This work is supported by NIH grants, K08135443 (Y.B), 1R01HL132991 (X.A).

All animal care was in accordance with the guidelines of the Institutional Animal Care and Use Committee of Massachusetts General Hospital. Wild-type C57/B6 male mice, 8 weeks of age, were used for the present study.
1. Experimental preparation
<ol>
	<li>Prepare the working solution.
	<ol>
		<li>Prepare 1x Hank&#39;s Balanced Salt Solution (HBSS, with Ca2+ and Mg2+, and pH balanced with 20 mM HEPES, see Table of Materials). Use the HBSS...

<ol>
	<li>Prakash, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+concepts+in+smooth+muscle+contributions+to+airway+structure+and+function:+implications+for+health+and+disease.">Emerging concepts in smooth muscle contributions to airway structure and function: implications for health and disease.</a> American Journal of Physiology Lung Cellular and Molecular Physiology. 311 (6), 1113-1140 (2016).</li><li>Lechartier, 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=Phenotypic+diversity+of+vascular+smooth+muscle+cells+in+pulmonary+arterial+hypertension:+implications+for+therapy.">Phenotypic diversity of vascular smooth muscle cells in pulmonary arterial hypertension: implications for therapy.</a> Chest. 161 (1), 219-231 (2022).</li><li>Doeing, D. C., Solway, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+smooth+muscle+in+the+pathophysiology+and+treatment+of+asthma.">Airway smooth muscle in the pathophysiology and treatment of asthma.</a> Journal of Applied Physiology. 114 (7), 834-843 (2013).</li><li>McGovern, T. K., 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=Evaluation+of+respiratory+system+mechanics+in+mice+using+the+forced+oscillation+technique.">Evaluation of respiratory system mechanics in mice using the forced oscillation technique.</a> Journal of Visualized Experiments: JoVE. (75), e50172 (2013).</li><li>Bikou, O., 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=Induction+and+characterization+of+pulmonary+hypertension+in+mice+using+the+hypoxia/SU5416+model.">Induction and characterization of pulmonary hypertension in mice using the hypoxia/SU5416 model.</a> Journal of Visualized Experiments: JoVE. (160), e59252 (2020).</li><li>Stenmark, K. R., 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=Dynamic+and+diverse+changes+in+the+functional+properties+of+vascular+smooth+muscle+cells+in+pulmonary+hypertension.">Dynamic and diverse changes in the functional properties of vascular smooth muscle cells in pulmonary hypertension.</a> Cardiovascular Research. 114 (4), 551-564 (2018).</li><li>Bai, Y., Zhang, M., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractility+and+Ca2++signaling+of+smooth+muscle+cells+in+different+generations+of+mouse+airways.">Contractility and Ca2+ signaling of smooth muscle cells in different generations of mouse airways.</a> American Journal of Respiratory Cell and Molecular Biology. 36 (1), 122-130 (2007).</li><li>Chin, L. Y., 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=Human+airway+smooth+muscle+is+structurally+and+mechanically+similar+to+that+of+other+species.">Human airway smooth muscle is structurally and mechanically similar to that of other species.</a> The European Respiratory Journal. 36 (1), 170-177 (2010).</li><li>Wang, 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=Inflammatory+mediators+mediate+airway+smooth+muscle+contraction+through+a+G+protein-coupled+receptor-transmembrane+protein+16A-voltage-dependent+Ca(2+)+channel+axis+and+contribute+to+bronchial+hyperresponsiveness+in+asthma.">Inflammatory mediators mediate airway smooth muscle contraction through a G protein-coupled receptor-transmembrane protein 16A-voltage-dependent Ca(2+) channel axis and contribute to bronchial hyperresponsiveness in asthma.</a> The Journal of Allergy and Clinical Immunology. 141 (4), 1259-1268 (2018).</li><li>Currigan, D. 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=Vasoconstrictor+responses+to+vasopressor+agents+in+human+pulmonary+and+radial+arteries:+an+in+vitro+study.">Vasoconstrictor responses to vasopressor agents in human pulmonary and radial arteries: an in vitro study.</a> Anesthesiology. 121 (5), 930-936 (2014).</li><li>Halayko, A. 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=Divergent+differentiation+paths+in+airway+smooth+muscle+culture:+induction+of+functionally+contractile+myocytes.">Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes.