Fig. 4

In vitro studies using bone marrow-derived mast cells confirm that mast cells store noradrenaline. A Representative images of bone marrow-derived mast cells (BMMCs) immunostained with antibodies against noradrenaline and c-kit, suggesting that the mast cells store noradrenaline. Note the heterogeneous staining pattern of noradrenaline (arrows). Scale bar: 50 µm. Four independent experiments were performed, and a representative image is shown. B BMMCs or HL-60 cells are stained with antibodies against noradrenaline. The negative control panel confirmed that signals were from the primary antibodies. Note that no signal was observed in HL-60 cells. Scale bars: 20 µm. Three independent experiments were performed, and the representative images are shown. C Representative image of BMMCs treated with NeuroSensor 521, a chemical fluorescent probe for noradrenaline, confirming that the mast cells store noradrenaline. Scale bar: 20 µm. Three independent experiments were performed, and a representative image is shown. D Representative image of BMMCs stained with antibodies against noradrenaline and mMCP-1 (left panel) or mMCP-4 (right panel). Noradrenaline-storing mast cells are negative for mMCP-1 but positive for mMCP-4 (yellow and white arrows), confirming that the noradrenaline-storing mast cells are CTMCs. Scale bars: 50 µm. Three independent sets of experiment were performed, and the representative images are shown. E Representative image of immunoelectron microscopic analysis of BMMCs using noradrenaline antibodies and secondary antibodies labeled with 10 nm gold nanoparticles. Note that the particles were abundantly detected in the secretory granule (arrow). Scale bar: 2 µm. Two independent experiments were performed, and a representative image is shown. F and G Gold nanoparticles indicate that noradrenaline was predominantly found in cells with bright cytoplasm (arrows). Scale bars: 5 µm or200 nm