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This Article Full Text Full Text (PDF) All Versions of this Article: 296/6/L1096    most recent 90613.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Google Scholar Articles by Fehrenbach, M. L. Articles by DeLisser, H. M. PubMed PubMed Citation Articles by Fehrenbach, M. L. Articles by DeLisser, H. M.

INNOVATIVE METHODOLOGY

Isolation of murine lung endothelial cells

Pulmonary, Allergy, and Critical Care Division, Department of Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Submitted 10 December 2008 ; accepted in final form 12 March 2009

ABSTRACT

Several protocols for the isolation of endothelial cells (ECs) from murine lung have been described in the literature. We, however, encountered a number of problems while using these procedures that prevented us from consistently or reliably obtaining pure populations of ECs from the lungs of mice. By incorporating specific elements from previously published protocols, as well as adding some novel features, we developed a new strategy for isolating ECs from murine lung. In this approach, a suspension of lung cells is initially prepared from the lungs of 7- to 14-day-old mouse pups using procedures that prevent intravascular clotting and leukocyte activation, minimize mechanical trauma to the lung tissue, and limit exposure to the digesting enzymes. The resulting cell suspension is cultured for 2–3 days, trypsinized to produce a suspension of single cells, and then subjected to fluorescence-activated cell sorting using an anti-ICAM-2 antibody. The sorted cells are then plated and split 1:2 at each passage to maintain a high density of the cells. Using this approach, we have been able to isolate pure populations of ECs that were sustainable for extended periods in culture without the emergence of fibroblast overgrowth or the development of senescence. We believe the success of this approach will provide opportunities to take advantage of the large and growing number of knockout and transgenic mouse lines to investigate the endothelial-specific roles of targeted molecules in the pulmonary vasculature.

fluorescence-activated cell sorting; mouse pups

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