In vitro staining of islets of Langerhans for fluorescence-activated cell sorting.

A previously described technique from the author's laboratories for purification of pancreatic islets by fluorescence-activated cell sorting used the dye neutral red (NR) to obtain specific fluorescence of islets sufficient to give a sorting signal. A major drawback with this technique was the need to inject the dye intravascularly before excision of the pancreas. Preliminary investigations showed that NR would produce selective staining of islets by topical application in vitro but only at low concentrations that were insufficient to give fluorescence strong enough for sorting. The chelating agent dithizone (DTZ) produces bright red staining of islets by topical application in vitro. Further studies showed that dithizone-stained islets exhibited moderately strong fluorescence that faded too quickly for reliable sorting. By combining both NR and DTZ staining in vitro, selective fluorescence of islets was obtained that was sufficient to allow efficient sorting. Using the combined DTZ/NR stain the yield of islets obtained by sorting from a single rat pancreas was 569 +/- 72 (n = 16), corresponding to 83% of the islets present in the digest. The mean purity of the preparation, confirmed by histologic examination, was 80%. The viability of the islets was shown to be good both by supravital staining and by the successful correction of streptozotocin diabetes in syngeneic rats following transplantation of sorted islets.