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%) Penicillin/Streptomycin.</li>
	<li>Prepare the PA Agarose - Mix 0.05 g agarose plus 0.05 g iron particles in 10 ml sterile, serum-free M199 media.</li>
	<li>Prepare the Lung Agarose - Mix 0.1 g agarose in 10 ml sterile, serum-free M199 media.</li>
	<li>Prepare the Collagenase - Mix collagenase into sterile, serum-free M199 media at a final concentration of 80 U/ml and then sterile filter this mixture.</li>
	<li>Set up all of the equipment ahead of time before euthanizing the mouse.</li>
	<li>Set PA agarose and lung agarose to boil right before euthanizing the mouse.</li>
	<li>Euthanize the mouse in the anesthetic jar with isoflurane overdose and be careful not to allow the mouse pup to come in contact with the isoflurane. The best cell yield is obtained when the cells are harvested immediately after death in order to avoid micro-clots that form in the circulation after death.</li>
	<li>Remove the mouse from the anesthetic jar, secure the mouse to the operating board, and sterilize the mouse, instruments, and gloves with alcohol.</li>
	<li>Load the scalpel with the blade and incise the mouse from the neck to the inferior abdomen.</li>
	<li>Open the abdomen on the mouse&#39;s left side, expose the kidney, and cut the renal artery to allow the mouse to exsanguinate, thereby avoiding clots in the small vessels of the lungs.</li>
	<li>Use scissors to cut along the mouse&#39;s left side of the sternum to open the thorax.</li>
	<li>Carefully use scalpel to cut the diaphragm on both sides and fully open the chest cavity. Use forceps or pins to hold the chest cavity open.</li>
	<li>Stick the RV with a 27 G &#189; inch needle directed towards the PA and perfuse the RV/PA/lungs with sterile PBS until the lungs appear white (all blood flushed out of the pulmonary circulation). This will take approximately 3-5 ml of PBS depending on the size of the mouse. It is important to flush first to prevent clot formation in the small lung vessels.</li>
	<li>Carefully use blunt dissection to expose the trachea and thread one suture underneath.</li>
	<li>Put the smallest forceps underneath the trachea for support and place the 24 G angiocatheter into the trachea. Place the angiocatheter as if placing an IV.</li>
	<li>Secure the angiocatheter into the trachea with the suture threaded in step 1.16.</li>
	<li>Take boiling PA agarose out of boiling water, and invert to mix well. Cool the agarose to approximately body temperature by swirling on ice. The agarose needs to remain in solution but not be so hot that it will damage the cells of the PA.</li>
	<li>Stick the RV with a 27 G &#189; inch needle and perfuse the RV/PA/lungs with PA agarose until the lungs appear grey. The iron particles in the agarose mixture will make the lungs appear grey in color. This will take approximately 3-5 ml of PA agarose depending on the size of the mouse. It is also important to go slowly as injecting too fast might cause the iron to leak into the airways.</li>
	<li>Take the lung agarose out of boiling water and invert to mix well. Then cool the agarose to approximately body temperature by swirling on ice. The agarose needs to remain in solution but not be so hot that it will damage the cells of the lung.</li>
	<li>Draw up the lung agarose and attach the syringe to the 24 G angiocatheter in the trachea. Infuse the agarose through the trachea to fully inflate the lungs. The lungs will be easier to mince (see later steps) if the lungs are appropriately inflated.</li>
	<li>Remove the heart-lung bloc, and submerge it into 25 ml of ice cold PBS to solidify the agarose (~5 min). Use this time to clean up the surgical area.</li>
	<li>For neonatal mice (P7-P21), the best PASMC yield comes from combining 2-3 pups into one batch of cells. PASMC prefer to be in contact with other cells for optimal cell health and growth. If one mouse is used per isolation, then the cells are very sparse and do not grow well. When combining animals, harvest and chill the heart-lung blocs together and then move to mincing once all heart-lung blocs have been removed from the animals.</li>
	<li>Move to the cell culture hood for sterility from this point onward.</li>
	<li>Place the chilled lungs into the lid of the 10 cm tissue culture dish and carefully remove the heart, thymus, trachea,&#160;and any connective tissue from the lungs.</li>
	<li>Move the lungs to the bottom of the 10 cm tissue culture dish and add approximately 1 ml ice-cold sterile PBS.</li>
	<li>Mince the lungs into very small pieces (1-2 mm) with 2 scalpels.</li>
	<li>Break the tip off of a sterile 5 ml serological pipette. Pipette minced lung slurry into sterile 50 ml conical tube. Use additional sterile ice-cold PBS to wash the plate to insure that all of the lung tissue gets into the 50 ml conical tube.</li>
