, Pankreatik acinar hücreleri ve salgı kapasitesinin değerlendirilmesi

The overall goal of the following experiment is to assess the secretion capacity of isolated pancreatic asin, our cells. This is achieved by first isolating intact asinine from the murine pancreas. As a second step, the amylase secretion assay is performed, which evaluates the global secretory capacity of the asinine.

Next direct imaging of the secretion process is carried out in order to obtain the characterization of secretion at a subcellular resolution, the results obtained offer the researcher a broad spectrum of tools to study pancreatic secretion and by that, identify novel participants. In this multi-step process, The we will set up of pancreatic as sins. Advantages is isolated as sin retained many characteristics of the intact pancreas and can be manipulated and monitored more readily than in the whole animal.

Using this setup, pancreatic secretion can be quantified by measuring the relative amount of Amys that was released following stimulation. Alternatively, secretion can be imaged live by the use of different sensors and dyes. Let’s start by seeing how to isolate pancreatic asinine from the mouse pancreas.

Begin the procedure by preparing 300 milliliters of fresh KRBH. Then oxygenate the KRBH medium for 30 minutes. Aliquot five to 10 milliliters of medium and supplement it with collagenase.

Add STI and BSA to the Resus suspension and digestion mediums and bring them to 37 degrees Celsius. Next place the sacrificed animal on its back on the dissection pad and clean the abdominal surface with ethanol. Make an incision on the abdominal skin and subcutaneous layer and identify the liver, stomach, intestine, pancreas, and spleen.

After that, use two blunt forceps to pull out the small intestine from its proximal end, which is near the exit from the stomach. Subsequently, separate the pancreas from the intestine and other internal organs and retain it within the body cavity. A pink, white connective tissue should be seen once reaching the large intestine.

Then clear as much pancreatic tissue as possible without collecting fat or connective tissue. Remove the pancreas by cutting the abdominal blood vessels beneath it. Rinse the pancreas a few times in Resus suspension.Medium.

Remove the remaining parts of the non pancreatic tissues. After that, immerse the pancreas in five milliliters of preheated digestion medium. In a 20 milliliter scintillation vial, mince the pancreas into one to three millimeter pieces.

Next, place the vial in a heated water bath to accelerate digestion. Pipette the digested tissue up and down once every few minutes to terminate digestion, transfer the digested tissue into a 50 milliliter tube filled with 45 milliliters of Resus suspension.Medium. Subsequently, filter the suspension through a coarse metal mesh into a fresh tube.

Then centrifuge the flow through at 100 times G for three minutes. After that, discard the supernatant and resuspend the digested pancreas. In 50 milliliters of Resus suspension medium, filter the suspension through a 70 or 100 micron nylon mesh and centrifuge again, as before, in order to remove floating damaged, asinine, discard the supernatant and resuspend the pancreatic tissue.

In three to five milliliters of Resus suspension medium apply one milliliter portions of the pancreatic asinine into 15 milliliter conical tubes filled with seven milliliters of resus suspension medium. Then allow the asinine to settle for two to three minutes. Collect the asinine that have settled at the bottom of the tube with a one milliliter pipette and repeat this.

Step two to three times the amylase secretion assay serves as a global measure for the asar cells secretion capacity. In this procedure, use at least three replicates for each experimental condition to be examined, and add a set of replicates to determine the total cellular amylase content and a set for the amylase activity of the medium. Label a multi-well plate in which the assay will be conducted, and two sets of micro tubes for each of the replicates to be used.

Wash the asinine twice in 30 milliliters of KRBH Resus medium and incubate them in it for 30 minutes at 37 degrees Celsius. Afterward, add a 10 microliter drop of secretagogue to the multi-well plate. Then wash and resus.

Suspend the asinine in Resus medium. Ensure that the asinine are distributed homogenously in the suspension and aliquot equal amounts of suspension into the multi-well plate and the total labeled micro tubes. Incubate the plate in a water bath under mild agitation for 30 minutes at 37 degrees Celsius.

During incubation, produce the total and zero samples by first centrifuging the total micro tubes for a few seconds at 5, 000 times G.Next, transfer 50 microliters of supernatant to the zero labeled micro tubes and place them at four degrees Celsius. Lice the asinine in the total samples by adding Triton X 100 to the micro tubes to reach a final concentration of 1%Vortex the micro tubes vigorously at the end of the incubation period. Collect the ASIN R suspension into the first set of the pre-labeled micro tubes.

Centrifuge the micro tubes for a few seconds at 5, 000 times G.Next, transfer the supernatant into the second set of the pre-labeled micro tubes. These micro tubes contain the secreted amylase, which can be assayed by a commercial kit. For imaging, prepare freshly oxygenated resus suspension medium and bring it to 37 degrees Celsius.

Wash the As in our cells in 30 milliliters of Resus suspension medium, and place them in a glass or plastic slide. Transfer the slide to the microscope. Add secretagogue dropwise, or using a perfusion chamber during or immediately prior to the initiation of imaging.

Then image the asinine with clear morphology and avoid imaging. The asinine, which display basal lateral blebs or intracellular vacuoles the properly isolated pancreatic asinine display. A stereotypic morphology.

Their basal lateral domains appear round and devoid of blebs. While their apical domains are surrounded by hundreds of secretory vesicles and appear darker cell debris and components of the pancreatic ductal system can be detected at early stages of asinine isolation. Improper isolation results in the generation of basolateral blebs and the breakage of cells, which discharge their digestive enzymes into the medium live imaging of asinine allows clear identification of the basal and apical domains of asin.

Our cells lipophilic dye can be used for distinguishing between the narrow apical domains and the lateral and basolateral aspects of the cells Ds.The ex vivo setup of the exocrine pancreas offers an accessible mean to study polarized secretory epithelium. The key methodological step is to isolate intact pancreatic AC without significant signs of cell damage and mortality once this is achieved. Assessment of the global secretory capacity by the amela secretion assay is fairly straightforward.

Direct imaging of secretion on the other hand, requires more experience with the tissue, but is manageable waste time. Good luck.