼠标等初级抗体在800nm​​的信道。在800纳米通道具有比700nm的信道背景少，并应保留用于所关注的最关键的蛋白质。 </li><li>孵育中的二抗1小时，在室温下，在抽屉避光保存。 </li></ol></li><li>以每孔，每10分钟洗涤3次的200微升PBS冲洗二次抗体。 </li><li>使DRAQ5 +蓝宝石的解决方案。对于该板的左半部分，稀DRAQ5 1:10000（0.5μM的最终浓度）和蓝宝石1:1000在PBS中与0.3％的Triton-X（列3 - 6， 图2B）。对于该板的右半部分，稀DRAQ5 1:20,000（0.25μM）和蓝宝石1:2000（列7 - 10， 图2B）。 注：用于在一个1毫米的股票被出售，而不是一个5毫米的股票DRAQ5。因此，一些此前公布的报告中使用1:4,000或1:2,000稀释DRAQ5 8。 <ol><li>为了节省成本，如果两个板同时被检测，同样DRAQ5 +蓝宝石解决方案可用于在序列中的两个板，吹打其关闭所述第一板和将其加入到第二个。如果相同的解决方案，将用这种方式被使用两次，使用它们在一天之内。摊薄DRAQ5 +蓝宝石解决方案不能被保存在4℃或冷冻以备后用。 </li><li>在培养这些解决方案为30分钟，在室温下，远离fROM光。如果时间很短，DRAQ5 +蓝宝石解决方案将不被重用在其他板块不同的次级，添加这些污渍在步骤3.5的二级抗体溶液孵育1小时。 注意：不要DRAQ5 +蓝宝石解决方案添加到背景减法井（ 图2B 2列），因为这些油井不应该在700 nm的通道被染色。 </li></ol></li><li>用200μlPBS中，每孔洗涤平板3次，每次10分钟。把0.2％叠氮化钠的PBS中最后清洗，如果板是将要染色的其它可见光范围的二级抗体后的红外成像完成。 </li><li>扫描平板上的奥德赛成像仪。通过扫描板的强度5和169 mm分辨率开始。无论是“中等质量”或“低质量”的设置就足够了。该公司没有公布详细的激发/发射过滤器的信息。然而，发射滤波器是约20纳米宽，都围绕720和820纳米，根据制造商。 注：这是可能的扫描板，同时还湿的调查人员可能希望继续污染其与其他标记。然而，该公司建议在其网上协议，干板导致较少的富裕以及信号传播。如果您扫描他们两个湿，然后干燥到比较数据时，一定要删除所有的PBS从井里因为蒸发后剩下的盐会导致沿边缘高背景荧光。 <ol><li>奥德赛成像仪扫描并通过该板的底部。来自不同制造商的板可要求不同的聚焦偏移，因为孔的底部可以在其厚度和深度变化的板。尝试扫描在不同的聚焦偏移，看看那里的最高信号噪声比和干脆的（最重点）信号达到2.5毫米，3.0毫米，3.5毫米，4.0毫米。也尝试测量井深从板用尺子底部确认发掘的GS上的奥德赛。请记住，在塑料中的井的底部可以添加额外的高度，以直尺测量。在本研究中使用的Costar平板要求以焦点为4.0mm的偏移。 </li><li>使用该软件的内嵌式西方函数将合适大小的网格将其安置在板的图像和数据导出到Microsoft Excel。考察在不同焦点的偏移量相同的井“积分强度”的价值观，以找到最高荧光值。焦点偏移产生（在免疫染色井信号对“无主阴性对照”孔）的最高信号与噪声的比值是1，选择以下。 </li><li>一旦一个焦点偏移后决定，以较小的增量再次扫描。例如，如果各路信号是在3.5和4.0毫米，试着扫描在3.6，3.7，3.8，和3.9毫米，看看是否有信号强度进一步提高。如果焦点偏移是错误的，跨在一个空PL的12列中的信号强度吃了（当所有列应该有平等的信号）将显示为一个U形曲线，而不是一条直线。如果这仍然是用塑料板有问题，尝试玻璃底板。 </li></ol></li><li>从在700和800纳米通道的每个对应的数据点;减去积分强度的平均值中减去背景井（2G孔2B - - 2D或井2E 图2B）。然后对各组油井的平均化积分信号强度。绘制的数据作为对细胞密度的散点图（ 见图3）。 <ol><li>比较第一和第二抗体和DRAQ5 +蓝宝石的两种不同的稀释度，以解决在每一个稀释用于随后的实验结果的两个不同稀释度的结果。如果线性度没有实现，尝试扫描以不同的强度。重新扫描与强度3和7，而不是5和重新分析数据。如果数据改善在这些强度的1，但仍然不能令人满意，重新扫描周围的强度增量。 </li><li>要小心，不要分析图像，其中软件显示白点在井;这些斑点表示饱和信号输出成像的范围。扫描以较低的强度，如果发生这种情况。 </li><li>如果线性度没有实现，也尝试修复在6小时镀层的细胞，因为它们可能会增长或在不同的列死不均匀过夜。也可以尝试不同的接种密度，不同的扫描强度，试剂的稀释因素，玻璃底船板，DRAQ5本身为核心，只有色斑，或者比抗α-微管蛋白或抗MAP2不同抗体的进一步优化。 </li></ol></li></ol>

