Isolation of Marginating Hepatic Leukocytes from a Mouse Liver

Marginating hepatic, MH, leukocytes, are found adhered to the walls of specialized liver blood vessels or sinusoids, which are lined by endothelial cells. MH leukocytes are recruited to the surrounding tissue in response to inflammation.

To harvest MH leukocytes, begin with a euthanized mouse in a supine position. Incise the peritoneal and chest cavities, exposing the liver and cardiopulmonary complex.

Displace the intestines to expose the portal vein, which carries blood from the spleen, gastrointestinal tract, and its associated organs to the liver. Insert a needle connected to a peristaltic pump into the portal vein.

Dissect the inferior vena cava — a major blood vessel carrying deoxygenated blood from the liver to the heart — to collect the perfusate during the perfusion.

Perfuse the liver through the portal vein with buffer containing anticoagulant at a low flow rate for a short duration. This step helps to remove blood with non-adherent cells from the liver's blood vessels through the inferior vena cava outflow. Discard this blood-contaminated perfusate.

Now, perfuse at a higher flow rate. The forced perfusion at an increased flow and shear rates disrupts the adhesion between the MH leukocytes and blood vessel inner lining, thus detaching the MH leukocytes from the sinusoidal wall.

Collect and transfer the perfusate containing MH leukocytes and other blood cells into a fresh tube. Centrifuge. Resuspend the pellet containing MH leukocytes for further downstream analysis.

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.