	<li>Place the 50 ml conical tube onto the magnet. Wait for the iron-containing tissue to move to side of the tube next to the magnet. Depending on the ratio of iron-containing vessel to lung tissue, some of the vessels may still be floating at this point. Aspirate off the PBS slowly and carefully trying not to aspirate floating lung tissue since it is unclear if it contains vessels.</li>
	<li>Wash the lung pellet 3x with 5 ml sterile PBS, again trying to minimize the amount of lung tissue that is aspirated.</li>
	<li>Resuspend the lung pellet in 3 ml collagenase and pour into 60 mm tissue culture dish. Do not try to pipette&#160;this; the lung chunks will stick to the interior of your pipette.</li>
	<li>Use an additional 3 ml to rinse the tube after pouring, and pour this into 60 mm tissue culture dish as well.</li>
	<li>Place into 37 &#176;C tissue culture incubator for 1 hr.</li>
	<li>Pipette tissue+collagenase slurry up and down with a 15 G blunt needle on a 5 ml syringe until all the large lung pieces are disrupted. The tissue+collagenase slurry will be cloudy at this point with no visible tissue chunks.</li>
	<li>Pour into a new sterile 50 ml conical tube and attach to the magnet.</li>
	<li>Wash the tissue culture dish with approximately 5 ml of complete M199 media to insure that all of the tissue has been collected, and pour this into the conical tube.</li>
	<li>Aspirate off the collagenase+media supernatant. This time there will be very few floaters, and a compact iron-containing pellet will be on the side of the conical tube next to the magnet.</li>
	<li>Wash with 5 ml complete media 3x. This is required to inactivate the collagenase.</li>
	<li>Add 3 ml of complete media, and mix up and down a few times to resuspend the iron-containing pellet.</li>
	<li>Pour the resuspended pellet into a 35 mm tissue culture dish. Using a microscope, there will be small vessel fragments floating around with iron particles contained in the vessel lumen.</li>
	<li>Place the tissue culture dish (labeled plate zero) in the tissue culture incubator overnight. The first cells to migrate off onto plate zero are approximately 50% fibroblasts and 50% PASMC, based on staining for smooth muscle markers. This plate is called plated zero and ultimately discarded after the subsequent enrichment steps.</li>
</ol>
2. Pulmonary Artery Isolation from Neonatal Mice - Day Two
<ol>
	<li>Pour the media supernatant from plate zero into a clean 50 ml conical tube.</li>
	<li>Aspirate off the media supernatant. This time there will be almost no floaters, and a compact iron-containing pellet will form on the side of the conical tube next to the magnet.</li>
	<li>Wash with 5 ml complete media 3x.</li>
	<li>Add 3 ml of complete media, and mix up and down a few times to resuspend the iron-containing pellet.</li>
	<li>Pour into a 35 mm tissue culture dish. Using a microscope, small vessel fragments floating around with iron particles contained in the vessel lumen are seen. This will be plate one (Figure 1A).</li>
	<li>Place the tissue culture dish in the tissue culture incubator.</li>
</ol>
3. Pulmonary Artery Isolation from Neonatal Mice - Day Six
<ol>
	<li>Pour the media supernatant from plate one into a clean 50 ml conical tube. Repeating this step multiple times insures that all of the smooth muscle cells resident in the wall of the vessel have an opportunity to migrate off onto a cell culture plate. There is a diminishing return with each step i.e. each plate will have fewer and fewer cells adhere.</li>
	<li>Refeed plate one with complete M199 media, and place back into the tissue culture incubator.</li>
	<li>Aspirate off the media supernatant. This time there will be almost no floaters, and a compact iron-containing pellet will form on the side of the conical tube next to the magnet.</li>
	<li>Wash with 5 ml complete media 3x.</li>
	<li>Add 3 ml of complete media, and mix up and down a few times to resuspend the iron-containing pellet.</li>
	<li>Pour into a 35 mm tissue culture dish. Using a microscope, small vessel fragments floating around with iron particles contained in the vessel lumen are seen. This will be plate two.</li>
	<li>Place the tissue culture dish in the tissue culture incubator.</li>
</ol>
4. Pulmonary Artery Isolation from Neonatal Mice - Day Nine
<ol>
	<li>Pour the media supernatant from the tissue culture dish plate two into a clean 50 ml conical tube.</li>
	<li>Refeed plate two with complete M199 media, and place back into the tissue culture incubator.</li>
	<li>Aspirate off the media supernatant. This time there will be almost no floaters, and a compact iron-containing pellet will form on the side of the conical tube next to the magnet.</li>
