Name: isolation of pulmonary artery smooth muscle cells from neonatal mice
Uploaded: 2013-10-19T19:00:00
Description: Watch this Scientific Journal Video about Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice at JoVE.com

This work was supported by NIH HL109478 (KNF). The authors acknowledge and thank Gina Kim and Joann Taylor for their assistance in isolating and maintaining the PASMC in culture.

The Institutional Animal Care and Use Committee at Northwestern University approved this protocol.
1. Pulmonary Artery Isolation from Neonatal Mice - Day One
<ol>
	<li>Prepare the Complete M199 media - Mix 400 ml M199 with 100 ml (final concentration =&#160;20%) heat inactivated FBS and 5 ml (final concentration =&#160;1%) Penicillin/Streptomycin.</li>
	<li>Prepare the Serum Free M199 Media - Mix 500 ml M199 with 5 ml (final concentration =&#160;1%) Penicill...

<ol>
	<li>Levin, D. L., Rudolph, A. M., Heymann, M. A., Phibbs, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+development+of+the+pulmonary+vascular+bed+in+fetal+lambs.">Morphological development of the pulmonary vascular bed in fetal lambs.</a> Circulation. 53, 144-151 (1976).</li><li>Walsh-Sukys, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+pulmonary+hypertension+of+the+newborn+in+the+era+before+nitric+oxide:+practice+variation+and+outcomes.">Persistent pulmonary hypertension of the newborn in the era before nitric oxide: practice variation and outcomes.</a> Pediatrics. 105, 14-20 (2000).</li><li>Khemani, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+artery+hypertension+in+formerly+premature+infants+with+bronchopulmonary+dysplasia:+clinical+features+and+outcomes+in+the+surfactant+era.">Pulmonary artery hypertension in formerly premature infants with bronchopulmonary dysplasia: clinical features and outcomes in the surfactant era.</a> Pediatrics. 120, 1260-1269 (2007).</li><li>Jobe, A. H., Bancalari, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchopulmonary+dysplasia.">Bronchopulmonary dysplasia.</a> Am. J. Respir. Crit. Care Med. 163, 1723-1729 (2001).</li><li>Mourani, P. M., Sontag, M. K., Younoszai, A., Ivy, D. D., Abman, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+utility+of+echocardiography+for+the+diagnosis+and+management+of+pulmonary+vascular+disease+in+young+children+with+chronic+lung+disease.">Clinical utility of echocardiography for the diagnosis and management of pulmonary vascular disease in young children with chronic lung disease.</a> Pediatrics. 121, 317-325 (2008).</li><li>Check, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+growth+restriction+and+pulmonary+hypertension+in+premature+infants+with+bronchopulmonary+dysplasia.">Fetal growth restriction and pulmonary hypertension in premature infants with bronchopulmonary dysplasia.</a> J. Perinatol. , (2013).</li><li>Farrow, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperoxia+increases+phosphodiesterase+5+expression+and+activity+in+ovine+fetal+pulmonary+artery+smooth+muscle+cells.">Hyperoxia increases phosphodiesterase 5 expression and activity in ovine fetal pulmonary artery smooth muscle cells.</a> Circ. Res. 102, 226-233 (2008).</li><li>Aschner, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+nitric+oxide+synthase+gene+transfer+enhances+dilation+of+newborn+piglet+pulmonary+arteries.">Endothelial nitric oxide synthase gene transfer enhances dilation of newborn piglet pulmonary arteries.</a> Am. J. Physiol. 277, 371-379 (1999).</li><li>Cornfield, D. N., Stevens, T., McMurtry, I. F., Abman, S. H., Rodman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+hypoxia+increases+cytosolic+calcium+in+fetal+pulmonary+artery+smooth+muscle+cells.">Acute hypoxia increases cytosolic calcium in fetal pulmonary artery smooth muscle cells.</a> Am. J. Physiol. 265, 53-56 (1993).</li><li>Bailly, K., Ridley, A. J., Hall, S. M., Haworth, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoA+activation+by+hypoxia+in+pulmonary+arterial+smooth+muscle+cells+is+age+and+site+specific.">RhoA activation by hypoxia in pulmonary arterial smooth muscle cells is age and site specific.</a> Circ. Res. 94, 1383-1391 (2004).</li><li>Black, S. M., DeVol, J. M., Wedgwood, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+fibroblast+growth+factor-2+expression+in+pulmonary+arterial+smooth+muscle+cells+involves+increased+reactive+oxygen+species+generation.">Regulation of fibroblast growth factor-2 expression in pulmonary arterial smooth muscle cells involves increased reactive oxygen species generation.</a> Am. J. Physiol. Cell Physiol. 294, 345-354 (2008).</li><li>Cogolludo, A., Moreno, L., Lodi, F., Tamargo, J., Perez-Vizcaino, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+maturational+shift+from+PKCzeta+and+voltage-gated+K++channels+to+RhoA/Rho+kinase+in+pulmonary+vasoconstriction.">Postnatal maturational shift from PKCzeta and voltage-gated K+ channels to RhoA/Rho kinase in pulmonary vasoconstriction.</a> Cardiovasc. Res. 66, 84-93 (2005).</li><li>Wedgwood, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+regulates+extracellular+superoxide+dismutase+activity+and+expression+in+neonatal+pulmonary+hypertension.">Hydrogen peroxide regulates extracellular superoxide dismutase activity and expression in neonatal pulmonary hypertension.