INVESTIGADORES
GRECCO Hernan Edgardo
artículos
Título:
Optimizing cell arrays for accurate functional genomics.
Autor/es:
FENGLER S; BASTIAENS PI; GRECCO HE; RODA-NAVARRO P
Revista:
BMC Research Notes
Editorial:
BioMed Central
Referencias:
Año: 2012 vol. 5 p. 358 - 364
ISSN:
1756-0500
Resumen:
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.