Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry

Abstract

Cell death is a stochastic process, often initiated and/or executed in a multi-pathway/multi-organelle fashion. Therefore, high-throughput single-cell analysis platforms are required to provide detailed characterization of kinetics and mechanisms of cell death in heterogeneous cell populations. However, there is still a largely unmet need for inert fluorescent probes, suitable for prolonged kinetic studies. Here, we compare the use of innovative adaptation of unsymmetrical SYTO dyes for dynamic real-time analysis of apoptosis in conventional as well as microfluidic chip-based systems. We show that cyanine SYTO probes allow non-invasive tracking of intracellular events over extended time. Easy handling and “stain–no wash” protocols open up new opportunities for high-throughput analysis and live-cell sorting. Furthermore, SYTO probes are easily adaptable for detection of cell death using automated microfluidic chip-based cytometry. Overall, the combined use of SYTO probes and state-of-the-art Lab-on-a-Chip platform emerges as a cost effective solution for automated drug screening compared to conventional Annexin V or TUNEL assays. In particular, it should allow for dynamic analysis of samples where low cell number has so far been an obstacle, e.g. primary cancer stems cells or circulating minimal residual tumors.

Real‐time flow cytometry for the kinetic analysis of oncosis

“…More recently the use of SYTO16 and viability dyes has allowed the discrimination of apoptosis and oncosis in that no reduction in SYTO16 was observed in cells undergoing oncosis compared to that observed in cells undergoing apoptosis (7). In another advance, propidium iodide has been used in a real-time image analysis study of apoptosis from the start of the induction process and continuously over a 24 h period (27)(28)(29). In this approach adherent cells were preloaded with Hoechst and grown in culture with a low concentration of PI (0.25 lg/ml) and then exposed to STS for 24 h with images taken every 15 min in a real-time manner (27).…”

“…Thus the measurement of mitochondrial membrane and plasma membrane potentials in real-time affords a rapid easy method to distinguish oncosis and apoptosis at the induction stage of these two different necrobiological processes. This approach to study oncosis could used in conjunction with that employed to study apoptosis in real-time and SYTO16 and thus enable differentiation between the induction of oncosis and apoptosis in vitro and potentially ex vivo (27)(28)(29).…”

“…More recently a major advance in the real-time study of apoptosis developed by Wlodkowic and colleagues has shown that a range of viability dyes, including PI and SYTO16 are not toxic to cells and can be used in Lab-on-a-Chip-based real-time imaging systems in the study of the induction of apoptosis by real-time fluorescence and morphological analysis (2,(27)(28)(29). In a similar vein a polarity-sensitive annexinbased biosensor (pSIVA) with switchable fluorescence states allows the real-time detection of apoptotic cells by time-lapse imaging from 5-42 h (30).…”

Rapid Quantification of Cell Viability and Apoptosis in B-Cell Lymphoma Cultures Using Cyanine SYTO Probes

“…This methodology, however, is slowly gaining appreciation as an easy to perform, live-cell assay (6–8). Importantly, we have recently showed that cyanine SYTO dyes represent a promising class of inert probes that do not adversely affect normal cellular physiology (9). When preloaded or continuously present in medium, SYTO do not interfere with cell viability, and their intracellular retention permits straightforward and kinetic analysis of investigational drug/compound cytotoxicity (9).…”

“…Importantly, we have recently showed that cyanine SYTO dyes represent a promising class of inert probes that do not adversely affect normal cellular physiology (9). When preloaded or continuously present in medium, SYTO do not interfere with cell viability, and their intracellular retention permits straightforward and kinetic analysis of investigational drug/compound cytotoxicity (9). Reduction of sample processing achieved with these protocols is important for preservation of fragile apoptotic cells.…”