</a> The American Journal of Physiology. 276 (1), 197-206 (1999).</li><li>Worth, N. 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=Vascular+smooth+muscle+cell+phenotypic+modulation+in+culture+is+associated+with+reorganisation+of+contractile+and+cytoskeletal+proteins.">Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins.</a> Cell Motility and the Cytoskeleton. 49 (3), 130-145 (2001).</li><li>Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+lung+physiology+in+health+and+disease+with+lung+slices.">Exploring lung physiology in health and disease with lung slices.</a> Pulmonary Pharmacology and Therapeutics. 24 (5), 452-465 (2011).</li><li>Placke, M. E., Fisher, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+peripheral+lung+organ+culture-a+model+for+respiratory+tract+toxicology.">Adult peripheral lung organ culture-a model for respiratory tract toxicology.</a> Toxicology and Applied Pharmacology. 90 (2), 284-298 (1987).</li><li>Fisher, G. L., Placke, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+lung+toxicity.">In vitro models of lung toxicity.</a> Toxicology. 47 (1-2), 71-93 (1987).</li><li>Perez, J. F., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contraction+of+smooth+muscle+cells+of+intrapulmonary+arterioles+is+determined+by+the+frequency+of+Ca2++oscillations+induced+by+5-HT+and+KCl.">The contraction of smooth muscle cells of intrapulmonary arterioles is determined by the frequency of Ca2+ oscillations induced by 5-HT and KCl.</a> The Journal of General Physiology. 125 (6), 555-567 (2005).</li><li>Li, G., 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=Preserving+airway+smooth+muscle+contraction+in+precision-cut+lung+slices.">Preserving airway smooth muscle contraction in precision-cut lung slices.</a> Scientific Reports. 10 (1), 6480 (2020).</li><li>Sanderson, M. J., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-rate+confocal+microscopy.">Video-rate confocal microscopy.</a> Methods in Enzymology. 360, 447-481 (2003).</li><li>Kolbe, U., 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=Early+cytokine+induction+upon+pseudomonas+aeruginosa+infection+in+murine+precision+cut+lung+slices+depends+on+sensing+of+bacterial+viability.">Early cytokine induction upon pseudomonas aeruginosa infection in murine precision cut lung slices depends on sensing of bacterial viability.</a> Frontiers in Immunology. 11, 598636 (2020).</li><li>Perez, J. F., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+frequency+of+calcium+oscillations+induced+by+5-HT,+ACH,+and+KCl+determine+the+contraction+of+smooth+muscle+cells+of+intrapulmonary+bronchioles.">The frequency of calcium oscillations induced by 5-HT, ACH, and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles.</a> The Journal of General Physiology. 125 (6), 535-553 (2005).</li><li>Rosner, S. R., 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=Airway+contractility+in+the+precision-cut+lung+slice+after+cryopreservation.">Airway contractility in the precision-cut lung slice after cryopreservation.</a> American Journal of Respiratory Cell and Molecular Biology. 50 (5), 876-881 (2014).</li><li>Bai, Y., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+Ca2++sensitivity+of+airway+smooth+muscle+cells+in+murine+lung+slices.">Modulation of the Ca2+ sensitivity of airway smooth muscle cells in murine lung slices.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 291 (2), 208-221 (2006).</li><li>Sanderson, M. 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=Fluorescence+microscopy.">Fluorescence microscopy.</a> Cold Spring Harbor Protocols. 10, 071795 (2014).</li><li>Sanderson, M. J., Bai, Y., Perez-Zoghbi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca(2+)+oscillations+regulate+contraction+of+intrapulmonary+smooth+muscle+cells.">Ca(2+) oscillations regulate contraction of intrapulmonary smooth muscle cells.</a> Advances in Experimental Medicine and Biology. 661, 77-96 (2010).</li><li>Perez-Zoghbi, J. F., Bai, Y., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+induces+airway+smooth+muscle+cell+relaxation+by+decreasing+the+frequency+of+agonist-induced+Ca2++oscillations.">Nitric oxide induces airway smooth muscle cell relaxation by decreasing the frequency of agonist-induced Ca2+ oscillations.