A technique for the isolation of highly viable pancreatic B-cells from ob/ob mice

A method has been developed to prepare free islet cells in suspension from adult ob/ob-mice. About 200 collagenase-isolated pancreatic islets were pooled in 4 ml of calcium-free Krebs-Ringer-HEPES buffer supplemented with 1 mM EGTA and 10 micrograms/ml DNAase. The islets were gently shaken in a water-bath for 10 min at 30 degrees C. Then, the cell suspension was filtered through a nylon screen and centrifuged through ice-cold, dense albumin. The isolated cells, of which more than 99% were B-cells, appeared well preserved both in light- and electron-microscopy. Out of the isolated cells, 7.1 +/- 0.5% took up Evans Blue and were thus considered non-viable.