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

Abstract

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 33342 before LCI, will 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 are optimized using a 4× objective, and the minimum required Hoechst concentration is evaluated, allowing LCI at 30‐min intervals over 5 days. Remarkably, a Hoechst concentration of only 57 × 10−9 m significantly inhibits proliferation and thus impairs cell viability. However, Hoechst concentrations between 7 × 10−9 and 28 × 10−9 m can be determined, which are 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.

Long-term in vitro monitoring of AAV-transduction efficiencies in real-time with Hoechst 33342

“…2.5 x10 3 hSFs were seeded in each well of 96-well plates with 100 μl complete medium. Four ng/10 4 cells of Hoechst, which corresponds to a concentration of 28 nM that can be used in live cells for longterm staining, according to our previous study [19], were added to stain the nuclei. For hRPE cells, self-complementary AAV-2-CMV-GFP, AAV-6-CMV-GFP, and AAV-DJ-CMV-GFP particles purchased from Charles River (Wilmington, MA, U.S.) were suspended in the same medium and added into cells at a multiplicity of infection (MOI) of 5 x10 4 GC/cell (the following units are identical).…”

“…In previous work, we have shown that by optimizing the acquisition parameters, a low Hoechst concentration in the medium is sufficient to visualize the nuclei over five days without significantly affecting cell activities such as cell proliferation or signaling cascades. Although the appropriate Hoechst concentration varies from cell type to cell type, a concentration of 7 to 28 nM is recommended based on our previous study [19]. Usually, cell bodies are gated for automated counting when nuclei are not stained.…”

“…With the assistance of Hoechst, fluorescent nuclei, which are usually more regular in size and spatially separated from each other, appear more prominent in the background and can be gated more efficiently. Considering that Hoechst also stains dead cells, other dyes that specifically stain dead cells or debris, such as propidium iodide (PI), can be applied simultaneously, as demonstrated in our prior publication [19]. PI-positive cells are detected with a red fluorescence filter and sub-masked within the primary Hoechst-stained objects.…”

“…However, according to our previous work, a concentration between 7 to 28 nM is neither cytotoxic nor inhibits cellular proliferation and provides a sufficient staining of cell nuclei. In addition, the acquisition parameters were optimized to exclude possible phototoxic effects due to the excitation light [19]. This method is efficient as it provides real-time transduction rates due to its arbitrary repeatability and is, therefore, suitable for high-throughput analyses.…”