An Assay for Apoptosis detection based on Quantification of Multi nuclei feature and Nucleus to Cytoplasm ratio in S. cerevisiae cells treated with Acetic Acid and Hydrogen peroxide

The programmed cell death, apoptosis is a complex universal biological process in all types of eukaryotes ranging from single cell to multi-cellular organisms. The markers for apoptosis have been studied by assays based on both biochemical as well as microscopy however most assays are not affordable for many smaller labs. Acetic acid and hydrogen peroxide both induce apoptosis at higher concentrations in S. cerevisiae. Here we describe an assay system for the detection of apoptosis features based on DAPI staining followed by fluorescence microscopy in the cells treated with apoptosis inducing concentration of acetic acid and hydrogen peroxide. In this assay both untreated and cells treated with acetic acid and hydrogen peroxide were stained with DAPI and observed for the late stage apoptosis feature, Nuclear DNA fragmentation based multi nuclei centers and increase in the nuclear region enlargement. Further the multi nuclei feature and enlarged nuclei region as nucleus to cytoplasm ratio was quantified using Image J software. We report that S. cerevisiae strain BY4741 cells when treated with apoptosis inducing doses of acetic acid (140mM) and hydrogen peroxide (10mM) for 200 minutes, showed apoptosis marker feature such as nuclear region enlargement with multi-nuclei feature due to nuclear DNA fragmentation and increased nucleus to cytoplasm ratio when compared with untreated cells. We propose that this assay can be utilized for scoring the quantitative apoptotic feature as increase in multi-nuclei centers due to DNA fragmentation and nucleus to cytoplasm ratio as an indicator of apoptosis in S. cerevisiae upon treatment with apoptosis inducing agents. The assay system described here is easy to perform and affordable for the smaller lab to analyze the apoptotic features in S. cerevisiae cells which can be applied to other system as well.