The protocol describes the isolation of pulmonary IMS and adoptive transfer after IL-33 determination in a mouse model, which can facilitate the in vivo study of idiopathic pulmonary fibrosis. The technique enables the researchers to explore the function of macrophages, simulated by traditional cytokines in developing IPF. To begin, take out the vial of clodronate liposomes from the refrigerator.
Allow it to warm for 30 minutes at room temperature and invert several times to ensure uniform mixing. Aspirate 60 microliters of clodronate liposomes using a pipette with a sterile suction tip. Administer the control and drug drop-wise into the nasal cavity of the anesthetized mouse.
After each drop, ensure the mouse inhales the drug completely and breathes evenly. After the depletion of alveolar macrophages in the recipient mouse, begin by disinfecting the skin of the anesthetized host mouse with 75%alcohol and iodine. Use scissors to cut through the skin and expose the cardiopulmonary tissue.
Aspirate 10 milliliters of PBS in a syringe equipped with a 20-gauge needle, and insert the tip of the needle into the right atrium of the mouse. Then cut the inferior vena cava of the mouse with surgical scissors and manually perfuse the mouse with PBS at a constant speed of 10 to 20 milliliters per minute until the lung tissue turns white. Further, excise the lung tissue and transfer it into ice cold PBS in a culture dish.
Cut the lung tissue into fragments and add 15 milliliters of DMEM medium containing 1%collagenase A to isolate the macrophages, then incubate it at 100 RPM for 30 minutes on a 37 degree Celsius shaking table. Aspirate the lung tissue suspension approximately 20 times through a 10-milliliter syringe to make it fine. Filter the suspension through a 40-micrometer cell strainer.
Centrifuge the filtrate at 400 G for 10 minutes. After discarding the supernatant, lyse the red blood with three milliliters of cell lysis buffer on ice for two to three minutes. Centrifuge at 150 G for five minutes and resuspend the cell pellet in 10 milliliters of DMEM.
Then count the cells using a hemocytometer. Seed the cells into a 10-centimeter adherent cell culture dish and incubate for one hour at 37 degrees Celsius and 5%carbon dioxide to adhere the lung interstitial macrophages to the dishes. After one hour, aspirate the supernatant and floating cells.
Add 10 milliliters of the fresh, complete culture medium and incubate for over eight hours or overnight. To stimulate the isolated interstitial macrophages, replace the spent culture medium with a complete medium interleukins 33. Also prepare a control plate by adding PBS instead of interleukins 33.
Incubate the plates for 24 hours at 37 degrees Celsius and 5%carbon dioxide. To dissociate the pulmonary interstitial macrophages, treat them with one milliliter of 0.25%trypsin for five minutes. Then add three milliliters of fresh culture complete medium to quench the digestion and centrifuge the cell suspension to harvest the pulmonary macrophages.
After counting the cells, resuspend the cells in PBS to a final concentration of five times 10 to the fifth cells per 50 microliters. To begin the transfer of interstitial macrophages, fix the limbs of the anesthetized recipient mouse on a vertical plate with medical tape and gently pull the mouse's tongue to one side, using a cotton swab. After turning the intubation lamp on, shine it on the throat of the mouse to see the trachea, which should be close to the base of the tongue.
Insert the indwelling needle cannula into the trachea using the 22-gauge intubation lamp and 0.4 millimeter diameter guide wire. To finish the intubation, remove the guide wire and push the cannula into the mouse trachea. Now inject 50 microliters of the cell suspension into the recipient mouse through the trachea when the cannula is seen to enter the trachea.
After 24 hours, administer bleomycin via the trachea to induce idiopathic pulmonary fibrosis. Also administer an equal volume of saline to the control group. After 21 days, determine the degree of severity of pulmonary fibrosis by evaluating the expression of markers for fibrosis and the Ashcroft scores.
The HE staining showed that the adoptive transfer of interleukins 33 stimulated macrophages exacerbated the degree of lung tissue destruction and increased fibroblast aggregation in the bleomycin-stimulated mice, compared to control. The increase in the Ashcroft score also illustrates the degree of pulmonary fibrosis. The expression levels of mRNA of alpha SMA and fibronectin in lung tissues of the recipient mouse containing adoptively transferred interleukins 33-stimulated interstitial macrophages and treated with bleomycin were higher, compared with those of the BLM-treated wild-type mice.
500 microliters of air should be immediately entered into the lungs with a one-millimeter syringe to ensure that the cell suspension in the cannula can completely enter the lung tissue.
