活性检测在培养细胞
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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…
Representative Results
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…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Cell Titer Glo | Promega | G7572 | Buy in 100 ml quantities and aliquot, instead of purchasing the more expensive 10 ml quantity. Reconstituted, unused reagents can be refrozen at -20 °C for at least 21 weeks |
| 18% Formalin | Thermo-Shandon | 9990244 | Buying this fixative avoids the weighing out of formaldehyde powders and boiling of the solution; exposure to vapors is thereby minimized |
| Sucrose | Sigma-Aldrich | S0389 | It is not essential to add this to formaldehyde solutions but it improves the appearance of the fixed cells |
| Odyssey Block | LI-COR | 927-40003 | This fish serum can be bought in bulk and frozen at -20 °C for long term use |
| Triton-X 100 | Sigma-Aldrich | 21568 | We store a stock solution of 10% Triton-X 100 in sterile water at 4 °C |
| Sodium Phosphate Monobasic | Fisher | S468 | One can also buy PBS tablets or 10x PBS solutions, but they are more expensive |
| Sodium Phosphate Dibasic | Fisher | S373 | See above |
| Sodium Azide (250x) | Ricca Chemical Company | 7144.8-16 | Do not buy the powder because sodium azide is very toxic. We store all our used antibodies in 1x sodium azide at 4 °C until they become contaminated with debris |
| Mouse anti-α-tubulin | Sigma-Aldrich | T5168 | This antibody is expensive but can be greatly diluted and is highly specific |
| Mouse anti-MAP2 | Sigma-Aldrich | M9942 | This antibody is expensive but is highly specific (a prerequisite for In-Cell Westerns) |
| 800 nm Goat anti-mouse IgG | LI-COR | 926-32210 | 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 |
| DRAQ5 | Biostatus | DR50200 | This compound used to be sold by LI-COR at 1 mM |
| Sapphire | LI-COR | 928-40022 | |
| Luminometer | PerkinElmer | VICTOR3 1420 multilabel counter | |
| Odyssey Imager | LI-COR | 9201-01 | |
| Shaker/Mixer | Research Products International | 248555 |
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Cite This Article
Posimo, J. M., Unnithan, A. S., Gleixner, A. M., Choi, H. J., Jiang, Y., Pulugulla, S. H., Leak, R. K. Viability Assays for Cells in Culture. J. Vis. Exp. (83), e50645, doi:10.3791/50645 (2014).