Intracellular calcium measurement

Eosinophils (1 x 10⁵cells/mL) were incubated with 2.5 μM Fura-2/AM for 50 minutes at room temperature. Extracellular Fura-2 was washed and then removed. Initially, the experiments were done using a photomultiplier from (Photon Technology, Princeton, NJ) in a modified 3-compartment superfusion chamber with the bottom formed by the coverslip (27). Eosinophils were chosen and excited at 340- and 380-nm wavelengths and measured the ratio of emission at 510 nm (340 nm/380 nm). Thus, the approximate intracellular calcium concentration was calculated from ratio of fluorescence at each wavelength excitation [27]. Eosinophils were perfused with standard saline at room temperature and drugs were applied in the initial mixing compartment, reaching the cells at a final concentration depicted in Fig legends.

Some experiments were performed, using a FlexStation III plate reader (Molecular Devices). For these assays, cells were loaded with 2 μM Fluo-4AM calcium indicator dye (Molecular Probes), with 0,02% pluronic acid F-127 (Molecular Probes), 250 μM sulfinpyrazone (Sigma) in Dmem F/12 (Sigma) with 10% FCS. Loading was performed for 60 minutes at 37°C. The medium was removed and wells were washed twice and replaced with Krebs buffer n (132 mM NaCl, 4 mM KCl, 1.4 mM MgCl2, 2.5 mM CaCl2, 6 mM glucose, 10 mM HEPES, pH 7.4). Cells were maintained for 5 minutes at 37°C before measurements started and P2 receptor agonists were injected automatically. Fluorescence was measured using a 494 nm excitation and 525 nm emission (cut off 515 nm) and fluorescence measurements were taken every 2 seconds.