</a> The Journal of General Physiology. 135 (3), 247-259 (2010).</li><li>Lam, M., Lamanna, E., Bourke, J. E. <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+airway+smooth+muscle+contraction+in+health+and+disease.">Regulation of airway smooth muscle contraction in health and disease.</a> Advances in Experimental Medicine and Biology. 1124, 381-422 (2019).</li><li>Patel, K. R., 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=Targeting+acetylcholine+receptor+M3+prevents+the+progression+of+airway+hyperreactivity+in+a+mouse+model+of+childhood+asthma.">Targeting acetylcholine receptor M3 prevents the progression of airway hyperreactivity in a mouse model of childhood asthma.</a> FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology. 31 (10), 4335-4346 (2017).</li><li>Aven, 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=An+NT4/TrkB-dependent+increase+in+innervation+links+early-life+allergen+exposure+to+persistent+airway+hyperreactivity.">An NT4/TrkB-dependent increase in innervation links early-life allergen exposure to persistent airway hyperreactivity.</a> FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology. 28 (2), 897-907 (2014).</li><li>Liu, G., 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=Use+of+precision+cut+lung+slices+as+a+translational+model+for+the+study+of+lung+biology.">Use of precision cut lung slices as a translational model for the study of lung biology.</a> Respiratory Research. 20 (1), 162 (2019).</li><li>Wu, X., 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=Mouse+lung+tissue+slice+culture.">Mouse lung tissue slice culture.</a> Methods in Molecular Biology. 1940, 297-311 (2019).</li><li>Bai, Y., 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=CD38+plays+an+age-related+role+in+cholinergic+deregulation+of+airway+smooth+muscle+contractility.">CD38 plays an age-related role in cholinergic deregulation of airway smooth muscle contractility.</a> The Journal of Allergy and Clinical Immunology. 6749 (21), 01760-01767 (2021).</li><li>Khan, M. 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=An+integrated+multiomic+and+quantitative+label-free+microscopy-based+approach+to+study+pro-fibrotic+signalling+in+ex+vivo+human+precision-cut+lung+slices.">An integrated multiomic and quantitative label-free microscopy-based approach to study pro-fibrotic signalling in ex vivo human precision-cut lung slices.</a> The European Respiratory Journal. 58 (1), (2021).</li><li>Kennedy, 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=Effects+of+rhinovirus+39+infection+on+airway+hyperresponsiveness+to+carbachol+in+human+airways+precision+cut+lung+slices.">Effects of rhinovirus 39 infection on airway hyperresponsiveness to carbachol in human airways precision cut lung slices.</a> The Journal of Allergy and Clinical Immunology. 141 (5), 1887-1890 (2018).</li><li>Bai, Y., 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=Cryopreserved+Human+precision-cut+lung+slices+as+a+bioassay+for+live+tissue+banking.+a+viability+study+of+bronchodilation+with+bitter-taste+receptor+agonists.">Cryopreserved Human precision-cut lung slices as a bioassay for live tissue banking. a viability study of bronchodilation with bitter-taste receptor agonists.</a> American Journal of Respiratory Cell and Molecular Biology. 54 (5), 656-663 (2016).</li><li>Mondo&#241;edo, J. R., 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=A+high-throughput+system+for+cyclic+stretching+of+precision-cut+lung+slices+during+acute+cigarette+smoke+extract+exposure.">A high-throughput system for cyclic stretching of precision-cut lung slices during acute cigarette smoke extract exposure.</a> Frontiers in Physiology. 11, 566 (2020).</li><li>Davidovich, N., Huang, J., Margulies, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+uniform+equibiaxial+stretch+of+precision-cut+lung+slices.">Reproducible uniform equibiaxial stretch of precision-cut lung slices.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 304 (4), 210-220 (2013).</li><li>Ram-Mohan, 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=Tissue+traction+microscopy+to+quantify+muscle+contraction+within+precision-cut+lung+slices.">Tissue traction microscopy to quantify muscle contraction within precision-cut lung slices.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 318 (2), 323-330 (2020).</li></ol>

