This MitoCASPred/SH-SY5Y cell line has been developed through stable transfection for monitoring the caspase-3 activity level through the turboFP602 protein distribution in cell-based assays. This cell line was obtained by transfection of an expression vector for a fusion protein of mitochondria localization signal from Cytochrome connected by the linker containing caspase-3 cleavage sequence DEVD and connected by the turboFP602 protein.In living cells the turboFP602 is localized in mitochondria. When the caspase-3 is activated, during early phase of apoptosis, the caspase-3 cleavage the specific DEVD sequence and the FP602 protein shows cellular dispersed localization including nuclear localization.Assay Details: To measure the apoptosis levels, MitoCASPred/SH-SY5Y were stimulated with staurosporine. In non-apoptotic cells the tFP602 appears localized in mitochondria in perinuclear localization due to de MTS (mitochondrial targeting sequence) situated in the N-terminal of the construct. After promoting apoptosis, caspases are activated and cleave the specific caspase-cleavage site (CCS) situated between the MTS and the tFP602 in the construct, resulting in liberation of tFP602 and entry into the nucleus. The assay was performed and optimized using the BD Pathway HCS Reader and Attovision compartimentalization Software. The parameter analyzed in order to check the apoptosis degree is the appearance of red fluorescence (intensity) in the nucleus. The Ec50 for staurosporine was 56 pM after a treatment of 24h. Activity was calculated relative to control. This assay was validated with an average Z prime of 0.62+/- 0.01 for High Content Screening. (See data sheet for more details)
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