The overall goal of this procedure is to force perfusion mouse or rat lungs for the selective harvest of marginating-pulmonary leukocytes, which adhere to the inner endothelium of lung capillaries. This method allows the study of this specific leukocyte population, which exhibits unique characteristics and distinct hematological functions. The main advantage of this technique is that it yields leukocytes exclusively from the lung capillaries, excluding cells from the parenchymal, interstitial, and bronchoalveolar compartments.

Demonstrating this procedure, will be Liat Sorski and Lee Shaashua, two graduate students from my laboratory. Before beginning the procedure, stream heparinized PBS through the peristaltic pump lines and butterfly needles to remove any air bubbles. Then immediately upon cessation of respiration, use sterile scissors and tooth tissue forceps to open the peritoneal and chest cavities of the euthanized rat by performing a midline abdominal incision up to the xiphoid process.

Next, use the forceps to lift the sternum and cautiously cut the ribcage on both sides. Clamp a hemostat on the sternum and fold the ribcage rostrally to expose the cardiopulmonary complex. Then insert a butterfly needle into the right ventricle of the heart and collect the maximal amount of blood.

When all of the blood has been collected, clamp the vena cava with a hemostat to avoid backward perfusion of the liver. Then, holding the butterfly needle within the right ventricle, replace the syringe of blood with the outflow pipe of the peristaltic pump and insert a second butterfly needle, connected to a five milliliter harvesting syringe, into the left ventricle. Now turn the peristaltic pump on to a speed of approximately five milliliters per minute and gently collect the first milliliters of blood contaminated perfusate into the harvesting syringe until the perfusate turns from dark to pale red.

Then, without cessation of the pump, quickly replace the five milliliter harvesting syringe with a 20 milliliter harvesting syringe and collect 20 milliliters of lung perfusate to harvest the rat marginating-pulmonary leukocytes at a rate of seven milliliters per minute. Notice the gradual discoloration of the lungs from a pale red to a clear white, indicating a successful perfusion. When the entire volume of perfusate has been collected, cease the flow of the peristaltic pump and transfer the collected perfusate to a tube.

Then centrifuge the perfusate and aspirate the supernatant. Stream heparinized PBS through the peristaltic pump lines and butterfly needles to remove any air bubbles. To collect mouse marginating-pulmonary leukocytes, immediately upon the cessation of respiration use sterile scissors and tooth tissue forceps to open the peritoneal and chest cavities by performing a midline abdominal incision up to the xiphoid process.

Next, use forcepts to lift the sternum and cut the ribcage on both sides. Clamp a hemostat on the sternum and fold the ribcage rostrally to expose the cardiopulmonary complex. Clamp the vena cava with a hemostat to avoid backward perfusion of the liver and insert the butterfly needle into the right ventricle of the heart.

Then, holding the butterfly needle within the right ventricle, connect the butterfly needle to the outflow pipe of the peristaltic pump and insert a second butterfly needle, connected to a two milliliter harvesting syringe, into the left ventricle. Turn on the pump to a speed of approximately two milliliters per minute. Gently collect the first milliliter of blood contaminated perfusate into the harvesting syringe until the perfusate turns from dark to pale red.

Then, without cessation of the peristaltic pump, rapidly replace the two milliliter harvesting syringe with a 10 milliliter harvesting syringe and collect 10 milliliters of lung perfusate at a perfusion rate of four milliliters per minute. Notice the gradual discoloration of the lungs from a pale red to a clear white indicating a successful perfusion. When all of the lung perfusate has been collected, cease the flow of the peristaltic pump and transfer the collected perfusate to a tube.

Then centrifuge the perfusate and aspirate the supernatant. The marginating-pulmonary compartment exhibits a different leukocyte subset composition than that observed for circulating leukocytes. Indeed, innate immunocytes, including granulocytes, monocytes, and natural killer cells constitute over 50%of the marginating-pulmonary leukocyte population.

Compared to less than 30%of the circulating cell compartment. In contrast, the T cell and dendritic cell populations are found in lower percentages in the marginating-pulmonary compartment compared to the circulating cells. A three fold higher percentage of large natural killer cells are present in the marginating-pulmonary compartment than in the circulating cell compartment, although the total number of natural killer cells overall is similar.

Moreover, the ability of natural killer cells from the marginating-pulmonary compartment to lyse tumor cells is greater than that of the circulating natural killer cells, as measured by a standard chromium release assay against at least two different tumor cell lines. In addition, immune stimulation via in vivo Poly I:C administration produces a more profound impact on natural killer cytotoxicity of marginating-pulmonary natural killer cells than on circulating natural killer cells when MADB106 targets are used. Once mastered, this technique can be performed in a few minutes if it is performed properly.

These techniques enable the selective harvesting of MP leukocytes. The study of these leukocytes will advance our understanding of the biological and clinical significance, specifically in respect to controlling circulating aberrant cells and lung related diseases. Following this procedure, the cells can be characterized and analyzed by flow cytometry, PCR, or any other assay of interest.

After watching this video, you should have a good understanding of how to collect marginating-pulmonary leukocytes.