</a> Antiox. Signal. 15, 1497-1506 (1089).</li><li>Farrow, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+oxidant+stress+increases+PDE5+activity+in+persistent+pulmonary+hypertension+of+the+newborn.">Mitochondrial oxidant stress increases PDE5 activity in persistent pulmonary hypertension of the newborn.</a> Respir. Physiol. Neurobiol. 174, 272-281 (2010).</li><li>Chester, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cinaciguat,+a+soluble+guanylate+cyclase+activator,+augments+cGMP+after+oxidative+stress+and+causes+pulmonary+vasodilation+in+neonatal+pulmonary+hypertension.">Cinaciguat, a soluble guanylate cyclase activator, augments cGMP after oxidative stress and causes pulmonary vasodilation in neonatal pulmonary hypertension.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 301, 755-764 (2011).</li><li>Konduri, G. G., Bakhutashvili, I., Eis, A., Gauthier, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+voltage+gated+potassium+channel+responses+in+a+fetal+lamb+model+of+persistent+pulmonary+hypertension+of+the+newborn.">Impaired voltage gated potassium channel responses in a fetal lamb model of persistent pulmonary hypertension of the newborn.</a> Pediatr. Res. 66, 289-294 (2009).</li><li>Olschewski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+K(Ca)+channel+to+membrane+potential+and+O2+sensitivity+is+decreased+in+an+ovine+PPHN+model.">Contribution of the K(Ca) channel to membrane potential and O2 sensitivity is decreased in an ovine PPHN model.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 283, L1103-L1109 (2002).</li><li>Aslam, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+attenuate+lung+injury+in+a+murine+model+of+neonatal+chronic+lung+disease.">Bone marrow stromal cells attenuate lung injury in a murine model of neonatal chronic lung disease.</a> Am. J. Respir. Crit. Care Med. 180, 1122-1130 (2009).</li><li>Balasubramaniam, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+angiogenic+cells+restore+lung+alveolar+and+vascular+structure+after+neonatal+hyperoxia+in+infant+mice.">Bone marrow-derived angiogenic cells restore lung alveolar and vascular structure after neonatal hyperoxia in infant mice.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 298, L315-L323 (2010).</li><li>de Visser, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphodiesterase-4+inhibition+attenuates+pulmonary+inflammation+in+neonatal+lung+injury.">Phosphodiesterase-4 inhibition attenuates pulmonary inflammation in neonatal lung injury.</a> Eur. Respir. J. 31, 633-644 (2008).</li><li>de Visser, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sildenafil+attenuates+pulmonary+inflammation+and+fibrin+deposition,+mortality+and+right+ventricular+hypertrophy+in+neonatal+hyperoxic+lung+injury.">Sildenafil attenuates pulmonary inflammation and fibrin deposition, mortality and right ventricular hypertrophy in neonatal hyperoxic lung injury.</a> Respir. 10, 30 (2009).</li><li>Ladha, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sildenafil+improves+alveolar+growth+and+pulmonary+hypertension+in+hyperoxia-induced+lung+injury.">Sildenafil improves alveolar growth and pulmonary hypertension in hyperoxia-induced lung injury.</a> Am. Respir. Crit. Care Med. 172, 750-756 (2005).</li><li>Farrow, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+hyperoxia+increases+mitochondrial+oxidation+and+increases+phosphodiesterase+5+activity+in+fetal+pulmonary+artery+smooth+muscle+cells.">Brief hyperoxia increases mitochondrial oxidation and increases phosphodiesterase 5 activity in fetal pulmonary artery smooth muscle cells.</a> Antioxid. Redox Signal. 17, 460-470 (2012).</li><li>Waypa, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+triggers+subcellular+compartmental+redox+signaling+in+vascular+smooth+muscle+cells.">Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells.</a> Circ. Res. 106, 526-535 (2010).</li><li>Waypa, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+Generated+at+Mitochondrial+Complex+III+Triggers+Acute+Responses+to+Hypoxia+in+the+Pulmonary+Circulation.">Superoxide Generated at Mitochondrial Complex III Triggers Acute Responses to Hypoxia in the Pulmonary Circulation.</a> Am. J. Respir. Crit. Care Med. 187, 424-432 (2013).</li><li>Marshall, C., Mamary, A. J., Verhoeven, A. J., Marshall, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+artery+NADPH-oxidase+is+activated+in+hypoxic+pulmonary+vasoconstriction.">Pulmonary artery NADPH-oxidase is activated in hypoxic pulmonary vasoconstriction.</a> Am. J. Respir. Cell Mol. Biol. 15, 633-644 (1996).</li><li>Farrow, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+Dismutase+and+Inhaled+Nitric+Oxide+Normalize+Phosphodiesterase+5+Expression+and+Activity+in+Neonatal+Lambs+with+Persistent+Pulmonary+Hypertension.">Superoxide Dismutase and Inhaled Nitric Oxide Normalize Phosphodiesterase 5 Expression and Activity in Neonatal Lambs with Persistent Pulmonary Hypertension.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 299, L109-L116 (2010).</li></ol>

