Ionwizard

DCs were isolated as described above and rested overnight in RPMI media. The cells were then loaded with membrane-permeable Ca²⁺-sensitive fluorescent indicator Fluo-4 AM. Cells were incubated in Tyrode’s solution (in mM: NaCl 140, KCl 5.4, MgCl₂ 1, glucose 10, HEPES 10), containing 1 mM Ca²⁺, 6.6 µM fluo-4 AM, and 0.16% Pluronic F127 for 20 min at room temperature to load indicator in the cytosol. Then the supernatant was removed, and cells were washed in Tyrode’s solution, containing 1 mM Ca²⁺, twice for 15 min. After that, the cells were placed into the experimental chamber superfused with 2 mM Ca²⁺ Tyrode’s solution. Intracellular Ca²⁺ was measured using fluorescence photometry setup (IonOptix). Ca²⁺ fluorescence was first measured after 3–5 min in normal salt (140 mmol/L Na⁺), and then solution was changed to high salt (190 mmol/L Na) and Ca²⁺ fluorescence was monitored in 3-min intervals for another 21–24 min. All experiments were conducted at room temperature (≈23°C). Intracellular Ca²⁺ was analyzed using commercial software (IonWizard; IonOptix). A total of 7–12 DCs from nine different animals were used.

Ca²⁺ measurement assays in adult cardiac myocytes have been described previously [33 (link)]. In brief, cells were loaded with 2 µM Fura-2 acetoxymethyl ester (Invitrogen, C-2938) for 15 min, and then placed in Tyrode’s solution containing: 130 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose and 10 mM HEPES (pH 7.4). The Fura-2 fluorescence ratio was determined with a Delta scan dual-beam spectrofluorophotometer (Photon Technology International) operated at an emission wavelength of 510 nM and excitation wavelengths of 340 and 380 nM. The myocyte stimulation frequency for Ca²⁺ transient measurements was 0.5 Hz. For caffeine-induced Ca²⁺ release, myocytes were perfused with a Tyrode’s solution and stimulated at 0.5 Hz until stabilization of the transients, after which caffeine was acutely added. Ca²⁺ traces from healthy myocytes sensitive to caffeine treatment were processed using a Savitzky-Golay filter and baseline Ca²⁺ levels, transient amplitude, caffeine-induced Ca²⁺ release, and Ca²⁺ decay kinetics were analyzed using Felix 1.1 and Ion Wizard (IonOptix) software. For cell shortening measurements in isolated adult myocytes, cells were bathed in media 199 (M199) at room temperature using an Ionoptix system as described previously [34 (link), 35 (link)] and myocytes were electrically paced at 0.5 Hz with 60V and the solution was refreshed between each measurement.