Isolation of pulmonary artery smooth muscle cells from neonatal mice
| Autor: | Kathryn N. Farrow, Gregory B. Waypa, Lyubov Czech, Keng Jin Lee |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Pathology
medicine.medical_specialty Vascular smooth muscle General Chemical Engineering Muscle Relaxation Population Cytological Techniques Biology Myosins Pulmonary Artery General Biochemistry Genetics and Molecular Biology Muscle Smooth Vascular Desmin Mice medicine.artery Myosin medicine Animals education education.field_of_study General Immunology and Microbiology General Neuroscience medicine.disease Pulmonary hypertension Culture Media Muscle relaxation Animals Newborn Pulmonary artery Cattle Vascular smooth muscle contraction Fetal bovine serum Muscle Contraction |
| Zdroj: | Journal of visualized experiments : JoVE. (80) |
| ISSN: | 1940-087X |
| Popis: | Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 μM in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin. |
| Databáze: | OpenAIRE |
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