ArticleAmerican journal of physiology. Lung cellular and molecular physiology2016
Flow-cytometric method for simultaneous analysis of mouse lung epithelial, endothelial, and hematopoietic lineage cells.
Article in American journal of physiology. Lung cellular and molecular physiology, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 47 papers.
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47 citing papers in PubMed, 69 citations in OpenAlex.
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- Lipid nanoparticle-mediated mRNA delivery in lung fibrosis.European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences · 2023Article
- Intratracheally administered LNA gapmer antisense oligonucleotides induce robust gene silencing in mouse lung fibroblasts.Nucleic acids research · 2022Article
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- Lack of Endothelial α1AMPK Reverses the Vascular Protective Effects of Exercise by Causing eNOS Uncoupling.Antioxidants (Basel, Switzerland) · 2021Article
- Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung.Journal of visualized experiments : JoVE · 2021Article
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
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.
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