The preparation of PCLS involves several critical steps. First, it is essential to inflate the lung lobe homogeneously to avoid the variation of tissue stiffness from uneven agarose distribution. As the liquid agarose rapidly gels in thin catheters or airways at a temperature below 37 &#176;C, the resultant filling defect in the distal lung field could increase the disparity of lung tissue stiffness and cause tissue tearing during the vibratome section. Therefore, keeping the low-melting agarose solution at 42 &#176;C in...

Smooth muscle cell (SMC) is a major structural cell type in the lung, primarily residing in the media wall of airways and pulmonary vessels. SMCs contract to alter the luminal caliber, thus regulating air and blood flow1,2. Therefore, contractile regulation of SMCs is essential to maintain the homeostasis of air ventilation and pulmonary circulation. In contrast, aberrant SMC contractility provokes obstructive airway or pulmonary vascular diseases like asthma and pulmonary arterial hypertension. However, the functional assessment of lung SMCs has been challenged by limited access to the lung tissue, especially those small airways and microvessels in the distal part of the lung2,3. Current solutions resort to indirect assays, such as measuring airflow resistance by Flexivent to reflect airway constriction, and checking pulmonary arterial blood pressure by right heart catheterization to assess pulmonary vasocontraction4,5. However, these indirect assays have multiple disadvantages, such as being confounded by structural factors, failing to capture the spatial diversity of airway or vascular responses in the whole lung scale6,7, and unfitting for the mechanistic study of contractile regulation at the cellular level. Therefore, alternative approaches using isolated primary cells, trachea/bronchi muscle strips8,9, or large vascular segments10 have been applied for the SMC study in vitro. Nevertheless, these methods also have limitations. For example, a quick phenotypical adaptation of primary SMCs in the culture condition11,12 makes it problematic to extrapolate findings from cell culture to in vivo settings. In addition, the contractile phenotype of SMCs in the isolated proximal airway or vascular segments may not represent the SMCs in the distal lung6,7. Moreover, the muscle force measurement at the tissue level remains dissociated from molecular and cellular events that are essential for mechanistic insight into contractile regulation.
Precision-cut lung slice (PCLS), a live lung tissue section, provides an ideal ex vivo tool to characterize pulmonary SMCs in a near in vivo microenvironment (i.e., preserved multi-cellular architecture and interaction)13. Since Drs. Placke and Fisher first introduced the preparation of lung slices from agarose-inflated rat and hamster lungs in the 1980s14,15, this technique has been advanced continuously to provide PCLSs with higher quality and greater versatility for biomedical research. One significant improvement is the enhancement of pulmonary arterial preservation by gelatin infusion in addition to lung inflation with agarose via the trachea. As a result, both the airway and pulmonary arteries are kept intact in the PCLS for ex vivo assessement16. Furthermore, the PCLS is viable for a prolonged time in culture. For instance, mouse PCLSs had no significant change in cell viability and metabolism for a minimum of 12 days in culture, as well as, they retained airway contractility for up to 7 days17. In addition, PCLS keeps different-sized airways or vessels for contraction and relaxation assays. Moreover, intracellular Ca2+ signaling of SMCs, the determinant factor of cell contractility, can be assayed with Ca2+ reporter dyes imaged by a confocal or 2-photon microscope13.
Considering the extensive application of the mouse model in lung research, a detailed protocol is described here for preparing mouse PCLS with intact airways and intrapulmonary arteries for ex vivo lung research. Using the prepared PCLSs, we subsequently demonstrated how to evaluate the airway and pulmonary arterial responses to constrictive or relaxant stimuli. In addition, the method of loading the PCLS with Ca2+ reporter dye and then imaging Ca2+ signaling of SMCs associated with contractile or relaxant responses are also described.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>BD</td>
			<td>309626</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile centrifuge tubes</td>
			<td>Celltreat</td>
			<td>229411</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD</td>
			<td>309585</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tubes</td>
			<td>Celltreat</td>
			<td>229422</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl-beta-methacholine</td>
			<td>Millipore Sigma</td>
			<td>62-51-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-anitmycotic</td>
			<td>Thermo Fisher</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD-camera</td>
			<td>Nikon</td>
			<td>Nikon Ds-Ri2 camera</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glassess</td>
			<td>Fisher Scientific</td>
			<td>12-548-5CP; 12-548-5PP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic vials</td>
			<td>Fisher Scientific</td>
			<td>430488</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom-built laser scanning confocal microscope</td>
			<td></td>
			<td></td>
			<td>Details in Reference 18</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Fisher Scientific</td>
			<td>MT-10-092-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelin 1</td>
			<td>Millipore Sigma</td>
			<td>E7764</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine dissecting scissor</td>
			<td>Fisher Scientific</td>
			<td>NC9702861</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing container</td>
			<td>Sigma-Aldrich</td>
			<td>C1562</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>9000-70-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>Thermo Fisher</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic forcep</td>
			<td>Fisher Scientific</td>
			<td>16-100-117</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>High vaccum silicone grease</td>
			<td>Fisher Scientific</td>
			<td>146355d</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>W292907-1KG-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal washers</td>
			<td>Home Depot Product Authority</td>
			<td>800442</td>
			<td>Everbilt Flat Washers #10</td>
		</tr>
		<tr>
			<td>Micro-dissecting forcep</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle scalp vein set (25 G)</td>
			<td>EXELINT</td>
			<td>26708</td>
			<td></td>
		</tr>
		<tr>
			<td>NOC-5</td>
			<td>Cayman Chemical</td>
			<td>16534</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh</td>
			<td>Component Supply</td>
			<td>U-CMN-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Oregon green 488 BAPTA-1 AM</td>
			<td>Life Technologies</td>
			<td>o-6807</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase-contrast microscope</td>
			<td>Nikon</td>
			<td>Nikon Eclipse TS 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Thermo Fisher</td>
			<td>P-6867</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Personna</td>
			<td></td>
			<td>Personna Double Edge Razor Blades in White Wrapper 100 count</td>
		</tr>
		<tr>
			<td>Sulfobromophthalein</td>
			<td>Sigma-Aldrich</td>
			<td>S0252</td>
			<td></td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>Krazy Glue</td>
			<td></td>
			<td>Krazy Glue, All purpose</td>
		</tr>
		<tr>
			<td>Ultrapure low melting point agarose</td>
			<td>Thermo Fisher</td>
			<td>16520050</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Precisionary</td>
			<td>VF 310-0Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome chilling block</td>
			<td>Precisionary</td>
			<td>SKU-VM-CB12.5-NC</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome specimen tube</td>
			<td>Precisionary</td>
			<td>SKU VF-SPS-VM-12.5-NC</td>
			<td></td>
		</tr>
		<tr>
			<td>Y shaped IV catheter</td>
			<td>BD</td>
			<td>383336</td>
			<td>BD Saf-T-Intima closed IV catheter</td>
		</tr>
	</tbody>
</table>

