Real-Time Fluorescent Imaging of Apoptosis using Chip-Based Technologies

Abstract

Validation of potential therapeutic targets necessitates the development of new assays that provide both spatial and temporal relationships in signalling networks. Cell-based assays are therefore becoming an important part of the postgenomic biomedical research. The present study was designed to provide a mechanistic rationale for the kinetic assays and to assess their potential to obtain dynamic and single-cell data on the stochastic process of cell death on Lab-on-a-Chip (LOC) technologies. We propose an innovative approach to dynamically trace cell death in real-time using fluorochromes such as propidium iodide (PI), SYTOX Green, SYTOX Red, YO-PRO 1, Annexin V and TMRM. We also demonstrate that, when used with innovative bioassays, microfluidic dynamic live-cell analysis is a practical alternative for multiparameter studies on a single-cell level. The LOC devices not only reduce the complexity of conventional cell culture protocols but also enabled time-resolved studies on apoptosis. The adaptation of non-toxic biomarkers that can continuously circulate inside enclosed microculture system will be beneficial for the advancement of up and coming LOC technologies. Our data supports the hypothesis that real-time bioassays in combination with LOC devices allow for rapid and simple analysis of cell death, particularly useful if the death pattern is a stochastic rather than deterministic process. As a result, they provide sensitivity that often cannot be achieved with conventional end-point analysis.