Abstract 4781: New generation of fluorescent probes for cell-based measurements of caspase activation and mitochondrial superoxide

Abstract

Abstract Cell health and stress readouts are critical indicators of altered or impaired function in normal and diseased states of cells, and work has been underway to develop improved small molecule sensor dyes compatible with traditional imaging and High Content Analysis (HCA) interrogation of apoptotic and mitochondrial stress pathways. The CellEvent™ Caspase Green dye effectively reports caspase activation, but suffers complications in assay configuration when attempting to multiplex with the Green Fluorescent Protein (GFP), calcein, or other 488 laser line tools in fluorescence microscopy. Here, we describe the testing and functional characterization of a new candidate molecule for measuring apoptosis in living cells. Our sensor is comprised of a fluorogenic reporter dye that is liberated from a DEVD peptide substrate by caspase activation, but operates in the Texas Red, 590nm excitation band, with an emission peak near 610 nm, permitting easy multiplex with GFP or calcein stained neurons in both traditional and HCA microscopy configurations. Similarly, mitochondrial superoxide accompanying cell stress is probed in microscopy with the MitoSOX™ Red Mitochondrial Superoxide Indicator dye, which localizes to mitochondria and reports superoxide generation, ignoring other Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS). This dye has an unusually long Stokes’ shift, requiring specialized microscopy and HCA filters that excite at 405nm, and capture emission at 610nm for specific superoxide detection. This unconventional spectroscopic profile prevents the dye’s use on many imaging platforms and promotes phototoxicity. To this end, our team has produced a dye with the same level of specificity for superoxide that will operate in one of the traditional fluorescence microscopy channels. Our candidate dye, here named MitoSOX™ Green Mitochondrial Superoxide Indicator also localizes to mitochondria of live cells and selectively reports superoxide generation, while ignoring other ROS and RNS species in ex vivo testing. With an Excitation/Emission profile in the GFP/FITC microscopy channel, a series of comparative studies in immortalized and neural cells are shown, highlighting photostability, specificity and signal amplitude from the dye. These reagents are research use only, not for diagnostic purposes Citation Format: Bhaskar S. Mandavilli, Daniel Beacham, Yi Zhen Hu, Jongtae Yang, Aimei Chen. New generation of fluorescent probes for cell-based measurements of caspase activation and mitochondrial superoxide. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4781.