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James R. johnson, Brandon Kocher, Edward M. Barnett, Jayne Marasa, David Piwnica-Worms; Characterization of Apoptosis In RGC-5 Cells Using Novel Caspase-Activated Cell-Penetrating Probes As a Model For Monitoring Retinal Neurodegeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):454.
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Caspase-activatable cell-penetrating peptide (CPP) probes show promise for characterizing retinal ganglion cell (RGC) apoptosis in vivo. These probes incorporate a CPP sequence (kkkrkv), and a fluorophore-quencher pair linked by a caspase-labile peptide sequence (DEVD). This scaffold allows efficient cell uptake and renders the probe optically silent until cleavage by executioner caspases. Here, these probes were applied to an RGC-5 cell culture model to characterize the mechanisms responsible for probe uptake, activation and to correlate with in vivo models of RGC degeneration.
To monitor cellular uptake and further study internalization mechanisms, nonquenched probes Kcap488 and Kcap647 (green and red fluorescence, respectively) were synthesized and applied to an RGC-5 cell culture model for analysis using high-throughput fluorescence microscopy and flow cytometry. For analysis of cell death, caspase-activatable probes KcapQ488 and KcapQ647 were synthesized and tested using ionomycin cytotoxicity models.
RGC-5 cells showed distinct uptake of nonquenched probes with a perinuclear distribution within the interior of the cells. Using small molecule inhibitors of endocytosis, the principle mechanisms responsible for probe uptake were determined to depend on clathrine, caveolin and pinocytosis mechanisms. KcapQ488 exposure to RGC-5 cells followed by ionomycin treatment produced distinct probe activation. Cells were monitored for 1 hour in real time using fluorescence microscopy and distinct probe activation was observed within 15 minutes. This imaging procedure allowed for the generation of real-time movies showing both the morphological changes of cells undergoing apoptosis and concurrent probe activation.
These experiments provide validation of self-quenched, caspase-activatable probes as apoptosis biosensors in cell culture models using RGC-5 cells. The availability of KcapQ probes in various emission windows may facilitate multispectral analysis when imaged in the presence of other probes and antibody labeling techniques. KcapQ488 and KcapQ647 facilitate detection of cells undergoing apoptosis using high-throughput imaging methods and allow for apoptosis detection on populational and single cell levels in real-time.
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