没有一个作家有任何冲突披露。

我们已经发现，在所有三个活力检测的信号强度是线性的，并与铺板密度相关。然而，并非所有的试验都是同样敏感的2倍或铺板密度1.5倍的变化。供的N2a细胞，红外线检测比ATP测定法较不敏感，特别是在较低的接种密度。虽然红外检测比ATP敏感性较低，在DRAQ5 +蓝宝石分析和α-微管蛋白检测有较好的一致性，因为它们揭示的N-乙酰半胱氨酸的高度保护的影响。有红外信号的剂量 - 响应损失与两种测...

在生物科学中最常见的活力测定包括细胞计数。这是由顶部（最近的）出版物200中出现的医学用关键字或者“ 体外 ”或“文化”对2013年4月29日和二〇一三年四月三十〇日的分析证实。这些出版物中，有23.5％的人使用细胞计数分析，包括人工细胞数计数，用图像处理软件自动细胞计数数和台盼蓝排斥。的活/死测定法用在这些出版物中的1％。使用MTT（3 - （4,5 - 二甲基吡啶-2 - 基）-2,5 - 二苯基溴化）的出版物的数量测定的代谢活力为11％。文学的这项调查还显示，使用试验，如MTT法与细胞计数分析相结合的出版物的数量仅为3.5％。尽管使用一个可行性分析本身，评估与细胞的号码组合细胞功能的趋势似乎评估蜂窝我最好的选择ntegrity。由自己的细胞计数是不够的，因为剩余的细胞可能不是功能性的或，即使它们是存在于井1,2 ​​健康。相反，功能性的措施如ATP可以增加或减少在没有在细胞的数量平行的变化。从细胞数的代谢读数的解偶联表明，ATP和MTT法不应该被用来作为唯一的可行性分析。在本报告中，三可行性分析的调查既细胞结构和代谢功能进行了描述，对细胞的完整性更全面的视图，而不是本身的任何一个检测可以负担得起。 我们的两个实验都需要一个红外成像仪测量荧光在700和800 nm的渠道。噪音低的红外线的波长，从而导致更高的信号-噪声比3。我们使用奥德赛成像仪拥有4.5日志的动态范围和位深度为16，translatin克至2 16或65,536色调红外线。这可以对比对8位彩色成像，这仅仅能提供2 8或256彩色浓淡的每个波长的光。因此，16位成像具有较高的分辨率。应当指出的是，原始红外图像通常伪彩色绿色（800纳米）和红色（700纳米），在列报发表的报告。奥德赛成像器通常既用于Western印迹和在细胞西部片4-7。在细胞西部片甲醛固定的细胞用第一抗体针对感兴趣的任何蛋白质和依次用红外荧光二抗标记它们。这种技术是已知的磷酸化端点6中特别有用。在我们的内嵌式西部片，我​​们固定染色细胞骨架蛋白α-微管蛋白或神经元微管相关蛋白2（MAP2）在800 nm的通道。这些蛋白质是足够丰富，得到高的信号噪声比。我们还弄脏我们的盘子在700纳米通道的原子核与DRAQ5染色和用于与蓝宝石染色细胞质中。无论是细胞骨架蛋白和DRAQ5 +蓝宝石污渍从而反映细胞结构。 第三生存试验测量的代谢功能，被称为“细胞滴度格洛”在此荧光素酶为基础的检测，发光值是成正比的ATP水平。 ATP测定法是常用来量化活菌8-12。然而，包括在测定中的名称的字“效价”是有些用词不当，因为每个细胞ATP的输出可以作为毒素处理的函数改变，因此不总是按比例细胞数8。 ATP水平也受昼夜节律13和细胞分裂14和细胞分化15。然而，这里显示的ATP检测是简单的执行和有用的，因为ATP是一个强大的测量代谢可行性16-21，如果不是细胞数本身。使用此法，以配合红外内嵌式西部片因此产生的细胞的完整性更全面的了解比任何单纯的一个实验。

