的三种不同方法对乳腺癌细胞株确定细胞增殖的比较
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Dulbecco's Modified Eagle Medium, no phenol-red | ThermoFisher Scientific | 21063-045 | Supplemented with 10% FBS, 200mM L-glutamine, 2µg/ml insulin and 1µg/ml puromycin |
| L-glutamine solution (100x) | ThermoFisher Scientific | 25030-081 | |
| Insulin solution human | Sigma-Aldrich | I9278-5ML | |
| Fetal bovine serum (FBS) | Bovogen Biologicals | SFBS-F-500ml | |
| Puromycin dihydrochloride | Sigma-Aldrich | P9620-10ML | |
| 0.5% trypsin-EDTA solution (10x) | ThermoFisher Scientific | 15400-054 | Dilute to 2x in DPBS |
| Dulbecco's Phosphate Buffered Saline (DPBS) (1x) | ThermoFisher Scientific | 30028-02 | |
| Tissue culture flask, 75cm2 growth area | Greiner Bio-One | 658175 | |
| Scepter 2.0 Cell Counter | Merck Millipore | Automated cell counter | |
| 96 well multiwell plate, flat bottom | Nunc | 167008 | |
| Improved Neubauer Hemocytometer | BOECO Germany | BOE 01 | |
| Olympus IX51 inverted microscope | Olympus | IX51 | |
| CellTiter-Glo 2.0 Assay | Promega | G9242 | Luminescence-based assay |
| Cytation 3 Cell Imaging Multi-Mode Reader | BioTek | Plate reader for luminescence, fluorescence and brightfield cell imaging | |
| Gen5 Data Analysis Software | BioTek | GEN5 |
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