This work was supported by the National Institutes of Health (R01HL056046 to PAL and K99HL116769 to AJT), and The Research Institute at Nationwide Children&#39;s Hospital (to PAL and AJT).

Ethics Statement: This study was conducted in accordance with the National Institutes of Health Guidelines, and it was approved by the Institution Animal Care and Use Committee at Nationwide Children&#39;s Hospital.
1. Preparation/Set up
Note: This isolation technique requires two Langendorff heating coils positioned side-by-side on a ring stand, and connected in parallel to a circulating water bath.
<ol>
	<li>Turn on circulating water bath and adjust temperature to achieve a final temperature of 33-34 &#176;C at the tip of the Langendorff cannula (the water bath used in this set up is set to 40 &#176;C due to a 6-7 &#176;C loss in temperature across tubing). Turn on the perfusion pumps and set perfusion rate to 0.5 ml/min.
	<ol>
		<li>Clean both of the water-jacketed heating coils, attached tubing and 25 gauge cannula by flushing each with 50 ml of 70% ethanol, followed by 100 ml of autoclaved ultrapure water.</li>
		<li>Clear the heating coils by pushing air with a syringe and wash with 10 ml of Hank&#39;s Balanced Salt Solution (HBSS) without phenol red. Leave HBSS in the heating coils and tubing.</li>
		<li>Attach a sterile 10 ml luer-lock syringe filled with room temperature HBSS without phenol red to the tubing. Ensure that no air bubbles are trapped inside the tubing and heating coils when attaching the HBSS-filled syringe as this could cause air emboli during the digestion to negatively impact the VSMC isolation.</li>
	</ol>
	</li>
	<li>Remove the 25 gauge cannula from the heating coils and place on another sterile 10 ml syringe filled with ice-cold HBSS without phenol red. Keep this on ice until ready to use. Repeat for the other cannula.</li>
	<li>Prepare Enzyme Digestion Solution.
	<ol>
		<li>Weigh collagenase type 2 according to specific lot number for 75 ml of digestion solution to achieve a final concentration of 300 Units/ml.</li>
		<li>Dissolve collagenase in 75 ml of HBSS with phenol red. Next, add 1.5 ml of 0.1 mg/ml soybean trypsin inhibitor and 75 &#181;l of 1 M CaCl2 to the mixture.</li>
	</ol>
	</li>
	<li>If using the digestion solution immediately, warm it to 37 &#176;C. If not, place it at 4 &#176;C for up to 6 hr. This solution must be made fresh daily.</li>
	<li>Prepare the stop solution/plating media.
	<ol>
		<li>Add 100 ml heat-inactivated FBS, 5 ml of L-glutamine, 5 ml of non-essential amino acids, 5 ml of HEPES, and 1 ml of primocin to 500 ml of high glucose (4.5 g/L) DMEM. Filter sterilization is recommended.</li>
		<li>Aliquot 6 ml of the stop solution into each of twelve 15 ml conical tubes, numbered A1-A6 and B1-B6, and place at 4 &#176;C. Aliquot 50 ml of stop solution into a 50 ml conical and place in 37 &#176;C incubator. 
		Note: This step can also be prepared the day prior to isolation if necessary. This stop solution will be used as the plating media following cell isolation.</li>
	</ol>
	</li>
	<li>Prepare two 1 ml syringes equipped with a 30 G needle with 100 &#181;l of heparin (1,000 units/ml).</li>
	<li>Cut a 5 cm piece of 5-0 surgical silk. Tie a loose overhand knot in the surgical silk with one end twice as long as the other. Place onto the 25-gauge cannula, but do not tighten. Repeat for the other 25 G cannula.</li>
	<li>Fill four 35 mm sterile Petri dishes (#1-4) with 2 ml of sterile, ice-cold, HBSS without phenol red. Keep on ice.</li>
</ol>
2. Euthanasia, Heart Isolation and Cannulation
<ol>
	<li>Before heart isolation, ensure that the digestion solution and 50 ml conical of stop solution are warm. Remove the HBSS-filled syringe with attached 25 G cannula from the ice and place in a burette clamp (or ring stand clamp) on a ring stand.</li>
	<li>Anesthetize mice using 3% isofluorane. Confirm the mouse is anesthetized by a toe touch on each hind limb. If the leg twitches, the mouse is not yet anesthetized so wait 1 min and repeat the toe touch.</li>
	<li>Expose the jugular vein by removing the fur and skin on the anterior of the neck with scissors. Next, use forceps to carefully remove any fat surrounding the jugular vein. After removing fat, slowly inject 100 &#181;l of heparin (1,000 units/ml). Place a surgical sponge over the vein to avoid excessive bleeding.</li>
	<li>After 1 min, open chest to expose heart, lift heart with curved forceps and excise it using scissors, making sure to keep the ascending aorta intact through the first brachiocephalic branch. Immediately place heart into a 35 mm dish (#1) with ice-cold HBSS without phenol red and rinse. 
	Note: Perform the following 4 steps using a dissecting scope.</li>
	<li>Position the 25 G cannula that is mounted on the ring stand at a 45&#176; angle so that the tip is located just under the surface of ice-cold HBSS in dish #2 and is clearly visible through the dissecting microscope. Transfer the heart to dish #2.</li>
	<li>Using Dumont forceps, expose the aorta by gently removing excess adipose via blunt dissection to prevent puncturing or tearing of the aorta. Next, use forceps to slide the aorta over the 25 G cannula.</li>
