Calcium Flux Dynamics in Eosinophils

Experiments to analyze changes in calcium flux were performed according to the same procedure as described recently [67 (link)]. Purified eosinophils were labeled with 3 µM Fluo-4 (Molecular Probes, Eugine, OR, USA) and stimulated with 0.1, 1, 10, or 100 µM capsaicin (Merck, Darmstadt, Germany) during the measurement. Ionomycin (500 nM) (ThermoFisher Scientific, Waltham, MA, USA) was used as the positive control and RPMI medium (VWR International, Leuven, Belgium) as the negative control. For statistical analysis, the factor of the intracellular fluorescence was calculated through comparing the intensity peak after application of stimulants at 60 s to the baseline (mean value from 40 to 45 s). This experiment was also conducted with 100 µM capsaicin after eosinophil priming with IL-3 (10 ng/mL) (PeproTech, Cranbury, NJ, USA) for 20 min at 37 °C and 5% CO₂.