Mouse Lung Preparation for Flow Cytometry: Generating Cell Suspension from Lung Tissue for Flow Cytometric Analysis

- 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