Development of Carboxy SNARF-1-AM and Annexin V Assays for the Determination of Apoptosis in Heterogeneous Cultures

Accurate identification and quantitation of apoptosis is essential for developing efficient strategies for optimisation of culture survivability and productivity. Flow cytometry in conjunction with several fluoroprobes is increasingly used to identify apoptotic cells. We have examined the possibility of using carboxy SNARF-1-AM, a pH sensitive fluoroprobe and FITC-labelled annexin V, a probe specific to phosphatidylserine exposed on the outer surface of apoptotic cells. Intracellular acidification was shown to precede the occurrence of apoptosis thereby proving to be an early indicator of cellular deterioration and cell death. Annexin V in combination with propidium iodide enabled identification of viable, transient apoptotic and necrotic cells in heterogeneous cultures. Metabolic activity (pHi), and cell death population dynamics (viable/ apoptotic/ necrotic fraction) were therefore effectively and reliably determined using flow cytometry.