rhod-2, ratiometric pericam and aequorin

The dynamics of mitochondrial [Ca 2+ ] ([Ca 2+ ] M) plays a key role in a variety of cellular processes. The most important methods available to monitor [Ca 2+ ] M are fluorescent dyes such as rhod-2 and specifically targeted proteins such as aequorin or pericam. However, significant discrepancies, both quantitative and qualitative, exist in the literature between the results obtained with different methods. We have made here a systematic comparison of the response of several fluorescent dyes, rhod-2 and rhod-FF, and two Ca 2+-sensitive proteins, aequorin and pericam. Our results show that measurements obtained with aequorin and pericam are consistent in terms of dynamic Ca 2+ changes. Instead, fluorescent dyes failed to follow Ca 2+ changes adequately, especially during repetitive stimulation. In particular, measures obtained with rhod-2 or rhod-FF evidenced the previously reported Ca 2+-dependent inhibition of mitochondrial Ca 2+ uptake, but data obtained with aequorin or pericam under the same conditions did not. The reason for the loss of response of fluorescent dyes is unclear. Loading with these dyes produced changes in mitochondrial morphology and membrane potential, which were small and reversible at low concentrations (1-2M), but produced large and prolonged damage at higher concentrations. In addition, cells loaded with low concentrations of rhod-2 suffered large changes in mitochondrial morphology after light excitation. Our results suggest that [Ca 2+ ] M data obtained with these dyes should be taken with care.