Murine Pancreatic Islet Isolation скачать в хорошем качестве

Murine Pancreatic Islet Isolation

Reference: https://app.jove.com/v/255/murine-pan... The process of isolating pancreatic islets from mice involves several steps to obtain a pure population of these specialized cells. Pancreatic islets, also known as islets of Langerhans, are clusters of cells within the pancreas that play a crucial role in regulating blood sugar levels by producing and releasing hormones such as insulin and glucagon. 1. Sacrifice and dissection: The first step is to sacrifice the mice using approved ethical protocols. Once euthanized, the pancreas is carefully dissected out from the surrounding tissues. 2. Tissue digestion: The dissected pancreas is then minced into small pieces to increase the surface area for enzymatic digestion. These small tissue fragments are transferred to a digestion solution containing enzymes such as collagenase and trypsin. The enzymes break down the connective tissue and dissociate the pancreatic cells. 3. Enzymatic digestion: The pancreatic tissue is incubated in the digestion solution at an appropriate temperature for a specific duration. During this time, the enzymes break down the extracellular matrix and release the individual cells. 4. Filtration and washing: After digestion, the cell suspension is filtered through a mesh or nylon filter to remove any undigested tissue fragments and obtain a single-cell suspension. The filtered cells are then washed multiple times with a buffer solution to remove any remaining enzymes and debris. 5. Density gradient centrifugation: To separate the pancreatic islets from other cell types, a density gradient centrifugation technique is employed. The cell suspension is layered onto a density gradient medium, such as Ficoll or Percoll, and centrifuged at a specific speed and duration. This process separates the islets, which have a different density compared to other cells, into distinct layers. 6. Islet collection: After centrifugation, the islets form a visible pellet at the bottom of the tube. The pellet is carefully collected using a pipette or a needle under a microscope. The collected islets are then transferred to a culture dish or tube for further analysis or experimentation. 7. Islet purification: To obtain a pure population of islets, additional purification steps may be performed. This can include handpicking individual islets under a microscope or using automated techniques such as fluorescence-activated cell sorting (FACS) based on specific markers expressed by islet cells.