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Journal of Histochemistry and Cytochemistry, Vol. 49, 519-528, April 2001, Copyright © 2001, The Histochemical Society, Inc.

ARTICLE

Identification and Purification of Functional Human ß-cells by a New Specific Zinc-fluorescent Probe

Bruno Lukowiaka, Brigitte Vandewallea, Rita Riachya, Julie Kerr–Contea, Valery Gmyra, Sandrine Belaicha, Jean Lefebvrea, and François Pattoua
a UPRS 1048/ERIT–M–INSERM, Université de Lille, Lille, France

Correspondence to: Brigitte Vandewalle, UPRES 1048/ERIT–M–INSERM, Université de Lille, 1 Place de Verdun, 59045 Lille, France. E-mail: bvandewalle@univ-lille2.fr

Pancreatic ß-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-sensitive fluorescent probe able to selectively label labile zinc in viable ß-cells and to exhibit excitation and emission wavelengths in the visible spectrum, making this technique exploitable by most instruments. We tested Newport Green, a probe excitable at 485 nm with a dissociation constant in the micromolar range corresponding to a low affinity for zinc. The loading of the lipophilic esterified form of Newport Green was easy, rapid, specific, and non-toxic to cells. Confocal microscopy highlighted an intense fluorescence associated with secretory granules. Regression analyses showed a good relationship between zinc fluorescence and islet number (r=0.98) and between 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 resulted in a clear separation of ß-cells, as judged by insulin content per DNA and immunocytochemical analysis. This zinc probe, the first able to specifically label living cells in the visible spectrum, appears very promising for ß-cell experimentation, both clinically and for basic research. (J Histochem Cytochem 49:519–527, 2001)

Key Words: human pancreatic ß-cell identification, human pancreatic islet purification, fluorescence-activated cell sorting, zinc-sensitive fluorescent probe


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