Fengler Sven, Bastiaens Philippe I H, Grecco Hernán E, Roda-Navarro Pedro
Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany.
BMC Res Notes. 2012 Jul 17;5(1):358. doi: 10.1186/1756-0500-5-358.
Cellular responses emerge from a complex network of dynamic biochemical reactions. In order to investigate them is necessary to develop methods that allow perturbing a high number of gene products in a flexible and fast way. Cell arrays (CA) enable such experiments on microscope slides via reverse transfection of cellular colonies growing on spotted genetic material. In contrast to multi-well plates, CA are susceptible to contamination among neighboring spots hindering accurate quantification in cell-based screening projects. Here we have developed a quality control protocol for quantifying and minimizing contamination in CA.
We imaged checkered CA that express two distinct fluorescent proteins and segmented images into single cells to quantify the transfection efficiency and interspot contamination. Compared with standard procedures, we measured a 3-fold reduction of contaminants when arrays containing HeLa cells were washed shortly after cell seeding. We proved that nucleic acid uptake during cell seeding rather than migration among neighboring spots was the major source of contamination. Arrays of MCF7 cells developed without the washing step showed 7-fold lower percentage of contaminant cells, demonstrating that contamination is dependent on specific cell properties.
Previously published methodological works have focused on achieving high transfection rate in densely packed CA. Here, we focused in an equally important parameter: The interspot contamination. The presented quality control is essential for estimating the rate of contamination, a major source of false positives and negatives in current microscopy based functional genomics screenings. We have demonstrated that a washing step after seeding enhances CA quality for HeLA but is not necessary for MCF7. The described method provides a way to find optimal seeding protocols for cell lines intended to be used for the first time in CA.
细胞反应源于一个复杂的动态生化反应网络。为了研究这些反应，有必要开发一些方法，以便能够灵活、快速地干扰大量基因产物。细胞阵列（CA）通过对生长在斑点状遗传物质上的细胞集落进行反向转染，从而在显微镜载玻片上进行此类实验。与多孔板不同，CA易受相邻斑点间污染的影响，这会妨碍基于细胞的筛选项目中的准确量化。在此，我们开发了一种用于量化和最小化CA污染的质量控制方案。
我们对表达两种不同荧光蛋白的方格状CA进行成像，并将图像分割为单个细胞，以量化转染效率和斑点间污染。与标准程序相比，当含有HeLa细胞的阵列在细胞接种后不久进行清洗时，我们测得污染物减少了3倍。我们证明，细胞接种过程中的核酸摄取而非相邻斑点间的迁移是污染的主要来源。未经过清洗步骤培养的MCF7细胞阵列显示污染细胞百分比降低了7倍，这表明污染取决于特定的细胞特性。
此前发表的方法学研究主要集中在实现密集包装的CA中的高转染率。在此，我们关注一个同样重要的参数：斑点间污染。所提出的质量控制对于估计污染率至关重要，污染率是当前基于显微镜的功能基因组学筛选中假阳性和假阴性的主要来源。我们已经证明，接种后进行清洗步骤可提高HeLA细胞阵列的质量，但对MCF7细胞则不必要。所描述的方法提供了一种途径，可为首次打算用于CA的细胞系找到最佳接种方案。
