Green Fluorescent Cytochrome c Mitochondrial Apoptosis Assay Cell Line
A novel HEK293/Cytc-tGFP cell line has been developed through stable transfection for monitoring the cellular apoptosis level trhougth the Cytochrome c behavior and morphologicall changes in cell-based assays.HEK293/Cytc-tGFP cell line was obtained by transfection of an expression vector for a fusion protein of mitochondria localization signal from Cytochrome and turboGFP.
Assay Details: To measure the apoptosis levels, HEK293/CytctGFP were stimulated with staurosporine and nucleus stained with DAPI. In non-apoptotic cells the tGFP appears localizated in mitochondria in perinuclear localization; after treatment tagged mitochondrias appear mislocalizated with a granular shape, and the nucleus stained with DAPI show morphological changes. The assay was developed and optimized using the BD Pathway HCS Reader and Attovision compartimentalization Software.
The parameters analyzed in order to check the apoptosis degree are the nucleus shape and the disaggregation of the mitochondria. The last parameter was analyzed as granularity of the tGFP. Both analysis were validated with an average Z prime of 0.61 +/- 0.02 for High Content Screening.