Describe the procedure for isolating endothelial cells from
mouse lung?
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Answer / Nitin Pratap Singh
The procedure for isolating endothelial cells from mouse lung involves several steps:
1. Anesthetize a mouse and perfuse the lungs with phosphate-buffered saline (PBS) to remove blood.
2. Digest the lung tissue with enzymes such as collagenase type IV, elastase, or dispase for 30 minutes at 37°C.
3. Mechanically dissociate the tissue by passing it through a series of decreasing gauge needles.
4. Centrifuge the cell suspension to pellet the cells, and resuspend the pellet in endothelial cell growth medium supplemented with heparin and basic fibroblast growth factor (bFGF).
5. Plate the cells on tissue culture dishes coated with extracellular matrix proteins such as fibronectin or collagen type I, and allow them to adhere for several hours.
6. Remove non-adherent cells by washing with PBS, and grow the adherent endothelial cells in endothelial cell growth medium containing antibiotics.
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