Fluorescent berberine binding as a marker of secretory activity in mast cells

We have previously shown that the weakly fluorescent cationic dye, berberine, forms a strongly fluorescent complex with the heparin of fixed mast cells and can be used for cytofluorometric measurement of heparin in both mast cells and individual mast cell granules. Now, we report on the use of berberine as a vital stain, demonstrating the secretory activity of mast cells. At a dye concentration of 0.025%, and after membrane stabilization with polyethylene glycol, normal mast cells exclude the dye while mast cells stimulated to secretion with polymyxin B show a strongly fluorescent dye binding to individual cytoplasmic granules. The mean fluorescence intensity (reflecting the number of stained granules) of the cell populations increased with increasing polymyxin B concentrations up to 2.0 micrograms/ml, thereafter remaining constant up to 10 micrograms/ml. Fluorescence intensity after vital staining with berberine was compared both to release of heparin measured by berberine binding to fixed cells (reflecting exocytosis of mast cell granules) and also to histamine release. The results strongly suggest that berberine specifically stains secreting granules that are located within the domains of the cells but may have released histamine. Vital berberine staining combined with histamine assays and cytofluorometric measurements of heparin is therefore of great potential interest for analyzing the dynamics of mast cell secretion.