utilizing the precision-cut lung slice to study the contractile regulation of airway and intrapulmonary arterial smooth muscle

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, respectively, hence playing a critical role in the homeostasis of the pulmonary system. Deregulation of SMC contractility contributes to several pulmonary diseases, including asthma and pulmonary hypertension. However, due to limited tissue access and a lack of culture systems to maintain in vivo SMC phenotypes, molecular mechanisms underlying the deregulated SMC contractility in these diseases remain fully identified. The precision-cut lung slice (PCLS) offers an ex vivo model that circumvents these technical difficulties. As a live, thin lung tissue section, the PCLS retains SMC in natural surroundings and allows in situ tracking of SMC contraction and intracellular Ca2+ signaling that regulates SMC contractility. Here, a detailed mouse PCLS preparation protocol is provided, which preserves intact airways and intrapulmonary arteries. This protocol involves two essential steps before subjecting the lung lobe to slicing: inflating the airway with low-melting-point agarose through the trachea and infilling pulmonary vessels with gelatin through the right ventricle. The PCLS prepared using this protocol can be used for bioassays to evaluate Ca2+-mediated contractile regulation of SMC in both the airway and the intrapulmonary arterial compartments. When applied to mouse models of respiratory diseases, this protocol enables the functional investigation of SMC, thereby providing insight into the underlying mechanism of SMC contractility deregulation in diseases.

