Primary Acinar Cell Isolation: A Method to Isolate Acinar Cells from Mouse Pancreas
筆記録
– A pancreatic acinar cell is an exocrine cell that contains a variety of digestive enzymes. Sometimes, in response to oncogenic signaling, the acinar cell converts to a ductal cell. This ductal cell can later become cancerous. Therefore, it's vital to isolate the acinar cells to study their role in pancreatic cancer progression.
Begin by isolating the pancreas from a euthanized mouse. Add the pancreas to a weigh boat containing a balanced salt solution supplemented with an antibiotic. Mince the pancreas into small pieces using scissors and add the pancreatic tissue to a tube. Next, add collagenase solution to the pancreas pieces in the tube.
Collagenase digests the collagen that holds the tissue together so that the tissue breaks into fine pieces. Now, add serum-containing media to the tube and place the tissue on ice. This helps to stop the action of collagenase.
Filter the tissue and media twice, progressively decreasing the mesh size of the filter each time to obtain fine tissue suspension. Finally, centrifuge the resulting suspension to collect the acinar cells in the pellet. Remove the supernatant and store the acinar cells for further analysis. In the following protocol, we will isolate acinar cells from the pancreas of a euthanized mouse.
– To dissect the pancreas from a euthanized mouse, first pin the paws of the mouse to the polystyrene lid, orienting it so that the tail is facing the researcher, and spray the abdomen with 70% ethanol. Use autoclaved forceps to lift the fur and skin at the midline, and with autoclaved scissors, make an incision through the fur and skin from the urethral opening to the diaphragm-rib cage area. Make additional incisions to the left and right to cut away the fur and skin, creating a clear view of the abdominal cavity.
Then, cut into the peritoneal lining down the middle and to the right and left, and pull it away from the organs. Use the forceps to lift the intestines and move them to the left side of the mouse, creating space to see the light pink pancreas attached to the dark red oval spleen. The pancreatic tissue will have soft, spongy texture. Cut out the pancreas, which runs along the stomach, intertwining with the intestines.
Separate the spleen from the pancreas and place the pancreas in a 50-milliliter tube containing 10 milliliters of HBSS with 1X penicillin-streptomycin and transfer it to the laminar flow hood. Pour the contents of the tube into an empty weigh boat and use forceps to wash the pancreas by swirling it. Then use the forceps to move the pancreas to a second weigh boat containing HBSS with penicillin-streptomycin and wash again by swirling.
Move the pancreas to the third weigh boat containing HBSS with penicillin-streptomycin and begin cutting the pancreas into 5-millimeter or smaller pieces. To pour the contents of the weigh boat into a fresh 50-milliliter tube, first use the forceps to move the pancreas pieces into the liquid as the boat is being tipped. Once the pieces are no longer attached to the boat, pour its contents into the tube. Pick up any remaining pieces with the forceps and wash the forceps in the tube.
After centrifuging the tube at 931 times gravity at 4 degrees Celsius for two minutes, use a 5-milliliter pipette to remove the HBSS and any floating fat. Add 5 milliliters of collagenase diluted in HBSS. Close the tube and ensure a sealed lid by wrapping the tube in plastic paraffin film. Place it in an incubator, shaking at 220 RPM at 37 degrees Celsius for 20 minutes, which will yield a turbid solution and few remaining tissue pieces.
To stop the dissociation, place the tube on ice and add 5 milliliters of cold HBSS with 5% FBS. After centrifuging at 931 times gravity at 4 degrees Celsius for two minutes, remove the supernatant using a 5-milliliter pipette. Resuspend the pellet in 10 milliliters of HBSS with 5% FBS and centrifuge again at 931 times gravity for two minutes at 4 degrees Celsius.
Remove the supernatant using a 5-milliliter pipette and repeat this step with another 10 milliliters of HBSS with 5% FBS. Then resuspend the pellet in 5 milliliters of HBSS with 5% FBS. Use a P1000 pipette to filter the cell suspension 1-milliliter at a time through a 500-micrometer mesh into a 50-milliliter tube.
To wash any remaining pancreatic cells through the mesh, add an additional 5 milliliters of HBSS with 5% FBS. Then use a P1000 pipette to pass this filtrate 1-milliliter at a time through the 105-micrometer mesh into a 50-milliliter tube. Gently pipette this cell suspension into a 50-milliliter tube containing HBSS with 30% FBS, which will form a layer of cell suspension at the top.
Centrifuge this cell suspension at 233 times gravity at 4 degrees Celsius for two minutes and remove the supernatant. Resuspend the pellet containing the acinar cells in 1X Waymouth's complete medium.