In this manuscript, we describe for the first time the isolation of PASMC from mice at P7, P14, and P21. In order to accomplish this, a slurry of agarose and 0.2 &#956;M iron particles are infused through the RV into the PA. Due to the small size of the iron particles, they cannot pass through the pulmonary capillary bed and are thus deposited in the small PA. The lungs are inflated, removed and dissociated. The iron-containing vessels are pulled out of solution using a magnet. Ultimately, the vessels are plated into a t...

Pulmonary hypertension is normal during intrauterine life since the placenta serves as the major organ of gas exchange and only 10% of the cardiac output is circulated through the pulmonary vascular bed. In utero, pulmonary pressures are similar to systemic pressures due to elevated pulmonary vascular resistance. As gestation progresses, there is rapid growth of the small PA within the lung, preparing the fetus for the dramatic increase in pulmonary blood flow that occurs at birth1. When the normal perinatal transition fails in near-term and full term infants, the result is persistent pulmonary hypertension of the newborn (PPHN). PPHN is a clinical syndrome caused by many different underlying pathologies. However, all of these infants share common pathophysiologic features such as elevated pulmonary vascular resistance, hypoxemia, and right-to-left shunting of blood flow across persistent fetal connections such as the ductus arteriosus or foramen ovale. PPHN affects 2-6 per 1,000 live births and conveys an 8-10% risk of mortality as well as significant short-term and long-term morbidity2. Additionally, very low birth weight premature infants may develop pulmonary hypertension as a result of their underlying lung disease. The most common underlying lung disease of premature infants is bronchopulmonary dysplasia (BPD). While the overall risk of BPD correlates with gestational age and birth weight, it remains unclear why a subset of these infants develops significant pulmonary hypertension and how to appropriately treat these infants. Poor outcomes, including prolonged hospital stays and increased mortality, are common3-6.
Historically, ovine fetal PASMC or porcine fetal PASMC from healthy animals have been used to study the signaling pathways involved in the normal pulmonary vascular transition after birth. These are typically isolated from fifth generation resistance PA of an ovine or porcine fetus that is delivered and euthanized prior to any spontaneous respiration7-9. Additionally, some investigators have isolated and utilized PASMC from slightly older and spontaneously breathing lambs and piglets at 3 days, 2 weeks, and 4 weeks10-12. More recently, some groups have successfully isolated and utilized PASMC isolated from lambs with PPHN to examine the derangements in signaling pathways in the disease state13-17. These cells have proved to be a valuable tool to examine which signaling pathways are crucial in both the normal and diseased near-term and term pulmonary vasculature. However, they do not give insight into the signaling pathways impacted in premature infants with pulmonary hypertension. Nor do they allow the possibilities of genetic manipulation seen in mouse models of disease.
Rat and mouse models have long been used to model BPD and more recently are being used to model pulmonary hypertension resulting from BPD18-22. Neonatal rats are enticing to work with due to their larger size, but they also suffer from lack of potential for genetic modification. Genetically modified animals have been extensively used to investigate the effects of specific gene targets on whole animal physiology in neonatal mice, but to date no one has previously successfully isolated PASMC from these small mice. By isolating PASMC, greater information can be obtained about how pathways change in response to environmental stimuli and/or genetic modification specifically in the pulmonary artery smooth muscle. Additionally, live PASMC can be imaged in real time to examine rapid changes in key signaling molecules such as calcium and reactive oxygen species23-25. We recently described the successful isolation of PASMC from adult mice using a variation of the technique of Marshall et al.26 used to isolate rat PASMC23,25,26. We now have adapted and extended this technique to small mice 7-21 days of age (P7, P14, and P21). The primary limitation to this new PASMC isolation technique is that it requires multiple mice to generate sufficient cells for experiments and that the cells grow very slowly, which is characteristic of primary smooth muscle cells. Despite these limitations, we believe this technique to isolate neonatal mouse PASMC will allow for the enhanced investigation of key signaling pathways involved in the development of pulmonary hypertension and represents a significant advance in this field.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Bard Parker surgical blade handle</td>
