mice islet isolation
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.