samples were concentrated and buffer exchanged using 5 kDa molecular weight cut-off spin concentrators

o glucose administration. Prior to the GSIS assay, mice were fasted for approximately 1518 hrs. Glucose was administered via intraperitoneal injection of a 30% dextrose SGI-1776 price solution at a dose of 3.0 g/ kg body weight, and blood was collected prior to and at combinations of 5, 10, 30, or 60 mins after glucose administration. Blood samples of approximately 2040 mL were collected by retroorbital sinus puncture and transferred to microtainer tubes containing blood/serum separation gel. Serum was collected after spinning these tubes at 100020006 g for 510 mins. The ELISA used for human C-peptide was the same as used previously,. We regard the lower limit of accurate quantitation of human C-peptide in serum samples to be 50 pM. The level of cross-detection of mouse C-peptide is,1%, or a maximum of 30 pM in glucose-stimulated control mice. The level of cross-detection of human insulin is,0.0006% and,1.8% for proinsulin. Streptozotocin Treatment of Mice Mice received 70 mg STZ per kg body weight, through intra-peritoneal injection on five consecutive days for a total dose of 350 mg/kg as described previously. Under these conditions non-implanted animals typically reach blood glucose levels greater than 300 mg/dL, indicating depletion of endogenous b-cells. For blood glucose measurements, tail vein samples were tested using glucometers specifically calibrated for measuring rodent blood glucose values. Statistical Analyses One-way ANOVA was performed for all statistical analyses using the StatPlus package for Excel, with p,0.01 considered significant. Graft Preparation and Implantation Pancreatic populations were grafted to the EFP as described previously. Most aggregates were collected and prepared at day 12 of differentiation, while some were maintained in Stage-4 medium until d14 or d16 before preparation. Briefly, the aggregates were allowed to settle by gravity and 5 mL of cell aggregate slurry was transferred onto 6 mm diameter by 0.81.0 mm thick gelatin foam disks that were prewetted with RPMI 1640 with 2% FBS. The implantable constructs were transferred to culture dishes with prewarmed medium until implantation, typically the same day. For implantation surgery, male SCID/Bg mice 613 wks of age were anesthetized by intra-peritoneal injection of an anesthetic cocktail and abdomens were shaved and swabbed with povidone iodine solution. A 1-cm midline incision was made through both skin and abdominal muscle layers, and one of the EFP was carefully externalized through the incision and placed on a salinedampened gauze pad. A single implant construct was wrapped in the EFP and fixed in place with a small amount of veterinary Histopathological Analysis Grafts from mice were explanted and fixed in 10% neutral buffered formalin for paraffin embedded blocks, and in 4% paraformaldehyde PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189542 for 4 hrs in 4uC for frozen blocks, followed by washing with PBS for 3 hrs and then equilibration in 30% sucrose at 4uC overnight. Samples of cell aggregates were made into frozen blocks by fixing with 4% paraformaldehyde for 30 mins and then with 30% sucrose at 4uC overnight. The paraffin embedded specimens were sectioned at 5 mm and OCT compound mounted frozen blocks were sectioned at 8 mm. Standard histological hematoxylin and eosin staining were performed on both graft and cell aggregates. The H&E images were captured by a Nanozoomer digital slide scanner. Immunofluorescence analyses of aggregates and grafts were performed as described previously,,,. The antibodies us

Leave a Reply