The isolation of peritoneal cavity cells begins with the mounting of a euthanized mouse on its back. The outer skin of the peritoneum is cut and to gently pulled back to expose the inner skin PBS with 3%FBS is injected into the peritoneal cavity, and the peritoneum is massaged gently. The fluid containing peritoneal cells is collected and placed in a 15 milliliter tube on ice.
The cells are washed once and counted on a hemo cytometer. Hi, I'm Oji from Dr.Bon's lab at the Blood Research Institute of Blood Center of Wisconsin. Today we will show you a procedure for isolating cells from the peritoneal cavity of a mouse.
We use this procedure in our laboratory to obtain cells for various phenotypic, bio biochemical and immunological assays. Primarily, we are interested in peritoneal B-cell populations. So let's get started.
Before beginning to isolate immune cells from the peritoneal cavity, prepare all necessary materials. Start by gathering an ice bucket, a five milliliter syringe with a 27 gauge needle, a five milliliter syringe with a 25 gauge needle, styrofoam block, and pins for mounting the mouse. A tray to hold the mounting block, scissors and forceps and collection tubes prior to the dissection.
Pre chill PBS containing 3%fetal calf serum. Keep these and the collection tubes on ice throughout the experiment following administration of anesthetic and appropriate euthanasia, spray the mouse with 70%ethanol and mount it on its back atop of styrofoam block or dissection pad. Using forceps and scissors, cut the outer skin of the peritoneum.
Gently pull the skin back to expose the inner skin that lines the peritoneal cavity. Being careful not to puncture any organs slowly push a 27 gauge needle into the peritoneum. Inject five milliliters of ice cold PBS into the peritoneal cavity.
Now gently massage the peritoneum to encourage cells to detach from the peritoneal wall and fall into the PBS solution. Then fit a five milliliter syringe with a 25 gauge needle bevel side up at this point, insert the needle into the peritoneum. Collect as much fluid as possible to boost your cell yield.
While you're doing this, gently move the tip to prevent fat tissue or other organs from clogging the needle. Remove the needle and empty the cell suspension into polypropylene collection tubes. Collect the remaining fluid from the cavity by making an incision in the inner skin of the peritoneum.
Use forceps to hold the skin apart while you use a plastic posterior pipette to collect any remaining fluid. Finally, centrifuge the cell suspension for eight minutes at 1500 RPM, discard the supernatant and resuspend the cells in five milliliters of PBS. Counting of the cells on a hemo cytometer.
Using trippen blue exclusion will allow the determination of cell numbers. Typically five to 10 million peritoneal cavity cells can be isolated from an unmanipulated mouse. 50 to 60%of the total cell population will be B cells.
About 40%will be macrophages and granula sites, and five to 10%will be T cells. We have just shown you how to isolate peritoneal cavity cells from a mouse. When using this procedure, it is important to limit the amount of blood in the peritoneal cavity cell population by euthanizing the mice carefully and not puncturing any organs in the peritoneal cavity.
Be sure to discard any samples that become contaminated with blood. So that's it. Thanks for watching and good luck with your experiments.
