Isolation of Marginating Hepatic Leukocytes from a Mouse Liver
Transcription
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.
