PURIFICATION OF PANCREATIC B-ISLET AND NON-B-ISLET CELLS LOADED WITH A FLUORESCENT CALCIUM INDICATOR

A new method was designed for the isolation of pancreatic B and non-B islet cells. It is based on flow cytometry of dispersed islet cells la belled with the calcium-sensitive fluorochrome fluo-3. In the presence of 16.7 mM D-glucose, two populations of cells were identified with a sevenfold difference in modal fluorescence intensity. They yielded a ratio in insulin/glucagon content about two thousand times higher in t he B than non-B cell population. The recovery of cells after sorting a mounted to 88.5 +/- 1.6%, and the cell viability exceeded 90%. In isol ated islets, the loading with fluo-3 did not alter the secretory respo nsiveness of the B cell to D-glucose. The purified B cells were furthe r characterized in terms of their intracellular space (757 +/- 49 fl/c ell) and the kinetics of 3-O-[C-14]methyl-D-glucose net uptake, which reached within 60 s an equilibrium value corresponding to 46 +/- 4% of the intracellular volume. This method thus allows to isolate with hig h recovery, purity and viability pancreatic islet B cells suitable for further metabolic and secretory investigations.