Collection of Peritoneal Cells
- Prepare a 10ml syringe fitted with a 26G short needle and filled with 5 to 7 ml of medium and 2 to 3 ml of air. Air is important.
- Prepare a Pasteur pipette with rubber pipetter.
This protocol has been orginated by Alan M Stall in the Leon and Lenore Herzenberg's Lab at Stanford (Ref). Shaking the mouse was found to be necessary for optimum yield of B1 cells (AM Stall personal communication).
- Euthanize by CO2 inhalation. Ask your Comparative Medicine Department for procedures and facilities. Make sure your mouse is dead before starting.
- If you need to collect blood from heart, take with a lateral approach to the thorax, so you don't spill blood in the peritoneum.
- Nick the abdominal skin below the sternum, taking care not to nick the peritoneum.
- Grab the two sides of the cut and gently rip apart the abdominal skin with a firm but gentle movement, exposing the sternum and the pelvis (A image). Be careful not to rip major abdominal vessels or you will get blood in peritoneum.
- Grab the sternum and gently fill the abdominal cavity with the medium and the air (B image). Make sure to keep the needle firm and steady so you make the smallest possible hole. Remove the needle. The peritoneum should self-seal.
- Hold the mouse in your hand and shake (image) it vigorously about ten-fifteen times. Put it back on the table.
- Grab the sternum and some peritoneum with a tweezer (C image). You should not loose the grip until you are done.
- With a swirling movement insert the air-filled Pasteur pipette in the peritoneal cavity ( C inset image).
- Express the air in the cavity, distending the peritoneum and start to aspirate the medium and the peritoneal cells (C image). Your sucess depends of the distension of the peritoneum.
- Collect all the fluid you inserted.
- While still holding the substernal peritoneum, open up the abdominal wall longitudinally (D image) and collect the remaining fluid.