Abstract
In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 x 106 ATII cells per mouse lung. The cell preps are highly pure and viable and can be used for genomic or proteomic analyses or cultured ex vivo to understand their roles in various biological processes.
Background
The internal surfaces of lungs are lined by epithelial cells, the type of epithelial cell varying morphologically and functionally with the location within the lung. ATII cells are one of the two types of epithelial cells that line the alveolar walls and have been described to play critical roles in surfactant synthesis and secretion. They are also part of the first line of defense within the lung and are involved in initiating and modulating immune responses during pulmonary infection or allergy. They are also thought to act as progenitor cells in the distal lung with proliferative capacity and the ability to repair the epithelium after injury. Available methods of ATII isolation did not yield cell preps that were more than 80-85% pure, making them unsuitable for reliable analyses of mRNA and protein expression. The method described here is an improvement over prior methods and yields mouse primary ATII cell preps with the highest purity that can thus be reliably used for expression analyses. For further discussion on the method, we refer the reader to the original publication from where this protocol originates (Sinha et al., 2016).
Materials and Reagents
Equipment
Software
Procedure
Note: Mice should be cared for and used in accordance with national and institutional policies. All protocols must be approved by the institutional animal committee. In our case, all procedures were approved by the Institutional Animal Care and Use Committee at the University of California San Francisco.
Data analysis
Notes
Recipes
Acknowledgments
This work was supported by the US National Institutes of Health (AI65495 and AI68150 to C.A.L.). Thanks to Pooja Mehta and James Mueller for help with pictures and video. This protocol was adapted from Sinha et al. (2016).
References
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