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.
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.
Authors: E D Hottz; M F Oliveira; P C G Nunes; R M R Nogueira; R Valls-de-Souza; A T Da Poian; A S Weyrich; G A Zimmerman; P T Bozza; F A Bozza Journal: J Thromb Haemost Date: 2013-05 Impact factor: 5.824
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Authors: Justyna Hajtuch; Maria Jose Santos-Martinez; Michal Wojcik; Ewelina Tomczyk; Maciej Jaskiewicz; Wojciech Kamysz; Magdalena Narajczyk; Iwona Inkielewicz-Stepniak Journal: Front Pharmacol Date: 2022-01-28 Impact factor: 5.810
Authors: Alessandro Protti; Anna Lecchi; Francesco Fortunato; Andrea Artoni; Noemi Greppi; Sarah Vecchio; Gigliola Fagiolari; Maurizio Moggio; Giacomo Pietro Comi; Giovanni Mistraletti; Barbara Lanticina; Loredana Faraldi; Luciano Gattinoni Journal: Crit Care Date: 2012-10-03 Impact factor: 9.097