Quantificação de apoptose e necrose mediante corantes fluorescentes e análise de imagens, no cultivo de células de inseto: o caso da Drosophila melanogaster S2
Abstract
In animal cell cultures there are two kinds of cell death: apoptosis
(programmed cell death) and necrosis (cell death due to external sources). The inovative
method of cell death identification that have attracted interest is the use of fluorescence
microscopy and fluorescent dye (to bind DNA and RNA). Image analysis techniques has
become a useful tool in cell death quantification, once they are non destructive for the culture
and have easy application with using adjusted softwares.
Based in these new technological trends, the present work considers the
development of a methodology for quantification of apoptotic and necrotic cell death in
cultures of insect cells Drosophila melanogaster (S2). This methodology involved the
development of a experimental procedure using new dyes (YO-PRO-1 and Propidium Iodide),
that have presented advantages over the others because they are non destructive to the culture
an are able to clearly discriminate between apoptotic and necrotic cell death. The use of this
dyes was compared with the method of exclusion of Trypan blue and fluorescent dyes
Acridine Orange and Ethidium Bromide. Besides being less toxic to cells S2, YO-PRO-1 and
Propidium Iodide showed more sensitivity in the identification of death and in the calculation
of cell viability. It also envolved the development of an algorithm based on image analysis
techniques for quantification of the two kinds of cell death, less subjective than the manual
methods currently used. The algorithm showed to be efficient, fast an of easy application with
a error around 2% when compared to manual methodology. The calculated cell concentration
by the algorithm was very close to experimental.