Background

A variety of cell-based assays are available to determine cell death, but most of them, including the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, crystal blue staining, and various flow cytometry-based methods, have the limitation of being end-point assays. Recently, we employed propidium iodide (PI) for live-cell assessment of cell death using an automated fluorescence imaging system (IncuCyte ZOOM from Essen Bioscience) (Sehgal et al., 2017). By combined analysis of phase-contrast images, cytostatic and cytotoxic effects can be simultaneously detected. This method proved to be reliable and reproducible, as well as very simple and cheap. In our recent publication, we used it in three different cancer cell lines (LNCaP, PC3 and MCF7) to efficiently determine and compare the toxicities of various drug analogs of the ER Ca²⁺ pump inhibitor thapsigargin (Tg) (Sehgal et al., 2017). The use of PI for real-time analysis of cell death has been described and validated by others (Wlodkowic et al., 2009; Zhao et al., 2010), but the analysis methods employed previously (e.g., flow cytometry, or lab-on-chip platforms) are more laborious and time-consuming than the simple analysis method we present here. In short, we quantify cell death using the integrated algorithms of the IncuCyte ZOOM software to calculate the confluence of PI-positive (red-fluorescent) cells and divide that by the total cell confluence (obtained by analysis of phase-contrast images). This simple analysis approach minimizes the time and efforts needed for data processing and analysis, and increases the throughput of the method. We have validated the method for cell death assessment through a side-by-side comparison with flow cytometry-based end-point measurements of PI-stained cells.