A fluorescence vital assay for the recognition and quantification of excitotoxic cell death by necrosis and apoptosis using confocal microscopy on neurons in culture

An automated fluorescence method for the detection of neuronal cell death by necrosis and apoptosis with sequential acridine orange (AO) and ethidium bromide (EB) staining using confocal microscopy is described. Since cell nuclei during apoptosis become acidic, AO staining was utilized to distinguish live neurons from neurons undergoing apoptosis, using the AO property to shift its fluorescence from green at normal pH toward brilliant orange-red in the process of acidification. Further EB application labels nuclei of necrotic neurons in red. Sequential treatment by AO and EB can be employed as an express vitality test to count fractions of live and dead cell via apoptosis and necrosis, respectively. An algorithm of automatic quantification of cell types is based on the image correlation analysis. Our conclusion is validated by experiments with the vital dye trypan blue and the pharmacological study of receptor subtypes involved in the excitotoxicity. The approach described here, therefore, offers an express, easy, sensitive and reproducible method by which necrosis and apoptosis can be recognized and quantified in a population of living neurons. Because this assay does not require any preliminary tissue treatment, fixation or dissociation in a cell suspension its utility is likely to be extended for measuring cell viability and cytotoxicity on a variety of living preparations (tissues, brain slices and cell cultures).