PhiPhiLux-GjD2 is a fluoroprobe that incorporates the group II cas-pase-recognition sequence DEVD into a bifluorophore-derivated peptide that mimics the structural loop conformation present in native protease cleavage sites. Group II caspases include caspase 3, caspase 2, and caspase 7. In this molecule, the core peptide, GDEVDGI, is coupled to a molecule of rhodamine on each side of the cleavage site. The two rhodamines interact as a dimer and emit a stable blue-green fluorescence. Cleavage of the substrate disrupts this interaction between rhodamine moieties to result in enhanced green fluorescence (excitation peak 490 nm and emission peak 520).
To measure caspase activity, embryos were removed from culture medium and washed three times in 50-|xl drops of prewarmed Hepes-TALP Embryos were incubated in 25-|xl microdrops of Hepes-TALP containing 5 |xM PhiPhiLux-G^ at 39°C for 40 min in the dark. Negative controls were incubated in Hepes-TALP only. Following incubation, embryos were washed twice in 50-|xl drops of Hepes-TALP and placed on poly-L-lysine coated slides and mounted with a coverslip. Caspase activity was determined immediately after the end of heat shock using a Zeiss Axioplan 2 fluorescence microscope with a 45X objective. Images were obtained using a Spot camera and software (Diagnostic Instruments, Inc., Sterling Heights, MI). Pictures were taken using a background subtract feature from the spot software, and digital images were stored as.tif files. Fluorescence intensity was analyzed using IPLab for MacIntosh version 3.5 (BioVision Technologies, Inc., Exton, PA). Each embryo represented a region of interest. Using the mouse, a circular draw function was manually performed for each region of interest, and the pixel intensity per unit area was determined.