Identification and purification of functional human beta-cells by a new specific zinc-fluorescent probe

Pancreatic beta -cells contain large amounts of zinc. We took advantage of this to try to localize, quantify, and isolate insulin-producing cells from islet preparations. Our study was designed to identify a non-toxic zinc-se nsitive fluorescent probe able to selectively label labile zinc in viable b eta -cells and to exhibit excitation and emission wavelengths in the visibl e spectrum, making this technique exploitable by most instruments. We teste d Newport Green, a probe excitable at 485 nm with a dissociation constant i n the micromolar range corresponding to a low affinity for zinc. The loadin g of the lipophilic esterified form of Newport Green was easy, rapid, speci fic, and non-toxic to cells. Confocal microscopy highlighted an intense flu orescence associated with secretory granules. Regression analyses showed a good relationship between zinc fluorescence and islet number (r=0.98) and b etween zinc fluorescence and insulin content (r=0.81). The determination of Zn fluorescence per DNA enabled us to assess the quality of the different islet preparations intended for islet allografting in terms of both purity and viability. Cell sorting of dissociated Newport Green-labeled cells resu lted in a clear separation of beta -cells, as judged by insulin content per DNA and immunocytochemical analysis. This zinc probe, the first able to sp ecifically label living cells in the Visible spectrum, appears very promisi ng for beta -cell experimentation, both clinically and for basic research.