Optimizing cell arrays for accurate functional genomicsReportar como inadecuado

Optimizing cell arrays for accurate functional genomics - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Research Notes

, 5:358

First Online: 17 July 2012Received: 21 February 2012Accepted: 14 June 2012DOI: 10.1186-1756-0500-5-358

Cite this article as: Fengler, S., H Bastiaens, P.I., Grecco, H.E. et al. BMC Res Notes 2012 5: 358. doi:10.1186-1756-0500-5-358


BackgroundCellular 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.

ResultsWe 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.

ConclusionsPreviously 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.

KeywordsAutomated microscopy Cell arrays Methods for systems biology Reverse transfection Single cell analysis AbbreviationCACell Array.

Electronic supplementary materialThe online version of this article doi:10.1186-1756-0500-5-358 contains supplementary material, which is available to authorized users.

Download fulltext PDF

Autor: Sven Fengler - Philippe I H Bastiaens - Hernán E Grecco - Pedro Roda-Navarro

Fuente: https://link.springer.com/

Documentos relacionados