A Technique to Isolate Primary Mast Cells from a Murine Peritoneal Cavity
Transcript
To begin this procedure, spray the euthanized mouse with 70% ethanol, and fix it on a foam block with its dorsal side down using pins. Using blunt-edge scissors, remove the skin over the abdomen of the mouse and avoid damaging the peritoneal cavity.
Next, inject 7 milliliters of ice-cold RPMI medium in the peritoneal cavity by inserting a 27-gauge needle carefully in the peritoneum. Do not perforate any organs and use a spot in the region of the epididymal fat to reduce the risk of an organ perforation.
After injection, shake the mouse for 1 minute to detach the peritoneal cells into the RPMI medium. Do not shake the mouse too strongly to avoid damaging the internal organs and contaminating the peritoneal cavity with blood. Then, re-use the syringe by equipping it with a new 20-gauge cannula.
Shift the inner organs to one side by tilting the foam block and gently tapping it on the bench to make medium aspiration easier from the other side. Subsequently, insert a 20-gauge needle, bevel up, to aspirate the fluid from the abdomen gently and slowly to avoid clogging by the inner organs.
Collect as much fluid as possible. Then, remove the needle from the syringe and transfer the collected cell suspension in a collection tube on ice. Discard a sample tube if there is visible blood contamination.
Centrifuge the tube with clean cell suspension at 300 x g for 5 minutes. Under a sterile hood, aspirate the supernatant. Resuspend the sample pellets in cold PMC medium, and transfer the cell suspension to a 25-square centimeter cell culture flask.
Subsequently, add the growth factors IL3 and SCF to the final concentrations of 10 nanograms per milliliter and 30 nanograms per milliliter respectively. Incubate the cells for approximately 48 hours at 37 degrees Celsius, until the next procedure.
