Isolation and purification of rat islet cells by flow cytometry

Flow cytometry has been employed as a method to study homogeneity of isolated islet subpopulations. After collagenase digestion of rat pancreas and elutriation of tissue fragments, islets were isolated and dissociated, and cells were analyzed and sorted according to their low forward angle light scattering properties by using automated flow cytometry. A standardized procedure was developed for the preparation of rat islet cell grafts for purification of islet cells. In this process, after collagenase digestion of pancreas, islets were isolated, dissociated, identification by dithizone method and then with enzymatic procedure by DNase and trypsin, the islet cells changed into single cells and beta cells were identified by immunofluorescence method and then assayed by flow cytometry. Methods have been developed for the preparation of suspension of viable rat pancreatic islet cells and their analysis and sorting in the fluorescence activated cell sorter (FACC IV, Becton Dickinson, Sunnyvale, Ca). Flow cytometry of these cells indicated that there were 91% of beta cells in cell suspension. Most of the exocrine particles were lost during digestion. Purified endocrine islet cell grafts were prepared by pure beta-cells, without endocrine non-beta cells. The purified aggregates were devoid of endocrine non-beta cells and damaged cells.