			<td>BD</td>
			<td>371030</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel surgical blades #10 (sterile)</td>
			<td>Miltex</td>
			<td>4-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (3 ml and 5 ml, sterile)</td>
			<td>BD</td>
			<td>309657 and 306646</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles (27 G, sterile)</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Angiocatheter (24 G, sterile)</td>
			<td>BD</td>
			<td>381412</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoject blunt cannula (15 G)</td>
			<td>Kendall</td>
			<td>SWD202314Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Fisher Scientific</td>
			<td>NC9782896</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynal magnet particle collector</td>
			<td>Invitrogen</td>
			<td>120-01D</td>
			<td>This is a critical tool for the protocol.</td>
		</tr>
		<tr>
			<td>Tissue culture plates (35 mm, 60 mm, and 10 cm, sterile)</td>
			<td>BD</td>
			<td>353001, 353004, and 353003</td>
			<td>Any brand of tissue culture plates will be fine.</td>
		</tr>
		<tr>
			<td>Iris Scissors (4 &#189; inch stainless steel)</td>
			<td>American Diagnostic Corporation</td>
			<td>3424</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (4 inch stainless steel)</td>
			<td>Quick Medical</td>
			<td>L5-5004</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Mediatech</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>M199 media</td>
			<td>Mediatech</td>
			<td>10-060-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>VWR</td>
			<td>TX16777-164NWU</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone/Thermo Scientific</td>
			<td>SH3091003</td>
			<td>Heat inactivate at 55 &#176;C for 45 min. For consistency in results, lot match all serum and obtain from same vendor.</td>
		</tr>
		<tr>
			<td>Iron particles (iron (II, III) oxide powder)</td>
			<td>Aldrich Chemical Company</td>
			<td>#31,006-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase (made to 80 U/ml)</td>
			<td>Sigma</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Butlet Schein</td>
			<td>NDC 11695-6776-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE2000-U with a Cascade Photometrics 12-bit camera</td>
			<td>Nikon</td>
			<td>TE2000-U</td>
			<td>Any good light microscope will be fine to observe PASMC in culture.</td>
		</tr>
		<tr>
			<td>Anti-desmin antibody</td>
			<td>Sigma</td>
			<td>D-8281</td>
			<td>Use at 1:200 dilution for immunostaining.</td>
		</tr>
		<tr>
			<td>Anti-smooth muscle myosin</td>
			<td>Biomedical Technologies</td>
			<td>BT-562</td>
			<td>Use at 1:2,000 dilution for immunostaining.</td>
		</tr>
		<tr>
			<td>Rhodamine-red anti-rabbit secondary</td>
			<td>Molecular Probes/Invitrogen</td>
			<td>R-6394</td>
			<td>Use at 1:200 dilution for immunostaining.</td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE-300 fluorescent microscope and Cool Snap digital camera</td>
			<td>Nikon</td>
			<td>TE300</td>
			<td>Any good epifluorescence microscope will be fine for immunostaining.</td>
		</tr>
		<tr>
			<td>Cyclic nucleotide phosphodiesterase assay kit</td>
			<td>Enzo Life Sciences</td>
			<td>BML-AK800-0001</td>
			<td>This is the only colorimetric PDE enzyme activity assay available.</td>
		</tr>
		<tr>
			<td>Sildenafil</td>
			<td>Sigma</td>
			<td>PZ-0003</td>
			<td>A PDE5-selective inhibitor is required for the PDE enzyme activity to determine specificity of cGMP hydrolysis.</td>
		</tr>
	</tbody>
</table>

isolation of pulmonary artery smooth muscle cells from neonatal mice

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 &#956;M 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.

During and after isolation, PASMC are examined both by light microscopy and by immunostaining for smooth muscle cell markers. By light microscopy early in the protocol, PASMC are seen migrating onto the tissue culture dish from the small iron containing vessels (Figure 1A). After pooling plates one through three on day thirteen, then iron particles are no longer seen as those have been pulled out in the final pooling step. Instead, a population of PASMC is seen on the tissue culture dish (Figure ...

Watch this Scientific Journal Video about Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice at JoVE.com

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Research

JoVE Journal

Biology

Focal Ca2+ Transient Detection in Smooth Muscle

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...