3.4. AO & EB Fluorescent Assay for the Assessment of Cell Death

Acridine orange and ethidium bromide staining was performed as described by Spector et al. [28] The cells (5 × 10⁵) were incubated with acridine orange and ethidium bromide solution (1 part each of 100 μg/mL acridine orange and ethidium bromide in PBS) and mixed gently, and examined in a fluorescent microscope (Carl Zeiss) using a UV filter (450–490 nm). Three hundred cells per sample were counted in duplicate for each dose point. Cells were scored as viable, apoptotic or necrotic, as judged from nuclear morphology and membrane integrity, and the respective percentages of apoptotic and necrotic cells were then calculated. The cells of interest were photographed.

There have been several approaches to detect cell death. In this study, double fluorescent staining of acridine orange (AO) and ethidium bromide (EB) has been used. Thus, the cells have classified into five types according to the fluorescence emission and the morphological feature of chromatin condensation in the stained nuclei: (i) viable cells have uniformly green fluorescing nuclei with a highly organized structure; (ii) early apoptotic cells have green fluorescing nuclei, but the peri-nuclear chromatin condensation has visible as bright green patches or fragments; (iii) late apoptotic cells have orange–red fluorescing nuclei with condensed or fragmented chromatin; (iv) necrotic cells have uniformly red nucleus and cytoplasm and (v) red and/or green stained cells with non-apoptotic morphological features i.e., fragmented nuclei with original cell morphology, vacuolated cytoplasm, cytoplasmic lesion, etc., have indicated that the terminal point in necro-apoptosis has reached. Fluorescent images of compounds 5a–m induced morphological features were observed using a high efficiency fluorescent microscope with apoptome fitted with time-lap and imaging facilities (Carl Zeiss) and analyzed using Axioscope and Image J Software (Version 1.36, Dresden, Germany). The data presented are representative of those obtained in at least three independent experiments conducted in triplicate.