1. Islets of Langerhans were isolated from 5- to 7-week-old nonobese diabetic (NOD) mice.
2. Which involves cannulation of the common bile duct and distension of the pancreas with 3 ml of 1.3 U/ml collagenase followed by purification of islets on a BSA gradient.
3. Approximately 200 islets per pancreas were obtained using this method, and usually 4–8 mice were used per experiment.
4. For flow cytometric analysis, islet cells were identified.
5. Islets were dispersed into single cells by brief incubation with 0.2% trypsin, 10 mM EDTA in HBSS.
6. Dispersed islets were then washed free of trypsin and allowed to recover in primary cell system plus 10% FCS for 0.5–1 h before staining.
7. Islet cells were usually analyzed on the day of isolation or occasionally at later times.
8. If analyzed after the day of isolation, they were incubated in low glucose (2.5 mM) medium for 16–48 h before being dispersed.
9. β cells under both these conditions have high autofluorescence due to increased intracellular FAD levels allowing them to be distinguished from other intraislet cells for analysis or sorting.
10. No differences were observed between islets dispersed on the day of isolation or subsequently.
11. All high autofluorescence islet cells stained with the monoclonal antibody A2B5 and >85% were positive for insulin by indirect immunofluorescence.
12. Pseudo-islets were made by dispersing isolated islets with trypsin, as above, and then incubating the islets undisturbed for 7 days.

References
1. Nadya Lumelsky, Olivier Blondel, Pascal Laeng, Ivan Velasco, Rea Ravin and Ron McKay. Differentiation of Embryonic Stem Cells to Insulin-Secreting Structures Similar to Pancreatic Islets. Science. 2001; 292: 1389-1394.

2. Leigh A. Stephens, Helen E. Thomas, Li Ming, Matthias Grell RIMA DARWICHE, Leonid Volodin and Thomas W. H. Kay. Tumor Necrosis Factor-α-Activated Cell Death Pathways in NIT-1 Insulinoma Cells and Primary Pancreatic β Cells. Endocrinology. 1999; 140: 3219-3227.

3. Kay T, Parker JL, Stephens LA, Thomas HE, Allison J. Rip-beta(2)-microglobulin transgene expression restores insulitis, but not diabetes, in β(2)-microglobulin(null) nonobese diabetic mice. J Immunol. 1996; 157: 3688–3693.

4. Lake SP, Anderson J, Chamberlain J, Gardner SJ, Bell PR, James RF. Bovine serum albumin density gradient isolation of rat pancreatic islets. Transplantation. 1987; 43: 805–808.

5. Pipeleers DG, in’t Veld PA, Van de Winkel M, Maes E, Schuit FC, Gepts W. A new in vitro model for the study of pancreatic A and B cells. Endocrinology 1985; 117: 806–816.