	<li>Guide the cannula downward through the aorta, close to, but not beyond the aortic valve, which will compromise the integrity of the valve and therefore, the retrograde perfusion. Once the cannula is in place, slide the pre-tied silk over the aorta, and tighten. Tie a second knot to form a square knot around the aorta to secure the heart.</li>
	<li>Once the aorta is securely tied, gently flush the remaining blood from the coronary circulation with the ice-cold HBSS in the syringe attached to the 25 G cannula. Take care to not push too much to avoid excessive coronary pressure. Only a very slow and gentle flush is required to complete the flush. Leak-free aortic cannulation can also be observed during this gentle flush.</li>
	<li>Turn on the perfusion pump to begin the flow of HBSS. Remove the 25 G cannula (with the heart attached) from the HBSS-filled syringe, taking care to keep the cannula hub filled with HBSS, and connect it to the warm HBSS-filled heating coils. Begin perfusion of the heart with the perfusion pump at a rate of 0.5 ml/min for 8 min.</li>
	<li>After the first heart is cannulated and is being perfused on the system, repeat steps 2.1-2.8 for the second heart. These isolations will be staggered for each heart therefore careful planning and timing are required.</li>
</ol>
3. Digestion
<ol>
	<li>After flushing with HBSS, change the 10 ml HBSS syringe currently on the pump to one filled with 10 ml of pre-warmed digestion solution. Perfuse each heart with digestion solution for 12 min at a rate of 0.5 ml/min. Do not collect fractions stemming from this first digestion as this will contain any remnant blood and endothelial cells.</li>
	<li>After 12 min, change the perfusion rate to 0.4 ml/min, place a 15 ml conical (tube A1 or B1, corresponding to the first or second heart for each digestion) filled with cold stop solution into an ice bucket beneath the heart in order to begin VSMC-enriched perfusate collection.</li>
	<li>After 15 min, change the collection conical to tube A2 or B2 and spin conical tube A1 and B1 at 89 x g for 10 min immediately after collection of both fractions. When the syringe filled with the digestion solution on the pump has less than 1 ml of solution, replace with a freshly-filled 10 ml syringe of digestion solution.</li>
	<li>After centrifugation, aspirate supernatant from each collection tube, leaving approximately 0.5 ml. Resuspend pellet from A1 with 2 mL of warm stop solution (or plating media). Add resuspension from A1 tube to B1 tube and place tube in 37 &#176;C incubator with the cap loose.</li>
	<li>Repeat steps 3.3 and 3.4 until all the tubes (through A6 and B6) are used for a total collection time of 90 min. At 80 min of digestion, cut open the heart apex to flush out any cells that may be trapped inside the ventricle and collect these cells in tube A6 and B6, respectively. 
	Note: Occasionally the heart will fall off the cannula prior to the 90 min time point. If this occurs, retrieve the heart and place into a sterile 35 mm Petri dish filled with 2 ml of digestion solution. Cut the apex of the heart and gently rinse the heart with digestion solution. Next, filter the 2 ml of digestion solution through a sterile 100 &#181;m cell strainer into a 50 ml conical and add to tube A6 or B6.</li>
	<li>After each spin of subsequent tubes (A2 and B2), add the resuspended VSMCs to the previous collection tube (combined A1 and B1). Also, take care not to let the digestion solution in the pump run out and switch the 10 ml syringes as necessary throughout the digestion.</li>
	<li>After all the tubes are combined, centrifuge the VSMC-rich suspension at 89 x g for 10 min.</li>
	<li>Aspirate as much supernatant from collection tubes as possible. Resuspend the remaining pellet in 2 ml of plating media and plate on one 35 mm culture dish. Place culture dish in 37 &#176;C incubator for 24 hr.</li>
</ol>
4. Cell Culture
Note: Complete the remaining steps in the biosafety cabinet.
<ol>
	<li>After 24 hr, gently aspirate the plating media. The culture will normally have a large amount of debris. To wash the cells, add 2 ml of warm, sterile PBS and gently tap the dish against two fingers while rotating in a clockwise motion.</li>
	<li>After rotating 360&#176;, aspirate the PBS and repeat a second time. Aspirate the PBS and add another 2 ml of sterile, warm PBS. Check the culture dish under the microscope to make sure there is little to no debris on the plate. Cell confluence can also be determined at this time.</li>
	<li>If debris remains in the culture dish, repeat step 4.2. If the culture dish is clear of debris, aspirate the PBS and add 2 ml of warm plating media.</li>
	<li>After 24 hr, wash the cells again with warm, sterile PBS to remove any final debris or dead cells from the culture. Aspirate the PBS and place another 2 ml of warm plating media on the cells.</li>
	<li>Check on the cells every day and feed every 48 hr with fresh warmed plating media. At this point, cells can be used for standard cell culture assays. VSMCs should be approximately 80% confluent 4-5 days after plating, although may take up to 7 days until the procedure is perfected. If splitting to subsequent passages, use a maximum of 1:4 35 mm dishes (or to a comparable area of culture dishes).</li>
</ol>