	<li>Wash with 5 ml complete media 3x.</li>
	<li>Add 3 ml of complete media and mix up and down a few times to resuspend the iron-containing pellet.</li>
	<li>Pour into a 35 mm tissue culture dish. Using a microscope, small vessel fragments floating around with iron particles contained in the vessel lumen are seen. This will be plate three.</li>
	<li>Place the tissue culture dish in the tissue culture incubator.</li>
</ol>
5. Pulmonary Artery Isolation from Neonatal Mice - Day Thirteen
<ol>
	<li>Aspirate the media off of plates 1-3.</li>
	<li>Gently wash the cells with warm, sterile PBS.</li>
	<li>Add a thin film of trypsin (~0.25-0.5 ml) trypsin to each plate to trypsinize off cells. Cells are very adherent and will need to be in trypsin in the tissue culture incubator for approximately 10 min to lift off the plate.</li>
	<li>Harvest cells off the plates into 5 ml complete media in a 50 ml conical tube attached to the magnet to pull out any residual iron particles.</li>
	<li>Wash each of the plates with an additional 0.5 ml of complete media and add that media to the 50 ml conical tube.</li>
	<li>This time, take the media supernatant (do not aspirate), and discard the iron particle pellet.</li>
	<li>Plate the cells in 5 ml complete M199 media in a 60 mm dish to have confluent cells in a few days. Alternatively, plate the cells in 10 ml complete M199 media in a 10 cm plate, and PASMC will take approximately another 1-2 weeks to reach confluence.</li>
</ol>
6. Pulmonary Artery Isolation from Neonatal Mice - Day Fourteen
<ol>
	<li>Change the media to fresh complete M199 media to remove any residual trypsin. There will be a thin population of PASMC on the tissue culture dish (Figure 1B).</li>
</ol>
7. Routine Care
<ol>
	<li>Change the media to fresh complete M199 media approximately 2x per week.</li>
	<li>When splitting these cells, approximately 10 min in the incubator in a film of trypsin (0.25-0.5 ml) is required for the cells to lift off the plate. Using the trypsin film allows replating of the PASMC without spinning down the cells. If too much trypsin is used, then the PASMC must be centrifuged to spin out the trypsin, or the cells will not readhere to the plate. Sometimes, after spinning down the cells, it is hard to get the PASMC resuspended into complete M199 media.</li>
	<li>In observations, the isolated PASMC behave like smooth muscle cells reliably for 4-5 passages, but the isolated cells will stain for smooth muscle cell markers up to passage 7. Count the first pooled 60 mm or 10 cm plate on day thirteen as passage 2 (plating after first exposure to trypsin).</li>
</ol>

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The authors declare that they have no competing financial interests.

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 ...

isolation-of-pulmonary-artery-smooth-muscle-cells-from-neonatal-mice

Research

JoVE Journal

Biology

18.2K Views.  Northwestern University Feinberg School of Medicine. Мы разработали новый и воспроизводимой техники для изоляции первичных культурах легочной артерии гладкомышечных клеток (PASMC) от мышей в возрасте P7, что позволяет лучше изучения сигнальных путей, участвующих в неонатальном сокращение гладких мышечных клеток ...

Video: Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

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+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

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)

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 and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

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 (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

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 regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

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 basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

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 nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

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 surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

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 from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

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 processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Proliferation is a critical part of cellular function, and a common readout used to assess potential toxicity of new drugs. Measuring proliferation is, therefore, a frequently used assay in cell biology. Here we present a simple, versatile method of measuring proliferation that can be used in adherent and non-adherent cells.

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 of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...