The influence of medium on Amplex Red as indicator for fluorescent hydrogen peroxide during high-voltage electric impulses

Abstract

Fluorescent dyes often use to study cell membrane electropermeabilization. When a high-voltage pulse is applied to the electrolyte solution, a variety of electrolysis reactions occur at the metal-electrolyte interfaces. Metal ions that released from the electrodes can react with the fluorescent dyes and quench their fluorescence. This may have an impact when estimating the efficiency of electropermeabilization. In this study, the influence of the medium treated by high-voltage pulse and Al3+, Fe2+, Fe3+, Cr6+, Ni2+ and Mn7+ ions on the fluorescence of fluorescent tracer molecules, including fluorescent indicator for hydrogen peroxide, in various solutions was studied. Cell culture medium consisted of Dulbecco‘s modified Eagle‘s medium supplemented with 9 % fetal bovine serum and 1 % L-glutamine solution (all Sigma–Aldrich Chemie, Steinheim, Germany). 50 μl of the culture medium or a solution of a fluorescent dye was treated with a square-wave electric pulse with the duration of 0,1– 2 ms and the amplitude 0,2–2,4 kV/cm. The fluorescence of calcein, meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS4), doxorubicin (Adriamycin) and H2O2 indicator Amplex Red was studied. The fluorescence was measured at room temperature using Tecan spectrofluorimeter (Tecan Group, Männedorf, Switzerland). The medium which was treated by the electric pulse with the amplitude of 1,2 kV/cm and the duration of 500 and 2000 μs can almost completely quench calcein fluorescence. When the concentration of the metal ions increase in solution, the intensity of the fluorescence of calcein, TPPS4, Adriamycin and Amplex Red decrease. For example, 1 mM of Fe3+ or Ni2+ ions suppressed fluorescence of calcein by 15 and 79 % respectively. Fe3+ at the concentration of 1 mM totally suppressed the fluorescence of porphyrin-sulphonate, by 30 % – the fluorescence of Adriamycin, and by 30–50 % the fluorescence of Amplex Red.[...]