Mouse Lung Preparation for Flow Cytometry: Generating Cell Suspension from Lung Tissue for Flow Cytometric Analysis
Transkript
– Begin by taking a euthanized mouse and secure it on a dissection board with lab tape. Use scissors to cut open the skin. Make a vertical incision through the diaphragm and cut ribs to reach the thoracic cavity exposing the heart and lungs. Next, make a small incision in the left ventricle of the heart. Subsequently, locate the right ventricle and insert a syringe containing PBS into the ventricle's lumen. Perfuse PBS through the right ventricle letting the blood flow out of the premade incision at the left ventricle till the lungs turn white. Take out the lung tissue containing tumor nodules and mince into small pieces.
Incubate the pieces in the lung digestion buffer. Collagenase in the lung digestion buffer helps isolate cells, and DNAse helps eliminate DNA from the dead cells. Strain the resulting suspension through a cell strainer to remove clumps and make the cell suspension uniform. Centrifuge and resuspend the pellet in the ammonium chloride potassium buffer to lyse residual erythrocytes. Centrifuge to collect the WBCs and tumor cells in the pellet. Resuspend the pellet in PBS supplemented with FCS, to maintain cell stability. In the following protocol, we will demonstrate the preparation of lung cell suspension for flow cytometric analysis.
After the appropriate experimental endpoint, soak the carcass in 70% ethanol and secure the animal to a dissection board. Make a ventral midline incision and gently invert the skin to expose the thoracic wall muscles and the abdominal organs. Use scissors to puncture the diaphragm and to cut the ribs exposing the thoracic cavity. Cut a small opening in the left ventricle and use a 27 gauge needle to perfuse the lung three times through the right ventricle with 6 to 8 milliliters of ice cold PBS per infusion. After the last perfusion the lungs should be completely clear of blood and appear white. After harvesting, use scissors to mince the lung tissue into small pieces.
Transfer the lung fragments into a 2 milliliter microcentrifuge tube containing 1.5 milliliters of lung digestion buffer for a 30 to 60 minute incubation at 37 degrees Celsius with shaking. At the end of the digestion, transfer the cell suspension through a 70 micron cell strainer into a 50 milliliter tube, and use the end of a sterile 10 milliliter syringe plunger to press any remaining tissue fragments through the filter. Rinse the strainer with 15 milliliters of PBS supplemented with 2% FCS, and collect the cells by centrifugation. Resuspend the pellet in 1 milliliter of ammonium chloride potassium lysis buffer for a 5 minute incubation at room temperature, and centrifuge the cells again. Then resuspend the white blood cell pellet in 1 milliliter of PBS supplemented with 2% FCS, and stain the cells for flow cytometric analysis according to the experimental protocol.
