28820 Single Oak Drive Temecula CA 9259 - PDF document

28820 Single Oak Drive • Temecula, CA 9259 0 www.millipore.comSCR045 The pancreas functions as both an exocrine and endocrine organ. Exocrine functions include the secretion of digestive enzymes (trypsinogen, chymotrypsinogen, pancreatic lipase, and amylase) from pancreatic acinar cells into the gastrointestinal tract. Endocrine functions occur within small clusters of heterogeneous cells, called the islets of Langerhans that are dispersed throughout the pancreatic tissue. The pancreatic islets comprise specific Gamma cells: Somatostatin Diabetes results from the loss or dysfunction of insulin-producing beta-cells in the pancreas. Cell replacement therapies through islet transplantation is considered a potential long term approach to the control of blood glucose levels. However obtaining sufficient quantities of insulin-producing tissues for islet transplantation remains a major impediment. Stem cells pancreatic progenitors) could potentially provide an Millipore’s Pancreatic Islet Cell Characterization Kit (SCR045) provides a convenient set of validated antibodies that allows researchers to reliably identify mature pancreatic islets cells. Al

ong with antibodies generated against discrete hormones secreted by alpha, beta, delta and gamma cells of the pancreatic islets, the kit includes PDX-1 (pancreatic duodenal homeobox gene-1), a master regulator of islet cell development and GLUT-2, a glucose 1. Culture the pancreatic cells on a 8-well chamber slide in the appropriate culture 2. Carefully aspirate the medium and fix the cells with 4% paraformaldehyde in 1X PBS for 15-20 minutes at room temperature. 28820 Single Oak Drive • Temecula, CA 9259 0 www.millipore.com3. Carefully aspirate the fixative and rinse three times (5-10 minutes each) with 1X 4. Apply a blocking solution (5% normal donkey serum 0.3% Triton X-100 in 1X PBS) for at least 2 hours at room temperature or overnight at 4C. IMPORTANT: Do not 5. Dilute the primary antibodies included in this kit to working concentrations in the blocking solution. For optimal results, the following antibody dilutions are 6. For a negative staining control, add an equivalent concentration (in 0.5 mL blocking 7. Incubate the cells in primary antibodies overnight at 48. The next day, wash the cells twice with9. At the completion of the last

wash, leave the cells in blocking solution for at least 30 10. Dilute secondary antibodies in the blocking solution just before use. We typically use donkey anti-mouse IgG Cy3 conjugated (Millipore Cat. No. AP192C) and donkey anti-rabbit IgG Cy3 conjugated (Millipore Cat. No. AP182C) antibodies at a 11. Overlay the cells with the appropriate donkey anti-mouse and anti-rabbit secondary antibodies that are conjugated to fluorescent molecules for 2 hours at room 12. Wash 3-5 times (5-10 minutes each) with 1X PBS. 13. Counterstain the cell nuclei with DAPI / 1X PBS Solution 14. Mount a glass coverslip over the chamber slides using antifading mounting solution 15. Visualize the cell staining with a fluorescent microscope. For further information on any of the included antibodies, please see the individual antibody oduct will occasionally become entrapped in products with volumes of 200 L or less, we recommend gently tapping tquid in the container’s cap. 28820 Single Oak Drive • Temecula, CA 9259 0 www.millipore.com 2006 - 2009: e works may be reproduced in any form without permission in writi *For color images, please view datasheet at http://www.m