title: Flow-cytometric method for simultaneous analysis of mouse lung epithelial, endothelial, and hematopoietic lineage cells.
abstract: Flow cytometry is a powerful tool capable of simultaneously analyzing multiple parameters on a cell-by-cell basis. Lung tissue preparation for flow cytometry requires creation of a single-cell suspension, which often employs enzymatic and mechanical dissociation techniques. These practices may damage cells and cause cell death that is unrelated to the experimental conditions under study. We tested methods of lung tissue dissociation and sought to minimize cell death in the epithelial, endothelial, and hematopoietic lineage cellular compartments. A protocol that involved flushing the pulmonary circulation and inflating the lung with Dispase, a bacillus-derived neutral metalloprotease, at the time of tissue harvest followed by mincing, digestion in a DNase and collagenase solution, and filtration before staining with fluorescent reagents concurrently maximized viable yields of epithelial, endothelial, and hematopoietic lineage cells compared with a standard method that did not use enzymes at the time of tissue harvest. Flow cytometry identified each population-epithelial (CD326(+)CD31(-)CD45(-)), endothelial (CD326(-)CD31(+)CD45(-)), and hematopoietic lineage (CD326(-)CD31(-)CD45(+))-and measured cellular viability by 7-aminoactinomycin D (7-AAD) staining. The Dispase method permitted discrimination of epithelial vs. endothelial cell death in a systemic lipopolysaccharide model of increased pulmonary vascular permeability. We conclude that application of a dissociative enzyme solution directly to the cellular compartments of interest at the time of tissue harvest maximized viable cellular yields of those compartments. Investigators could employ this dissociation method to simultaneously harvest epithelial, endothelial, and hematopoietic lineage and other lineage-negative cells for flow-cytometric analysis.
authors.affiliation: Johns Hopkins University Division of Pulmonary and Critical Care Medicine, Baltimore, Maryland benjamin-singer@northwestern.edu.
authors.forename: Benjamin D
authors.identifier: 0000-0001-5775-8427
authors.initials: BD
authors.lastname: Singer
authors.affiliation: Johns Hopkins University Division of Pulmonary and Critical Care Medicine, Baltimore, Maryland.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Mock
authors.affiliation: Johns Hopkins University Division of Pulmonary and Critical Care Medicine, Baltimore, Maryland.
authors.forename: Franco R
authors.initials: FR
authors.lastname: D'Alessio
authors.affiliation: Johns Hopkins University Division of Pulmonary and Critical Care Medicine, Baltimore, Maryland.
authors.forename: Neil R
authors.initials: NR
authors.lastname: Aggarwal
authors.affiliation: Johns Hopkins University Division of Pulmonary and Critical Care Medicine, Baltimore, Maryland.
authors.forename: Pooja
authors.initials: P
authors.lastname: Mandke
authors.affiliation: Johns Hopkins University Division of Pulmonary and Critical Care Medicine, Baltimore, Maryland.
authors.forename: Laura
authors.initials: L
authors.lastname: Johnston
authors.affiliation: Johns Hopkins University Division of Pulmonary and Critical Care Medicine, Baltimore, Maryland.
authors.forename: Mahendra
authors.initials: M
authors.lastname: Damarla
mesh_terms: D000818:Animals; D019070:Cell Lineage; D002470:Cell Survival; D042783:Endothelial Cells / Q000502:physiology*; D004847:Epithelial Cells / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D008168:Lung / Q000166:cytology; D008297:Male; D008810:Mice, Inbred C57BL
keywords: cell death; flow cytometry; lung; tissue digestion; tissue processing