Isolation of Human Pulmonary Epithelial Cells
Isolation of Human Pulmonary Epithelial Cells
1. Pulmonary epithelial cells from human lung tissue were isolated.
2. Lungs were perfused with 10 ml of primary cell medium (supplemented with 1% antibiotics and 2 mM L-glutamine) through the pulmonary artery until they were cleared of blood.
3. Bronchoalveolar lavages were performed using 5 × 1 ml of PBS, 1 mM EDTA to remove alveolar leukocytes.
4. One ml of primary cell isolation solution was instilled into the lungs through the trachea.
5. Lungs were then removed and placed in a sterile tube containing 2 ml of primary cell medium (supplemented with 1% antibiotics and 2 mM L-glutamine) and incubated at 4 °C overnight for enzymatic digestion to occur.
6. Lung tissues were further teased apart in primary cell medium, 10% FCS in a dish.
7. The cell isolate was passed through a 100-μm filter, and pulmonary epithelial cells were purified as follows.
8. Collected cells were counted and incubated for 15 min at 4 °C at the appropriate ratios.
9. Cells were then washed and diluted in primary cell medium, 0.5% FCS.
10. Pulmonary epithelial cells thus obtained were plated on 6-well plate (4 × 105 cells/well) in primary cell medium, 10% FCS.
