New generation of fluorescent probes for cell-based measurements of caspase Activation and mitochondrial superoxide

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, with ex/em spectra of 590/610 nm, permitting easy multiplex with GFP or calcein stained neurons in both traditional and HCA microscopy configurations. MitoSOX™ Green that has specificity to superoxide like MitoSOX™ Red dye. In addition, it has better spectral properties and works with GFP filters in microscopy, and allows for measurement of mitochondrial superoxide in RFP-expressing cells. 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