Isolation of leukocytes infiltrating the islets of Langerhans of diabetes-prone mice for flow cytometric analysis.

A simple method was developed for flow cytometric immunofluorescence analysis of cells infiltrating the islets of Langerhans in diabetes-prone rodents. Pancreases were submitted to collagenase P digestion and, in order to minimize collagenase action on leukocytes, islets were isolated before digestion was completed, that is, when exocrine tissue still remained around the islets. Islets, maintained in medium supplemented with sodium azide to prevent modulation of surface markers, were then pressed through a metal sieve and the cell suspension filtered through two different nylon screens. Cells were washed before immunofluorescence staining. Comparative phenotyping of spleen cells submitted to a similar treatment showed that, provided the collagenase P batch was screened, this procedure did not alter leukocyte surface markers and allowed multicolor analysis of the islet infiltrating cells.