Isolation of Marginating Hepatic Leukocytes from a Mouse Liver

To collect mouse marginating-hepatic leukocytes, immediately upon the cessation of respiration, use sterile scissors and tooth tissue forceps to make a midline abdominal incision up to the xiphoid process. Next, use the forceps to lift the sternum and carefully cut the diaphragm and rib cage on both sides.

Clamp a hemostat on the sternum and fold the rib cage rostrally to expose the cardiopulmonary complex. Move the intestines outside of the abdominal cavity to the left of the animal, exposing the portal vein. Then, connect a 30-gauge needle to a peristaltic pump, and insert it into the portal vein, rostral to the splenic vein.

Cut the inferior vena cava above the diaphragm to allow the drainage and collection of the perfusate from the chest cavity. Then, turn on the peristaltic pump at a speed of approximately 3 milliliters per minute, and collect the first milliliters of blood-contaminated perfusate from the chest cavity pool. When 3 milliliters have been collected, turn off the pump.

Then, after inserting a 21-gauge butterfly needle connected to a 10-milliliter syringe, re-initiate the perfusion at a speed of up to 4 milliliters per minute until 10 milliliters of perfusate are collected from the chest cavity. When the liver turns light brown, terminate the perfusion. Next, transfer the perfusate to a 15-milliliter tube. Centrifuge it, and then, aspirate the supernatant.