Mouse PCLS preparation preserving intact intrapulmonary airways and arteries 
A 150 &#181;m thick PCLS was observed under the inverted phase-contrast microscope. In mouse lungs, conductive airways are accompanied by intrapulmonary arteries, running from the hilus to the peripheral lung. A representative pulmonary airway-artery bundle in a mouse PCLS is shown in Figure 2B. The airway can be easily identified by cuboidal epithelial cells with active cilial beating lining ...

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Utilizing the Precision-Cut Lung Slice to Study the Contractile Regulation of Airway and Intrapulmonary Arterial Smooth Muscle

The present protocol describes preparing and utilizing mouse precision-cut lung slices to assess the airway and intrapulmonary arterial smooth muscle contractility in a nearly in vivo milieu.

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, ...

The PCLS supports the assessment of airway and vascular smooth muscle cell activity in a nearly intact tissue environment, thereby providing an invaluable ex vivo model for pulmonary research. In term of smooth muscle research, the precision-cut lung slices preserve the in vivo phenotype of smooth muscle cells and their interaction with surrounding structures while allowing access to smooth muscle cell, which is important for a mechanistic study at the cellular level. To begin, place the mouse body on a dissection board in the supine position.Pin down the tail, front paws, and head with 25G syringe needles and sanitize the body with 70%ethanol. Open the chest cavity carefully along the sternum and bilateral inferior rib cage above the diaphragm. Then, point the sharp scissor tip away from the lung tissue and remove part of the bilateral ventral rib cages to expose the heart.Observe that the lung lobes collapse when the chest cavity opens. Use scissors to remove soft tissue in the mouse neck to expose the trachea. At the upper end of the trachea make a small hole of diameter 1.2 millimeters that should allow the passing of a 20G Y-shaped IV catheter tip.Connect one adapter port of the Y-shaped catheter with a 3 milliliter syringe prefilled with 0.5 milliliters of air and the other port with a 3 milliliter syringe prefilled with 2 milliliters of 1.5%agarose solution warmed to 42 degree Celsius. Inject agarose solution to fill the catheter and then push the catheter through the opening into the trachea to 5 to 8 millimeters. Slowly inject the agarose solution at a rate of 1 milliliter per 5 seconds.Observe that the lung expands along the proximal to distal axis. Stop the injection when the edge of each lung lobe is inflated. Then, inject 0.2 to 0.3 milliliters of air from the other syringe to push the residual agarose into conductive airways to the distal alveoli space.Clip closed the trachea with a pair of curved hemostatic forceps. To infill the pulmonary vasculature, fill a 1 milliliter syringe with warm 6%gelatin and connect it to a needle scalp vein catheter. Infill the catheter with gelatin solution and then puncture the right ventricle close to the inferior wall with the needle.Push the needle for 2 to 3 millimeters into the right ventricle and point the needle tip to the main pulmonary artery. Inject approximately 0.2 milliliters of gelatin solution slowly into the right ventricle and pulmonary arterial vessels. Keep the needle in place for five minutes after injection and cool the lung lobes by pouring ice cold HBSS solution on the heart and lung and place the body in a refrigerator.After this step, remove the mouse lung and heart from surrounding connective tissues with scissors. Then, separate each lung lobe and keep them in HBSS solution on ice. Trim the lung lobe and orient it such that the cutting direction is perpendicular to most airways from the hila to the lung surface.Attach it on top of the specimen column with super glue. Use a vibratome with a fresh thin razor blade to cut the lung lobe into 150 micrometer slices. Collect the slices in sterile Petri dishes prefilled with cold HBSS solution.Transfer the slices to Petri dishes filled with DMEM/F-12 culture medium. Place the dishes in a 37 degree Celsius incubator overnight before the experiments. Place a single