The overall goal of this procedure is to selectively harvest marginating-hepatic leukocytes, immune cells that adhere to the sinusoids of the liver, by forced perfusion from mouse or rat livers. This method allows the study of the hepatic leukocytes which exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. The main advantage of this technique is that it yields leukocytes exclusively from the liver sinusoid uncontaminated by cells from other liver compartments such as parenchyma.
Demonstrating the procedure will be Liat Sorski and Lee Shaashua, two amazing graduate students from my laboratory. Before beginning the liver perfusion procedure flush heparinized PBS through the peristaltic pump lines and the butterfly needles to remove any air bubbles. Then, immediately upon the cessation of respiration use sterile scissors and tooth tissue forceps to make a midline abdominal incision starting at the lower abdomen of the euthanized rat up to the xiphoid process taking care not to damage the internal organs.
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. Then, prepare a 21 gauge butterfly needle connected to a 10 milliliter syringe and insert it into the right ventricle of the heart.
Next, use the 10 milliliter syringe to collect as much blood as possible. When all of the blood has been collected, use a hemostat to clamp the caudal vena cava as close as possible to the heart. Then, move the intestines out of the abdominal cavity to the left side of the animal to expose the portal vein.
Next, prepare a 25 gauge IV catheter connected to the outflow pipe of the peristaltic pump and insert it into the portal vein as caudal as possible while remaining rostral to the splenic vein. Then, ready a 21 gauge butterfly needle connected to a five milliliter syringe and insert it into the inferior vena cava caudal to the hemostat. Turn on the peristaltic pump at a speed of approximately three milliliters per minute and collect the first milliliters of perfusate contaminated with blood into the syringe.
Place the five milliliter harvesting syringe with the 20 milliliter harvesting syringe without stopping the pump when the perfusate becomes a pale red color. Then, collect at least 20 milliliters of liver perfusate at seven milliliters per minute. When the whole liver is light brown, terminate the perfusion.
Then, centrifuge the perfusate and aspirate the supernatant. 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 three milliliters per minute and collect the first milliliters of blood contaminated perfusate from the chest cavity pool. When three milliliters have been collected, turn off the pump.
Then, after inserting a 21 gauge butterfly needle connected to a 10 milliliter syringe, reinitiate the perfusion at a speed of up to four milliliters per minute until 10 milliliter 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. Whereas marginating-hepatic NK cells exhibit a profound cytotoxicity against allogeneic target cell lines both the entire liver cell population and the circulating leukocytes display profoundly lower levels of cytotoxicity and almost no killing against a syngeneic target cell line.
The same relative lower levels of cytotoxicity are evident when mechanically processed livers undergo washing and density gradient separation. Facts analysis of specific leukocyte populations reveals profound differences in the composition of the immune cell populations from the circulating and liver cell compartments. Once mastered, this technique can be completed in a few minutes if it is performed properly.
Following this procedure, the cell can be used for flow cytrometry, PCR, or any other methods for characterizing this important leukocyte population. This technique enables the selective harvesting of marginating-hepatic leukocytes to advance our understanding of the biological and clinical significance. Specifically we expect to controlling circulating aberrant cells and liver related diseases.
After watching this video, you should have a good understanding of how to collect margating-hepatic leukocytes.
