Chip-Based Dynamic Real-Time Quantification of Drug-Induced Cytotoxicity in Human Tumor Cells

Abstract

Cell cytotoxicity tests are among the most common bioassays using flow cytometry and fluorescence imaging analysis. The permeability of plasma membranes to charged fluorescent probes serves, in these assays, as a marker distinguishing live from dead cells. Since it is generally assumed that probes, such as propidium iodide (PI) or 7-amino-actinomycin D (7-AAD), are themselves cytotoxic, they are currently generally used only as the end-point markers of assays for live versus dead cells. In the current study, we provide novel insights into potential applications of these classical plasma membrane integrity markers in the dynamic tracking of drug-induced cytotoxicity. We show that treatment of a number of different human tumor cell lines in cultures for up to 72 h with the PI, 7-AAD, SYTOX Green (SY-G), SYTOX Red (SYR), TO-PRO, and YO-PRO had no effect on cell viability assessed by the integrity of plasma membrane, cell cycle progression, and rate of proliferation. We subsequently explore the potential of dynamic labeling with these markers in real-time analysis, by comparing results from both conventional cytometry and microfluidic chips. Considering the simplicity of the staining protocols and their low cost combined with the potential for real-time data collection, we show how that real-time fluorescent imaging and Lab-on-a-Chip platforms have the potential to be used for automated drug screening routines.

Real‐time flow cytometry for the kinetic analysis of oncosis

“…Previously SYTO16 has been used to differentiate oncosis and apoptosis in a time dependent manner (7). However, the real-time imaging of cells loaded with Hoechst in the presence of PI allowed the detection of apoptosis an hour after induction (27). This major advance in the study of apoptosis could be used to study the oncotic process too by the employment fluorescent mitochondrial dyes as tested in this study (27).…”

“…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).…”

“…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). The use of annexin V in real-time image analysis has also allowed the study of apoptosis by real-time live cell microscopy but only covering the 5-42 h period after the induction of apoptosis (30).…”

“…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).…”