Intracellular Ca2+ mobilization assay

LAD2 cells were loaded with 2 µM Fura-2 AM (Invitrogen) for 30 min at 37°C. The cells were washed three times and then resuspended in HEPES buffer with human albumin prior to challenging with different stimuli. Fura-2-loaded mast cells were viewed with an Olympus inverted IX51 microscope. The images were captured with a CCD camera at every 10 s intervals. Fluorescence images were obtained at wavelengths of 340 and 380 nm with an emission wavelength of 510 nm. The fluorescence ratio of 340 to 380 nm was measured and the overall levels of calcium influx were compared with area under the curve analysis. F₁/F₀ was the fluorescence ratio of time point X divided by the ratio of time point zero.