Live Cell Painting: New nontoxic dye to probe cell physiology in high content screening.

High-content imaging approaches, in combination with the use of perturbing agents such as small molecules or CRISPR-driven gene editing, have widely contributed to the identification of new therapeutic compounds. Thanks to recent advances in image-analysis methods, the use of high-content screens is increasingly gaining popularity and thus accelerating the discovery of new therapeutics. However, due to the lack of fully biocompatible fluorescent markers, large-scale high-content screens are mostly performed on fixed cells, which complicates the monitoring of changes in cell physiology over time. Here we present a novel fluorescent nontoxic dye that displays intensity and staining pattern changes in response to different physiological states. With multiparametric image analysis, these unique properties allow not only for the detection of distinct phenotypic fingerprints, but also for the quantification of more traditional disease-relevant phenotypes such as apoptosis, autophagy, ER stress and more. Since the dye only gets fluorescent when incorporated into cellular membranes, it is typically used without washing steps, therefore making it ideal to include in automation workflows. In this work, we present ​​relevant data on its biocompatibility and its potential to quantitatively assess subtle cellular phenotypes. Applications such as live kinetic imaging, and live image-based morphological profiling are also discussed. The rich information this fluorescent probe provides facilitates unbiased quantitative phenotypic analysis at larger scale, and ultimately paves the way for more discoveries of new therapeutic agents.