Mitochondria are unique cellular organelles that possess their own DNA, encoding thirteen subunits of the respiratory complexes in mammals as well as twenty-two tRNAs and two rRNAs. Cytochrome c (Cytc) is an evolutionarily conserved nuclear-encoded mitochondrial protein, which contains 104 amino acids in mammals. Cytc is a multi-functional enzyme that plays an important role in life and death decisions of the cell. It participates in electron transfer as part of the mitochondrial electron transport chain (ETC) and is thus an indispensable part of the energy production process. It is the key protein that initiates the intrinsic apoptosis pathway. Fluorescent Cytochrome c HEK293 Cell Line developed by Innoprot is a vaulable tool to monitor mitochondrial apoptosis in living cells.
To measure the apoptosis levels, we stain nucleus following a stimulation of HEK293/Cytc-tGFP cell line with staurosporine. 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 parameters analyzed in order to check the apoptosis degree are the nucleus shape and the disaggregation of the mitochondria. Both analysis showed an average of Z´=0.61 +/- 0.02 for High Content Screening.