LysoTracker is a marker of differentiated alveolar type II cells.
BACKGROUND
LysoTracker Green DND-26 is a fluorescent dye that stains acidic compartments in live cells and has been shown to selectively accumulate in lamellar bodies in alveolar type II (AT2) cells in the lung. The aim of this study was to determine whether the accumulation of LysoTracker in lamellar bodies can be used to isolate viable AT2 cells by flow cytometry and track their differentiation in live-cell culture by microscopy.


METHODS
Mouse lung cells were sorted on the basis of CD45(neg)CD31(neg)EpCAM(pos)LysoTracker(pos) expression and characterized by immunostaining for SP-C and cultured in a three-dimensional epithelial colony-forming unit (CFU-Epi) assay. To track AT2 cell differentiation, lung epithelial stem and progenitor cells were cultured in a CFU-Epi assay with LysoTracker-supplemented media.


RESULTS
The purity of sorted AT2 cells as determined by SP-C staining was 97.4% and viability was 85.3%. LysoTracker(pos) AT2 cells generated SP-C(pos) alveolar epithelial cell colonies in culture, and when added to the CFU-Epi culture medium, LysoTracker marked the differentiation of stem/progenitor-derived AT2 cells.


CONCLUSIONS
This study describes a novel method for isolating AT2 cells from mouse lungs. The high purity and viability of cells attained by this method, makes them suitable for functional analysis in vitro. The application of LysoTracker to live cell cultures will allow better assessment of the cellular and molecular mechanisms that regulate AT2 cell differentiation.
Joanne L
JL
Van der Velden
Ivan
I
Bertoncello
Lung Health Research Centre, Department of Pharmacology and Therapeutics, University of Melbourne, Melbourne, Victoria, Australia. jlmcq@unimelb.edu.au.
Jonathan L
JL
McQualter
D000588:Amines; D000818:Animals; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008168:Lung / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D023421:Models, Animal; D011650:Pulmonary Alveoli / Q000166:cytology*
D000588:Amines; D005456:Fluorescent Dyes; C493330:Red DND-99