<ol>
	<li>Katz, P. 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=Coronary+arterioles+in+type+2+diabetic+(db/db)+mice+undergo+a+distinct+pattern+of+remodeling+associated+with+decreased+vessel+stiffness.">Coronary arterioles in type 2 diabetic (db/db) mice undergo a distinct pattern of remodeling associated with decreased vessel stiffness.</a> Basic Res Cardiol. 106 (6), 1123-1134 (2011).</li><li>Husarek, K. 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=The+angiotensin+receptor+blocker+losartan+reduces+coronary+arteriole+remodeling+in+type+2+diabetic+mice.">The angiotensin receptor blocker losartan reduces coronary arteriole remodeling in type 2 diabetic mice.</a> Vascul Pharmacol. , (2015).</li><li>Teng, B., Ansari, H. R., Oldenburg, P. J., Schnermann, J., Mustafa, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+coronary+endothelial+and+smooth+muscle+cells+from+A1+adenosine+receptor-knockout+mice.">Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice.</a> Am J Physiol Heart Circ Physiol. 290 (4), H1713-H1720 (2006).</li><li>Oldenburg, P. J., Wyatt, T. A., Sisson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+attenuates+contraction+of+primary+cultured+rat+airway+smooth+muscle+cells.">Ethanol attenuates contraction of primary cultured rat airway smooth muscle cells.</a> Am J Respir Cell Mol Biol. 43 (5), 539-545 (2010).</li><li>Xu, 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=Intracellular+two-phase+Ca2++release+and+apoptosis+controlled+by+TRP-ML1+channel+activity+in+coronary+arterial+myocytes.">Intracellular two-phase Ca2+ release and apoptosis controlled by TRP-ML1 channel activity in coronary arterial myocytes.</a> Am J Physiol Cell Physiol. 304 (5), C458-C466 (2013).</li><li>Skrzypiec-Spring, M., Grotthus, B., Szelag, A., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+heart+perfusion+according+to+Langendorff---still+viable+in+the+new+millennium.">Isolated heart perfusion according to Langendorff---still viable in the new millennium.</a> J Pharmacol Toxicol Methods. 55 (2), 113-126 (2007).</li><li>Owens, G. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+phenotype+and+function+of+vascular+smooth+muscle+cells.">Diversity of phenotype and function of vascular smooth muscle cells.</a> J Lab Clin Med. 127 (6), 524-529 (1996).</li><li>Souza-Smith, F. 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=Mesenteric+resistance+arteries+in+type+2+diabetic+db/db+mice+undergo+outward+remodeling.">Mesenteric resistance arteries in type 2 diabetic db/db mice undergo outward remodeling.</a> PLoS One. 6 (8), e23337 (2011).</li><li>Thuroff, J. 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The authors declare that they have no competing financial interests.

The purpose of this study was to adapt existing cell isolation protocols to increase the yield of coronary vascular smooth from murine hearts. Most of the pioneering work in vascular smooth muscle biology was performed with cultured rat aortic smooth muscle cells. These studies provided fundamental knowledge of molecular mechanisms that control VSMC growth, migration and hypertrophy7. However, as the field progressed, it became apparent that VSMC phenotype and function was controlled by a number of vascular be...

