Temporal heterogeneity in apoptosis determined by imaging flow cytometry

Abstract

Apoptotic process is highly heterogeneous, and a long-standing question is how many parameters define time and reversibility of the apoptotic response at a population and single-cell levels. Cell death analysis applications have greatly expanded since the introduction of flow cytometry. Classical approach for evaluation of apoptosis is en masse analysis of cells treated with different stimuli, but these methods cannot demonstrate heterogeneity in the population. Single-cell heterogeneity is now usually assessed by multicolor fluorescence microscopy; however obtaining reasonable statistics is time consuming and laborious. Therefore we combined flow cytometry, imaging flow cytometry, and fluorescent microscopy to characterize at a single-cell and population level sequence of apoptotic events induced by a variety of treatments (Vorobjev, Barteneva, J Histochem Cytochem 63:494-510, 2015). We show that simultaneous use of membrane potential dye TMRE, caspases 3/7 sensor, Annexin V and nuclear staining along with morphological parameters demonstrate heterogeneity of the whole process and is a valuable method for quantitative study of the apoptosis execution. Imaging flow cytometry allowed us to analyze correlation between TMRE, caspases 3/7, and Annexin V staining and morphological characteristics providing valuable information on the process of apoptotic execution. Importantly, comparisons of different data sets obtained by three methods allowed us to achieve temporal resolution of the whole process superior to that had been obtained by only one method.