Optimizing cell arrays for accurate functional genomics.
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.
Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany.
Sven
S
Fengler
Philippe I H
PI
Bastiaens
Hernán E
HE
Grecco
Pedro
P
Roda-Navarro
D023281:Genomics / Q000379:methods* / Q000592:standards; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D009696:Nucleic Acids / Q000032:analysis / Q000235:genetics*; D046888:Tissue Array Analysis / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D014162:Transfection
D009696:Nucleic Acids