Fluorescent Berberine Binding as a Marker of Secretory Activity in Mast Cells

Abstract

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 μg/ml, thereafter remaining constant up to 10 μg/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.

Different concentrations of berberine result in distinct cellular localization patterns and cell cycle effects in a melanoma cell line

“…Although it has been utilized as a Xuorescent stain for mast cells [15,16], sperm and oocytes [17], DNA and RNA [18][19][20][21], and mitochondria [22], the kinetics of its uptake and distribution in living cells has not been extensively characterized. In this study, 25 and 50 M berberine strongly labeled mitochondria, with little detectable cytoplasmic or nuclear Xuorescence.…”

On the Heterogeneity of Mast Cells

“…This dye forms a highly fluorescent complex with heparin and may be used both to visualise rat connective tissue mast cells and for the quantitative cytofluorometric de termination of their content of the proteogly can [14,15]. Again, gut mucosal mast cells of the rat do not stain with this reagent.…”

Mast Cell Secretion

“…The optical conditions used for fluorescence measurements af ter vital berberine staining (Berlin and Enerback, 1983], and for cytofluorometric measurement of heparin | Berlin and Enerback, 1978], were as described previously.…”

“…1980]. Heparin does not appearto be released from the gran ular matrix in the early phase of the secretory re sponse [Gustavsson and Enerback, 1980], We have recently reported the use of the cationic dye berberine as a vital stain for demonstrating in tracellular granules in contact with the extracellular environment [Berlin and Enerback, 1983]. Vital ber berine staining was combined with cytofluorometric heparin quantitation by berberine binding to fixed cells [Enerback.…”