2.7. Size and Enumeration of Recovered Islets Individual mouse islets may range from smaller reference 4 than 50 to over 400 microns in size (Figure 2). For the purpose of this study, islets larger than 50 microns were counted by direct enumeration. Islets smaller than 50 microns were counted by combining 2�C4 islets depending on size, and counting them as 1 islet. The number and size of islets recovered varies with strain and age of the animal with retired female breeders having Inhibitors,Modulators,Libraries the largest and most per animal. The adult animals used in this study generally had islets in the same proportionate size ranges. In general, the size range of the individual islets Inhibitors,Modulators,Libraries are approximately 20% less than 50��m, 30% between 50 and 100��m, 35% between 100 and 250��m, and 15% greater than 250��m.

On average, this isolation method reliably recovers 150�C300 islets Inhibitors,Modulators,Libraries from an adult mouse (25g body weight or larger). Islets were counted immediately after isolation. For transplantation experiments, islets of size 100�C200��m were used to assure uniform islet mass between recipients. Figure 2 Normal mouse pancreatic Inhibitors,Modulators,Libraries islets after collagenase digestion and Ficoll separation with further purification by handpicking. Islets were stained with dithizone (red). The left panel shows islets isolated with BSA; the right panel shows islets isolated without … 2.8. Islet Functional Assessment-Perifusion Assay Insulin release in response to changing glucose conditions was determined with a perifusion assay for at least three different islet preparations [6].

For the assay, 75 islets of similar size from each of the BSA and non-BSA-treated islet groups Inhibitors,Modulators,Libraries were handpicked and cultured identically overnight in complete medium at 37��C and 5% CO2. After a 30 minute preincubation period, the islets were perifused for 30 minutes with KRB containing a subphysiological (low) glucose concentration (2.8mmol/l) followed by a 30 minute exposure to high-glucose conditions (20mmol/l) and returned to subphysiologic conditions for a 30 minute recovery period. Elution samples were taken once per minute for 90 minutes. Insulin concentration of the elution samples was determined by mouse insulin ELISA (Alpco, Windham, NH, USA). 2.9. Viability Assay Calcein-AM (1��L) (Molecular Probes, Eugene, OR) and propidium iodide (PI, 10��L) (Sigma-Aldrich, St Louis, MO, USA)are added to 1mL PBS and protected from light.

Islets are added to the mixture and incubated at 37��C for 25 minutes. Islets are examined by fluorescent microscopy immediately following incubation. Carfilzomib Image analysis of the percentage of dead cells (PI stained red) to live cells (Calcien-AM stained green) is calculated using Metaphor Image Analysis software (Molecular Devices, Downingtown PA, USA). The area of PI positive stain to the total area of the islets is measured. Results are expressed as the percentage of dead cells in the islet group. 2.10. Antibody Staining of Islets Islets are cytospun onto gelatin-coated slides.