To measure intracellular ROS production the cells growing on coverslips were loaded with 5 μM dihydrofluorescein diacetate (H2DCF-AC, Sigma; 20 min, 37 °C), thoroughly rinsed with Hank’s Balanced Salt Solution, and mounted on slides. Live cells were examined with the Olympus IX71 fluorescence microscope equipped with the Cell^F software (Olympus). The fluorescence of the dye was excited at 488 nm for 500 ms58 (link).
The Ca2+-sensitive fluorescent dye Fluo-3-AM was used to detect the relative change of the Ca2+-dependent fluorescence in cells. Cells were loaded for 30 min with 5 µM dye in amino-acids- and serum-free medium, washed twice and incubated for 15 min in full culture medium (de-esterification of the dye). The fluorescence was measured with the FV-1000 confocal microscope (Olympus) using excitation and emission wavelengths of 488 and 525 nm, respectively. The measurements were taken from at least 70 cells (usually over 100 cells) from randomly selected areas. All cells observed in the areas were used for measurements. The corrected total cell fluorescence (CTCF) of individual cells was calculated using the Cell^F software (Olympus) and presented in percentage frequency graphs (“cumulative distribution”) graphs. The experiments were performed in triplicate, with similar results.
The Ca2+-sensitive fluorescent dye Fluo-3-AM was used to detect the relative change of the Ca2+-dependent fluorescence in cells. Cells were loaded for 30 min with 5 µM dye in amino-acids- and serum-free medium, washed twice and incubated for 15 min in full culture medium (de-esterification of the dye). The fluorescence was measured with the FV-1000 confocal microscope (Olympus) using excitation and emission wavelengths of 488 and 525 nm, respectively. The measurements were taken from at least 70 cells (usually over 100 cells) from randomly selected areas. All cells observed in the areas were used for measurements. The corrected total cell fluorescence (CTCF) of individual cells was calculated using the Cell^F software (Olympus) and presented in percentage frequency graphs (“cumulative distribution”) graphs. The experiments were performed in triplicate, with similar results.
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