The authors thank T. Yamamoto and Y. Mori for their assistance in the morphologic experiments. This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (18K06871 to M.O.K. and 17K08536 to H.M.).

All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the institutional Review Board of the Shiga University of Medical Science Animal Care and Use Committee (approved No. 2019-3-7). The methods were carried out in accordance with approved guidelines.
1. Instruments and solution
NOTE: An outline of the experimental procedure is illustrated in a flow diagram (Supplementary Figure 1). An infusion pump (or syringe pump) should be used for the antegrade perfusion of the heart with a one-way flow. A peristaltic pump that creates a pulsating flow is not recommended.
<ol>
	<li>Before the experimental
	<ol>
		<li>Mark the injection needle at a site approximately 3 mm from the tip with nail polish. After allowing it the air dry, keep it in the container at room temperature. A red or bright color is desirable to confirm the depth of insertion into the myocardium during perfusion.</li>
		<li>Make the heart stand by cutting the lids off of 1.5-, 0.5- and 0.2-mL sample tubes and attaching the lids to the bottom of a 60-mm culture dish with double-sided tape or adhesive. Fixation of three different-sized lids in one dish makes it possible to choose the appropriate one according to the size of the mouse heart. This heart stand can be reused after washing.</li>
		<li>Make stock solution as shown in Table 1. Store the stock solutions at 4 &#176;C.</li>
	</ol>
	</li>
	<li>On the experiment day 
	NOTE: Cell isolation buffer (CIB) contains (in mM) 130 NaCl, 5.4 KCl, 0.5 MgCl2, 0.33 NaH2PO4, 22 glucose, 40 units/mL insulin and 25 HEPES (pH adjusted to 7.4 with NaOH); and Tyrode solution contains (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.33 NaH2PO4, 5.5 glucose and 5.0 HEPES (pH adjusted to 7.4 with NaOH).
	<ol>
		<li>Prepare the CIB. Warm 160 mL of distilled water (DW) using a microwave to around 32 &#176;C and then add 20 mL of 10X CIB. After the addition of 0.79 g glucose and 10 &#181;L of insulin solution, adjust the pH using 1 M NaOH and bring to 200 mL with DW.</li>
		<li>Prepare the enzyme mix solution (enzyme mix). Add 30 mg of collagenase, 1.8 mg of trypsin, 1.8 mg of protease and 90 &#181;L of 100 mM CaCl2 stock solution to 30 mL of CIB (final Ca2+ concentration is 0.3 mM), mix, and keep it on ice. In mice &#60;4 weeks old, reduce trypsin and protease to 0.9 mg10. Warm at 37 &#176;C in a water bath before use.</li>
		<li>Prepare the CIB-Ca2+-BSA solution. Add 30 mg BSA and 90 &#181;L of 100 mM CaCl2 stock solution to 15 mL of CIB (final Ca2+ concentration is 1.2 mM), mix, filter through a 20-&#181;m filter, and keep it on ice.</li>
		<li>Prepare the CIB-EGTA solution. After making the solutions described in steps 1.2.2 and 1.2.3, add 400 mM EGTA stock solution to the remaining CIB at a 1:1000 dilution (final EGTA concentration is 0.4 mM), and mix. Fill the 35-mm culture dish and heart stand dish with CIB-EGTA and keep them on ice.
		<ol>
			<li>Pour approximately 20 mL of CIB-EGTA into a 30-mL glass beaker and stand a plastic transfer pipette in the beaker, keeping it on ice.</li>
		</ol>
		</li>
		<li>Prepare Tyrode solution. Add 100 mL of 10X&#160;Tyrode to 800 mL of DW and warm it using a microwave to around 32 &#176;C. After adding 0.99 g of glucose and 1.8 mL of 1 M CaCl2, adjust the pH using 1 M NaOH and bring to 1000 mL with DW.</li>
		<li>Prepare the cell resuspension solution. Add 30 mg of BSA and 300 &#181;L of 50X&#160;antibiotics to 15 mL of Tyrode solution.</li>
		<li>Prepare syringes. Fill the 20-mL syringe connected with the flexible extension tube and the marked injection needle with CIB-EGTA. Fill the 30-mL syringe with the warmed enzyme mix. Hold them both at 37 &#176;C until just before use.</li>
		<li>Prepare the perfusion plate. Prewarm the heater mat under a stereoscopic microscope. Place the perfusion plate (lid of a multi-well culture plate) on the prewarmed heater mat. Prewarm the vascular clamp by placing it in the perfusion plate until use. Place the 60-mm culture dish for pipetting and the cell strainer on the prewarmed heater mat as well.</li>
	</ol>
	</li>
</ol>
2. Antegrade perfusion of the mouse heart
NOTE: The plastic transfer pipette used for sucking the heart should be soft and not be sharply tapered towards the tip. Choose a small vascular clamp with serration. The recommended instruments are listed in the Table of Materials.
<ol>
	<li>Excision of the mouse heart and clamping the aorta 
	NOTE: The adult mice (&#62;8 weeks old) should be euthanized by an overdose of sodium pentobarbital (&#62;300 mg/kg, intraperitoneal [i.p.] injection) with heparin (8000 unit/kg).
	<ol>
		<li>Excise the mouse heart quickly by sucking.
