Efficient and rapid isolation and purification of mouse alveolar type II epithelial cells
| Autor: | Elise M. Messier, Robert John Mason, Beata Kosmider |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Male
Pulmonary and Respiratory Medicine Cell type Pathology medicine.medical_specialty Pulmonary Surfactant-Associated Proteins Clinical Biochemistry Population Cell Separation Lamellar granule Biology Flow cytometry Mice Pulmonary surfactant Antigen Dispase medicine Animals education Molecular Biology Cells Cultured education.field_of_study medicine.diagnostic_test 1-Acylglycerophosphocholine O-Acyltransferase Epithelial Cells respiratory system Microspheres Epithelium Cell biology Mice Inbred C57BL Pulmonary Alveoli medicine.anatomical_structure Antigens Surface Female |
| Zdroj: | Experimental Lung Research. 38:363-373 |
| ISSN: | 1521-0499 0190-2148 |
| DOI: | 10.3109/01902148.2012.713077 |
| Popis: | The alveolar surface is covered by an epithelium composed of 2 main cell types: type I and type II cells. Alveolar type II (ATII) cells have a distinct morphology with apical microvilli and characteristic lamellar bodies, which are the intracellular storage form of pulmonary surfactant. ATII cells play an important role in innate immunity and produce and secrete pulmonary surfactant. They proliferate to restore the epithelium after damage to the more sensitive type I cells. We developed an efficient and rapid method to isolate and purify ATII cells from mice. Alveolar epithelial cells were dissociated in the murine lung with dispase and lung tissue was gently minced with a GentleMACS Dissociator. ATII cell purification was performed using negative depletion with CD45 MicroBeads and positive selection for the epithelial-cell adhesion molecule (Ep-CAM) by magnetic labeling with Streptavidin MicroBeads in MACS LS columns. The purity of these cells as measured by flow cytometry was up to 92.1% and 91.1% for co-staining with Ep-CAM and cytokeratin and co-staining with Ep-CAM and SP-A, respectively. The resulting ATII cell population has a high purity, viability, and yield. The phenotype of isolated and cultured ATII cells was confirmed by electron micrographs, expression of surfactant proteins (SP-A, proSP-B, mature SP-B, proSP-C, SP-D), and lysophosphatidylcholine acyltransferase (LPCAT) by western blotting and immunocytofluorescence. This protocol is based on surface antigens and our data demonstrated that murine ATII cells can be rapidly isolated, efficiently purified, and effectively cultured. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|