Breaking a Dogma: High-Throughput Live-Cell Imaging in Real-Time with Hoechst 33342.

Automated high-throughput live cell imaging (LCI) enables investigation of substance effects on cells in vitro. Usually, cell number is analyzed by phase-contrast imaging, which is reliable only for a few cell types. Therefore, an accurate cell counting method, such as staining the nuclei with Hoechst 33 342 before LCI, would be desirable. However, since the mid-1980s, the dogma exists that Hoechst can only be used for endpoint analyses because of its cytotoxic properties and the potentially phototoxic effects of the excitation light. Since microscopic camera sensitivity has significantly improved, this study investigates whether this dogma is still justified. Therefore, exposure parameters were optimized using a 4x objective, and the minimum required Hoechst concentration was evaluated, allowing LCI at 30-minute intervals over 5 days. Remarkably, a Hoechst concentration of only 57 nM significantly inhibits proliferation and thus impairs cell viability. However, Hoechst concentrations between 7 and 28 nM could be determined, which were neither cytotoxic nor impacting cell viability, proliferation, or signaling pathways. The method can be adapted to regular inverted fluorescence microscopes and allows, for example, to determine the cytotoxicity of a substance or the transduction efficiency, with the advantage that the analysis can be repeated at any desired time point. This article is protected by copyright. All rights reserved.