Real-time cytotoxicity assays

Abstract

Validation of new therapeutic targets calls for the advance in innovative assays that probe both spatial and temporal relationships in signaling networks. Cell death assays have already found a widespread use in pharmacological profiling of anticancer drugs. Such assays are, however, predominantly restricted to end point DEAD/LIVE parameter that provides only a snapshot of inherently stochastic process such as tumor cell death. Development of new methods that can offer kinetic real-time analysis would be highly advantageous for the pharmacological screening and predictive toxicology. In the present work we outline innovative protocols for the real-time analysis of tumor cell death, based on propidium iodide (PI) and SYTOX Green probes. These can be readily adapted to both flow cytometry and time-lapse fluorescence imaging. Considering vast time savings and kinetic data acquisition such assays have the potential to be applied in a number of areas including accelerated anticancer drug discovery and high-throughput screening routines.

Fig. 1

Dynamic analysis of cytotoxicity using simplified real-time protocols: (a) Workflow of a modified real-time (no-wash) protocol. Note that viability marker SYTOX Green is continuously present in the culture medium as opposed to a standard, end-point staining procedure. (b) Viability marker SYTOX Green was applied to dynamically track drug-induced cytotoxicity using flow cytometry. Human promyelocytic leukemia HL60 cells were exposed to a pro-apoptotic drug cycloheximide (CHX; 50 μg/ml) for 24 h in the continuous presence of SYTOX Green (100 nM). Fluorescent probe was excited using 488 nm Argon-ion laser. SYTOX Green fluorescence signal was logarithmically amplified using 530 nm band-pass filter. Debris was excluded electronically. Analysis based on bivariate dot plots FSC vs. SYTOX Green is shown. LIVE – viable cells, APO – apoptotic cells, DEAD – late apoptotic/necrotic cells. (c) Comparison between percentages of cell death estimated using standard SYTOX Green end-point vs. new kinetic protocol. Human promyelocytic leukemia HL60 cells were exposed to a range of pro-apoptotic drugs cycloheximide (CHX), campthothecin (CAM), and staurosporine (STS) for 24 h. Data were acquired using BD FACS Calibur flow cytometer equipped with 488 nm excitation line and 530 nm band-pass filter. Note excellent agreement between results obtained with both assays (R² ≥ 0.98 for p < 0.05 in Pearson and Lee linear correlation test).