The aim of our work was to establish flow cytometry methods for the characterization of mitochondrial Ca(2+) levels, plasma membrane potential, and superoxide generation and to relate kinetics to that of cytoplasmic Ca(2+) levels during short-term activation of T-lymphocytes. We monitored the change of fluorescence absorbance of sequentially measured Jurkat cells for 12?min. The cells were stained with the fluorescent dyes Fluo3-AM, Rhod2/AM, di-BA-C4-(5), or dihydroethidium and then were stimulated with increasing doses of phytohemagglutinin (PHA) or were treated with rotenone. Double-logistic function was fitted to cytoplasmic Ca(2+) signal and mitochondrial Ca(2+) levels, whereas logistic function was fitted to plasma membrane potential and superoxide levels. The calculated function parameters were area under the curve (AUC), maximum (Max), time to reach maximum (t(max)), slope at the first 50% value of Max (Slope), and ending (End) values, respectively. We found significant dose-response relationship between PHA dose and cytoplasmic Ca(2+) signals (AUC, Max, Slope: P<0.05), mitochondrial Ca(2+) levels (AUC and Max: P<0.05), and plasma membrane potential (AUC and End values: P<0.05). In rotenone-treated cells, superoxide generation increased in a dose-dependent manner (P<0.05 for AUC and End values, respectively). The present methodology provides an opportunity for monitoring and characterizing mitochondrial Ca(2+) levels, plasma membrane potential, and superoxide generation in PHA-activated or rotenone-treated Jurkat cells with flow cytometry.
