Optimizing cell arrays for accurate functional genomics

BMC Research Notes

Pedro Roda-Navarro¹  Hernán E Grecco²  Philippe I H Bastiaens²  Sven Fengler² 

[1] Immunology Research Institute Hospital 12 de Octubre (i+12), Faculty of Medicine, Universidad Complutense de Madrid, Madrid, Spain;Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany

关键词: Single cell analysis;    Reverse transfection;    Methods for systems biology;    Cell arrays;    Automated microscopy;   

Others  :  1166137
DOI  :  10.1186/1756-0500-5-358

 received in 2012-02-21, accepted in 2012-06-14,  发布年份 2012

【 摘 要 】

Background

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.

Results

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.

Conclusions

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.

【 授权许可】

   
2012 Fengler et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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