Background: Deterioration of platelet (PLT) quality during storage is accompanied by an increase in lactate production, indicating a decrease in mitochondrial function. In this study, the optimal conditions under which the fluorescent dye JC-1 can be used to detect changes in mitochondrial function in PLTs were established.
Study design and methods: PLTs were incubated at 37 degrees C in synthetic medium under various conditions of JC-1 loading. In the presence of a high membrane potential, this dye accumulates in the mitochondria with a concomitant increase in red fluorescence. After JC-1 loading, the ratio of red (FL2) to green (FL1) fluorescence was determined by flow cytometry.
Results: The FL2-to-FL1 ratio of PLTs (3 x 10(7)/mL, loaded with 0.5 micromol/L JC-1) amounted to about 5 in 1-day-old PLTs. At higher dye concentrations, the FL2-to-FL1 ratio was significantly lower, suggesting uncoupling by the dye itself. Plasma concentrations above 3 percent significantly affected the JC-1 signal. The FL2-to-FL1 ratio showed a dose-dependent decrease to an uncoupler of oxidative phosphorylation or to inhibition of the respiratory chain. JC-1-loaded PLTs showed a clear decrease in FL2-to-FL1 ratio after prolonged storage or upon ultraviolet (UV) illumination. Only after UV treatment did changes in JC-1 signal correlate with changes in CD62P expression.
Conclusion: The FL2-to-F1 ratio of PLTs loaded with JC-1 is a reliable and sensitive indicator of the mitochondrial membrane potential, provided that the proper experimental conditions have been applied.