The goal of this method is to isolate vascular smooth muscle cells (VSMCs) from the murine coronary circulation for use in cell culture and standard cell culture assays. We developed this technique to assess the molecular mechanisms of vascular remodeling in diabetes. We have previously reported inward hypertrophic remodeling in the septal coronary arterioles in the db/db mouse model of diabetes1. Due to the limited amount of tissue found in the murine septal coronaries, standard experimental techniques investigating protein changes (e.g. western blot) in db/db and control mice are difficult at best. In addition, we have previously shown that the angiotensin receptor blocker (ARB) losartan reduces the remodeling observed in db/db mice2. Therefore, isolation of primary VSMCs from the coronary circulation allows us to further investigate changes in VSMC phenotype or activated signaling pathways in diabetic mice, which may be contributing to adverse coronary arteriole remodeling.
Numerous studies have elucidated canonical signaling pathways using VSMCs isolated from rodent aorta, rather than in each specific vascular bed. However, we have demonstrated vascular-bed specific remodeling in coronary, aortic, and mesenteric circulations of db/db mice1, suggesting the VSMCs in each vascular bed may be different. Therefore, it is necessary to isolate VSMCs from each vascular bed in order to better understand the pathological changes occurring in each set of VSMCs. There are a plethora of different methods for isolating and culturing aortic VSMCs. However, currently, there is only one study that has been published on the isolation of VSMCs from the mouse coronary circulation3. Teng et al. was the first to report a method for isolating VSMCs from the mouse coronary circulation; however, we have amended the protocol significantly as they also isolated endothelial cells. Other labs have also used the protocol from Teng et al. to isolate coronary arterial myocytes and airway smooth muscle cells4,5. The alterations we have incorporated will yield a population of cells highly enriched for VSMCs from the coronary circulation.
The retrograde perfusion of the isolated mammalian heart, or Langendorff technique, was established in 18976 by Oscar Langendorff and is still widely used today for the isolation of cardiovascular cells. The technique presented here, coupled with the advancement of modern murine genetic modifications, provides a valuable tool for closer investigation of the molecular behavior of VSMCs from the coronary circulation.

<table border="0" cellpadding="0" cellspacing="0" width="758">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Fetal bovine serum</td>
			<td style="width:143px;">Life Technologies</td>
			<td style="width:119px;">16140-071</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">HEPES 1M solution</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">MT-25-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primocin - 20mL</td>
			<td>Invivogen</td>
			<td>ant-pm-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (High Glucose, Sodium Pyruvate, L-Glutamine)&#160;</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM NEAA 10 mM 100X</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glut 200 mM - Gibco</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Cell Strainer 100um nylon mesh</td>
			<td>Fisher</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunclon Polystrene dish with lid, sterile, 35 mm</td>
			<td>Fisher</td>
			<td>12-565-91</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Harvard Apparatus black silk suture 5-0</td>
			<td>Fisher</td>
			<td>14-516-124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase Type-2&#160;</td>
			<td>Worthington Biochemical</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor 25mg</td>
			<td>Sigma</td>
			<td>T6522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid (clear)</td>
			<td style="width:143px;">Life Technologies</td>
			<td>14175-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid (phenol red)</td>
			<td style="width:143px;">Life Technologies</td>
			<td>14170-161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5.0 ml heating coil with degassing bubble trap</td>
			<td>Radnoti</td>
			<td>158830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">11 plus pump</td>
			<td style="width:143px;">Harvard Apparatus</td>
			<td style="width:119px;">70-2208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Circulating heated water pump</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td>any brand will work</td>
		</tr>
	</tbody>
</table>

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.

Due to the novel aspect of our coronary VSMC isolation technique, we sought to determine the purity of the cell isolation. Mouse coronary VSMCs were identified based on their morphology and immunofluorescence staining up to passage 2. Based on morphology of the cells in culture after the first wash, the isolation procedure effectively removes cardiac myocytes and endothelial cells. The VSMCs retain their morphology up to passage 2 (Figure 1). However, there is a possibili...

Watch this Scientific Journal Video about Isolation of Murine Coronary Vascular Smooth Muscle Cells at JoVE.com

Isolation of Murine Coronary Vascular Smooth Muscle Cells

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 ...

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14.0K Views.  The Ohio State University College of Medicine. 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.

Video: Isolation of Murine Coronary Vascular Smooth Muscle Cells

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 ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

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 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.

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 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 and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...