Isolation of Marginating Hepatic Leukocytes from a Mouse Liver

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Mouse Protocol

1. Perfusion of the Liver and Collection of MH-Leukocytes
   1. Euthanize the animal with an overdose of 8% isoflurane. As a precaution, place a 15 ml tube containing an isoflurane-soaked pad around the mouse's head until opening its chest cavity.
   2. Fix the limbs of the mouse to a bed.
   3. Open the peritoneal and chest cavities to expose the liver and the cardiopulmonary complex immediately upon cessation of respiration, keeping the lower aspect of the diaphragm intact (to create a draining chest cavity pool). Avoid bleeding through this process.
      1. Specifically, start the incision at the lower abdominal midline point, without damaging internal organs, and progress to both sides diagonally toward the ribs, cutting through them to upper chest-cavity levels.
   4. Pull out the intestines and place them outside the animal to the experimenter's right, to expose the portal vein.
   5. Insert a 30 g needle, connected to a peristaltic pump into the portal vein rostral to the splenic vein.
   6. Cut the inferior vena cava above the diaphragm to allow drainage and collection of the perfusate from the chest cavity.
   7. Turn on the peristaltic pump at a speed of approximately 3 ml/min and gently collect the first 3 ml of perfusate that is contaminated with blood from the chest cavity pool.
   8. Discard this perfusate. Repeat such a washing again if needed, by using saline, and only then start collecting liver perfusate.
   9. Re-initiate the perfusion at a speed of up to 4 ml/min, and collect 10 ml of perfusate from the chest cavity using a butterfly connected to a syringe.
   10. Terminate perfusion when the color of the liver turns light brown.

2. Leukocytes Extraction

1. Centrifuge the perfusate for 10 min at 400 x g.
2. Aspirate the supernatant.