Figure 6.

Altered activation phenotype in Fcgr2^−/− BMMCs. (A) Murine WT and Fcgr2b^−/− BMMCs were challenged with increasing concentrations of DNP-HSA for 4 h. IL-6 release in the culture supernatant was measured by ELISA. (B) WT and Fcgr2^−/− BMMCs were challenged with increasing Ag concentrations for 30 min and β-hexosaminidase release was measured. (C) LAMP-1 assay was performed to analyze differences in the kinetics of vesicle fusion between WT and Fcgr2^−/− BMMCs. Cells were stimulated as depicted, stained with a FITC-conjugated α-LAMP-1 antibody and analyzed by flow cytometry. (D,E) SPE-7 sensitized WT and Fcgr2^−/− BMMCs were stained with the Ca²⁺-sensitive fluorophores Fluo-3 and Fura Red. (D) Steady state fluorescence was measured for 1 min, to determine the Ca²⁺ signal in unstimulated cells. Indicated Ag concentrations were added and changes in cytosolic Ca²⁺ levels were measured for another 4 min. (E) Differentiation between intra- and extracellular Ca²⁺. Steady-state fluorescence in WT and Fcgr2^−/− MCs was measured for 1 min. 1 mM EDTA was added to chelate extracellular Ca²⁺ and the fluorescence signal was measured for another minute. After 2 min cells were stimulated with DNP-HSA at either 20 or 2000 ng/ml and the resulting increase in cytosolic Ca²⁺ was measured for 2.5 min. Finally, 2 mM CaCl₂ was added to refill extracellular Ca²⁺ stores and the resulting SOC influx was measured for 2.5 min. Results shown in this figure are representatives of at least 3 independent experiments and in the diagrams depicted as mean ± SD. ^*p < 0.05, ^**p < 0.005, ^***p < 0.0005.