Defective Ca²⁺ flux in FcϵRI-stimulated 4-1BB^-/- mast cells. (A) Wt and 4-1BB^-/- BMMCs that had been sensitized overnight with IgE were loaded with Indo-1 am. Continuous monitoring of the fluorescence ratio (525:405 nm) was performed on washed cells using a flow cytometer. Baseline fluorescence ratios were collected for 100 seconds before different concentrations of DNP₂₃-HSA was added (indicated by ↓). Ionomycin was added to all samples after 300 seconds of observation of calcium mobilization by IgE + Ag stimulation (indicated by ↑). Results shown are representative of 3 experiments. (B) Wt and 4-1BB^-/- BMMCs that had been sensitized overnight with IgE were stimulated by the indicated concentrations of ionomycin alone or ionomycin and DNP₂₃-HSA for 45 minutes (histamine release) or 8 hours (cytokine production). Ratios of histamine release and cytokine production in 4-1BB^-/- versus wt BMMCs are plotted as function of ionomycin concentrations. Results shown are representative of 2 experiments.