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Efficient and rapid isolation and purification of mouse alveolar type II epithelial cells.
MedLine Citation:
PMID:  22888851     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
ABSTRACT 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.
Authors:
Elise M Messier; Robert J Mason; Beata Kosmider
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Experimental lung research     Volume:  38     ISSN:  1521-0499     ISO Abbreviation:  Exp. Lung Res.     Publication Date:  2012 Sep 
Date Detail:
Created Date:  2012-08-14     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8004944     Medline TA:  Exp Lung Res     Country:  England    
Other Details:
Languages:  eng     Pagination:  363-73     Citation Subset:  IM    
Affiliation:
Department of Medicine, National Jewish Health, Denver, Colorado, USA.
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