Back to Search
Start Over
A simple protocol for isolating mouse lung endothelial cells
- Source :
- Scientific Reports, Scientific Reports, Vol 9, Iss 1, Pp 1-10 (2019)
- Publication Year :
- 2019
- Publisher :
- Nature Publishing Group UK, 2019.
-
Abstract
- Endothelial dysfunction is the common molecular basis of multiple human diseases, such as atherosclerosis, diabetes, hypertension, and acute lung injury. Therefore, primary isolation of high-purity endothelial cells (ECs) is crucial to study the mechanisms of endothelial function and disease pathogenesis. Mouse lung ECs (MLECs) are widely used in vascular biology and lung cell biology studies such as pulmonary inflammation, angiogenesis, vessel permeability, leukocyte/EC interaction, nitric oxide production, and mechanotransduction. Thus, in this paper, we describe a simple, and reproducible protocol for the isolation and culture of MLECs from adult mice using collagenase I-based enzymatic digestion, followed by sequential sorting with PECAM1 (also known as CD31)- and ICAM2 (also known as CD102)-coated microbeads. The morphology of isolated MLECs were observed with phase contrast microscope. MLECs were authenticated by CD31 immunoblotting, and immunofluorescent staining of established EC markers VE-cadherin and von Willebrand factor (vWF). Cultured MLECs also showed functional characteristics of ECs, evidenced by DiI-oxLDL uptake assay and THP-1 monocyte adhesion assay. Finally, we used MLECs from endothelium-specific enhancer of zeste homolog 2 (EZH2) knockout mice to show the general applicability of our protocol. To conclude, we describe here a simple and reproducible protocol to isolate highly pure and functional ECs from adult mouse lungs. Isolation of ECs from genetically engineered mice is important for downstream phenotypic, genetic, or proteomic studies.
- Subjects :
- 0301 basic medicine
CD31
Angiogenesis
lcsh:Medicine
Cell Separation
Lung injury
Article
03 medical and health sciences
Gene Knockout Techniques
Mice
0302 clinical medicine
Von Willebrand factor
Antigens, CD
von Willebrand Factor
medicine
Animals
Enhancer of Zeste Homolog 2 Protein
Microscopy, Phase-Contrast
Collagenases
Endothelial dysfunction
Mechanotransduction
lcsh:Science
Lung
Cells, Cultured
Multidisciplinary
ICAM2
biology
Chemistry
lcsh:R
Endothelial Cells
medicine.disease
Cadherins
3. Good health
Cell biology
030104 developmental biology
Knockout mouse
biology.protein
lcsh:Q
Cell Adhesion Molecules
030217 neurology & neurosurgery
Subjects
Details
- Language :
- English
- ISSN :
- 20452322
- Volume :
- 9
- Database :
- OpenAIRE
- Journal :
- Scientific Reports
- Accession number :
- edsair.doi.dedup.....46a557ebd319fca119b7a502b1fabde5