precision-cut lung slice in each well of a 24 well culture plate filled with HBSS.Locate the slice in the middle of the well and then remove the HBSS solution with a pipette. Under a microscope, find the target airway and vessel in the slice and then cover it using a nylon mesh with a precut central hole to expose the targeted airway vessel area. Put a hollow metal washer on top of the mesh to hold the slices in place.Then, add 600 microliters of HBSS solution to submerge the slices. After 10 minutes, record the baseline images. To induce the airway or vascular contraction, cautiously remove the HBSS solution with a pipette and add 600 microliters of HBSS with an agonist.To prepare the calcium dye loading buffer, first dissolve 50 micrograms of dye with 10 microliters of DMSO and 0.2 grams of Pluronic F-12 powder in 1 milliliter of DMSO. Mix 10 microliters of the Pluronic solution with 10 microliters of calcium dye solution. Add this mixture to 2 milliliters of HBSS solution containing 200 micromolar of sulfobromophthalein.Then, place 15 precision-cut lung slices in 2 milliliters of calcium loading buffer and incubate at 30 degrees Celsius for one hour, followed by incubating in HBSS containing 100 micromolar sulfobromophthalein for 30 minutes. Then, place calcium dye loaded slices on a large cover glass. Fill high vacuum silicone grease in a 3 milliliter syringe attached to 18G blunt needle or a 200 microliter pipette tip and draw two parallel lines across the cover glass, above and below the slice.Cover the slice using an nylon mesh between two grease lines. Place the second cover glass on top of the mesh to generate a chamber. Add HBSS or a agonist solution into the chamber from one end with a pipette.Remove the fluid from the chamber by suctioning from the other end with tissue paper. The slice's chamber is now ready for the calcium imaging with a high speed laser scanning confocal microscope. Pulmonary airway artery bundles were observed in precision-cut lung slices of thickness 150 micrometers under a phase contrast microscope.At resting state, an airway was identified by cuboidal epithelial cells with a nearby pulmonary artery. Following exposure to methacholine, the luminal area was reduced while the pulmonary artery showed no response to the stimuli. Airway contractile responses were quantified by the percentage of luminal area reduction and showed similar dose dependent responses in one day and five day cultures.Upon reaching the peripheral lung field, the conductive airways branch into respiratory ducts and sacs surrounding the small intra-acinar arterioles. When exposed to endothelii both airways and pulmonary arteries constrict, which is followed by NOC-5 induced relaxation. At the resting state, the calcium dye loaded slices showed low fluorescence in the smooth muscle cells of the airway and vascular system under a confocal fluorescent microscope.Upon exposure to agonists, the calcium fluorescence intensity elevated in the cells and propagated to the entire cell, which correlated with the oscillatory signals. Technique wise, it is essential to inflate the lung with agarose homogeneously and avoid quick or excessive agarose injection. Always push air at the end to flush the agarose from the conductive airway to the distal alveoli space.In summary, using the precision-cut lung slices culture, one can provide a special variety of pulmonary smooth muscle functions and model the smooth muscle deregulation in vitro. It also provides an ideal platform to screen novo vasodilatory or bronchodilator medications.

utilizing-precision-cut-lung-slice-to-study-contractile-regulation

Research

JoVE Journal

Biology

3.2K visualizações.  Massachusetts General Hospital and Harvard Medical School. O PCLS apoia a avaliação da atividade celular lisa das vias aéreas e vasculares em um ambiente tecidual quase intacto, fornecendo assim um modelo ex vivo inestimável para pesquisa pulmonar. Em termos de pesquisa muscular suave, as fatias pulmonares cortadas com precisão preservam o fenótipo in vivo de células musculares lisas e sua interação com estruturas circundantes, permitindo o acesso a células musculares lisas, o que é importante para um estudo mecanicista no nível celular. ...