<table border="0" cellpadding="0" cellspacing="0" width="821">
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	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Cell Titer Glo</td>
			<td style="width:112px;">Promega</td>
			<td style="width:93px;">G7572</td>
			<td style="width:507px;">Buy in 100 ml quantities and aliquot, instead of purchasing the more expensive 10 ml quantity. Reconstituted, unused reagents can be refrozen at -20 &#176;C for at least 21 weeks</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">18% Formalin</td>
			<td style="width:112px;">Thermo-Shandon</td>
			<td style="width:93px;">9990244</td>
			<td style="width:507px;">Buying this fixative avoids the weighing out of formaldehyde powders and boiling of the solution; exposure to vapors is thereby minimized</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sucrose</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">S0389</td>
			<td style="width:507px;">It is not essential to add this to formaldehyde solutions but it improves the appearance of the fixed cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Odyssey Block</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">927-40003</td>
			<td style="width:507px;">This fish serum can be bought in bulk and frozen at -20 &#176;C for long term use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Triton-X 100</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">21568</td>
			<td style="width:507px;">We store a stock solution of 10% Triton-X 100 in sterile water at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Phosphate Monobasic</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:93px;">S468</td>
			<td style="width:507px;">One can also buy PBS tablets or 10x PBS solutions, but they are more expensive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Phosphate Dibasic</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:93px;">S373</td>
			<td style="width:507px;">See above</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Azide (250x)</td>
			<td style="width:112px;">Ricca Chemical Company</td>
			<td style="width:93px;">7144.8-16</td>
			<td style="width:507px;">Do not buy the powder because sodium azide is very toxic. We store all our used antibodies in 1x sodium azide at 4 &#176;C until they become contaminated with debris</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Mouse anti-&#945;-tubulin</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">T5168</td>
			<td style="width:507px;">This antibody is expensive but can be greatly diluted and is highly specific</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Mouse anti-MAP2</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">M9942</td>
			<td style="width:507px;">This antibody is expensive but is highly specific (a prerequisite for In-Cell Westerns)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">800 nm Goat anti-mouse IgG</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">926-32210</td>
			<td style="width:507px;">Other companies also sell infrared secondary antibodies. Be sure to purchase the highly cross-adsorbed antibodies and note that concentrations of IgGs may vary with the source</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">DRAQ5</td>
			<td style="width:112px;">Biostatus</td>
			<td style="width:93px;">DR50200</td>
			<td style="width:507px;">This compound used to be sold by LI-COR at 1 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sapphire</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">928-40022</td>
			<td style="width:507px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Luminometer</td>
			<td style="width:129px;">PerkinElmer</td>
			<td style="width:189px;">VICTOR3 1420 multilabel counter</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Odyssey Imager</td>
			<td style="width:129px;">LI-COR</td>
			<td style="width:189px;">9201-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Shaker/Mixer</td>
			<td style="width:129px;">Research Products International</td>
			<td style="width:189px;">248555</td>
			<td></td>
		</tr>
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viability assays for cells in culture

在显微镜手动细胞计数评估细胞活力的敏感装置，但费时，因此价格昂贵。计算机化的可行性分析是昂贵的设备方面，但可以更快，更客观的比手工细胞计数。本报告介绍了使用三个这样的可行性分析。两个这些测定是红外线和1是发光。两个红外检测依赖于一个16位的奥德赛成像仪。一个红外检测使用的DRAQ5染色的细胞核结合蓝宝石染色为细胞​​质和可视化在700 nm的通道。其他红外测定中，一个胞内western，使用抗细胞骨架蛋白（α-微管蛋白或微管相关蛋白2）和标识它们在800 nm处的通道。第三个可行性分析是对ATP一种常用的发光检测，但是我们用四分之一的推荐量，以节省成本。这些测量都是线性的，并与CE的数量关联LLS镀金，但有所不同的敏感性。所有这三个实验规避耗时显微镜和样品的整个井，从而减少抽样误差。最后，所有的测定法可以很容易地一天实验结束时内完成，从而允许更大数目的要在短的时间范围进行了实验。然而，它们都依赖于假定的细胞数目保持在比例处理，即有时不能满足，特别是对细胞内ATP的假设后的信号强度。此外，如果细胞增加或治疗后减少的规模，这可能会影响信号强度，而不影响细胞数量。我们的结论是所有的可行性分析，包括手动计数，从一些需要注意的地方受苦，但计算机化的可行性分析是非常值得的初始投资。使用所有这三个实验同时产生细胞结构和功能的全面视图。

在这些实验中的限速因素是红外线染色，作为ATP测定法的持续时间相对短暂。用于红外测定法，我们预计8 96孔板可染色和扫描在一天之内由交错两批各4板（ 见图1）。这个估计假定固定20分钟，洗涤30分钟，阻断，2小时初级抗体孵育后洗涤30分钟的30分钟，1小时的第二抗体温育后洗涤30分钟，30分钟DRAQ5 +蓝宝石后跟30清洗，以及扫描时间为4片34分钟的分钟。另外十五分钟分解成图...

Watch this Scientific Journal Video about Viability Assays for Cells in Culture at JoVE.com

Viability Assays for Cells in Culture

治疗性化合物通常先在体外用可行性分析研究。盲细胞计数由人类观察者可以是在细胞数量的微小变化高度敏感，但不评估功能。计算机化的可行性分析，如下所述，可以客观地评估在结构和功能。

活性检测在培养细胞

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Research

JoVE Journal

Biology

46.0K 次观看.  Duquesne University. 治疗性化合物通常先在体外用可行性分析研究。盲细胞计数由人类观察者可以是在细胞数量的微小变化高度敏感，但不评估功能。计算机化的可行性分析，如下所述，可以客观地评估在结构和功能。 

视频: Viability Assays for Cells in Culture

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

计数和确定培养细胞的可行性

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

科研

生物学

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit检测癫痫发作癫痫突变果蝇</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

在培养的哺乳动物细胞大规模并行报告基因分析

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

测定法错误折叠的蛋白质在细胞中的降解

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...