Quantitative analysis of intracellular calcium and mitochondrial kinetic fluorescence changes in GSNO-induced...

A fluorescence microscopy imaging technique was applied to observe the single-cell kinetic changes of intracellular Ca(2+) concentration ([Ca(2+)](i)) and mitochondrial membrane potential (ΔΨ(m)) during the early stage of S-nitrosoglutathione (GSNO)-induced thymocytes apoptosis. The kinetic features of [Ca(2+)](i) and ΔΨ(m) were quantitatively analyzed and compared by fitting the fluorescence intensity data. The mathematical parameter, inflection point which indicated the time point when [Ca(2+)](i) or ΔΨ(m) changed the most rapidly, was proposed to analyze the fitting curve. The results revealed that the inflection point of [Ca(2+)](i) always appeared prior to that of ΔΨ(m) during apoptosis induced by a certain GSNO concentration. Both the [Ca(2+)](i) and ΔΨ(m) changed in a GSNO concentration-dependent manner. Another parameter, half-max effect point was also employed and displayed the similar results. Such quantitative analyses of real-time observations at the single-cell level are useful for interpreting the sequence of the biological events operating in GSNO-induced thymocyte apoptosis.