		<ol>
			<li>Open the thoracic cavity quickly to expose the heart. Cut the plastic transfer pipette, the tip of which is approximately the same size as, or slightly smaller than, the exposed heart (usually at a site approximately 1 cm from the 0.5-mL mark towards the tip, but it depends on the heart size).</li>
			<li>Suck the heart into the pipette, raise the pipette to create enough space to insert scissors, and excise the heart with curved scissors from the dorsal side, avoiding damaging the atria.</li>
			<li>Immediately transfer the excised heart to the 30-mL glass beaker containing ice-chilled CIB-EGTA to stop the contraction. This procedure usually takes &#60;1 min.</li>
		</ol>
		</li>
		<li>Cleaning around the aorta
		<ol>
			<li>Transfer the heart to a 35-mm culture dish filled with ice-chilled CIB-EGTA and remove the lung and other visible tissues, and then transfer the roughly cleaned heart to the heart stand filled with chilled CIB-EGTA and place it with the apex down.</li>
			<li>Under the stereoscopic microscope, remove the fat and connective tissues to clean around the aorta. If the length of the cut aorta is too long including the brachiocephalic artery, the left common carotid artery or the left subclavian artery, cut off the aorta just under the brachiocephalic artery to shorten it in order to proceed to the next step. This procedure usually takes approximately 4 min.</li>
		</ol>
		</li>
		<li>Clamping the aorta and placing the clamped heart on the perfusion plate
		<ol>
			<li>Under the microscope, place the heart in the heart stand. The operator should face the anterior surface of the heart, pick up the end of the aorta with tweezers, and clamp the aorta near the atria with a small vascular clamp while gently pushing down on the aorta a bit.</li>
			<li>Place the clamped heart on the perfusion plate with the anterior side up, and then cover it with a few drops of CIB-EGTA to keep it from drying out. This procedure usually takes &#60;20 s.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Antegrade perfusion of the heart 
	NOTE: First, perfuse the heart with CIB-EGTA to discharge blood and prevent clotting.
	<ol>
		<li>Insert the injection needle and start perfusion to discharge blood
		<ol>
			<li>Set the 20-mL syringe filled with prewarmed CIB-EGTA connected to the flexible extension tube and a marked injection needle on the infusion pump. Start the pump at a slow rate of 0.5 mL/min to carefully fill the needle and tube with CIB-EGTA and be sure to prevent any air from entering the tube.</li>
			<li>Place the injection needle on the perfusion plate with the shorter side of the diagonal shape in front. Slide the needle towards the apex of the heart until it is touching it, and then carefully insert the needle near the apex of the left ventricle into the ventricular chamber without twisting. Do not detach the needle from the plate while performing insertion.</li>
			<li>Watch the red mark to estimate the depth of the needle insertion. When the needle insertion is completed, blood flowing from the coronary artery should start to be discharged.</li>
			<li>Fix the injection needle on the plate with tape, and increase the pump speed to 1 mL/min. This procedure usually takes approximately 30 s. If the heart is perfused successfully, the flow of the CIB-EGTA in the capillary just under the epicardium can be seen under the microscope.</li>
		</ol>
		</li>
		<li>Perfusion the heart with enzyme mix 
		NOTE: During enzymatic perfusion, the depth of the inserted needle can be monitored by checking the red mark on the needle.
		<ol>
			<li>After perfusing 2-3 mL of CIB-EGTA to completely discharge blood from the coronary artery, change the perfusate to enzyme mix. Avoid allowing air bubbles to enter the tube. Check the flow of the enzyme mix, and ensure that the injection needle has not come out by checking the position of the red mark.</li>
			<li>After 1-2 mL has been perfused, increase the pump speed to 1.5 mL/min. Remove the accumulated perfusate containing blood that has flowed out of the heart with a pipette from time to time. 
			NOTE: Over time, the myocardial wall will become translucent in some places and appear mottled, which is a sign of digestion of the extracellular matrix following successful perfusion. Another sign is the restarting of atrial beating caused by the presence of Ca2+ in the enzyme mix.</li>
			<li>Stop perfusion when the total volume of the enzyme mix perfused is 10 mL.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Isolation of individual heart cells
<ol>
	<li>Dissociating heart cells
	<ol>
		<li>After perfusion with the enzyme mix, transfer 10 mL of the enzyme mix remaining in the syringe to a 60-mm culture dish placed on the heater mat and add 20 mg of BSA (0.2% BSA in enzyme mix). The dropped BSA powder should dissolve immediately by gently swirling with a hand. Remove the injection needle and the vascular clamp from the heart.</li>
		<li>Separate the ventricles and atria and transfer each into the enzyme mix supplemented with 0.2% BSA on the heater mat.</li>
	</ol>
	</li>
	<li>Isolation of ventricular myocytes
	<ol>
		<li>Grab the epicardium with two tweezers, and gently tear and pull the ventricles from side to side in the enzyme mix supplemented with 0.2% BSA into small pieces. Disperse the cells with gentle pipetting (approximately 30 times).</li>
		<li>Filter the undigested debris through a 100-&#181;m mesh cell strainer, and transfer the filtrate to a 15-mL centrifuge tube for centrifugation at 50 &#215; g for 3 min. Resuspend the pelleted cardiomyocytes in prewarmed CIB-Ca2+-BSA solution, incubate it for 5 min at 37 &#176;C, and then centrifuge it at 14 &#215; g for 3 min.</li>
		<li>Resuspend the final precipitated cardiomyocytes in cell resuspension solution (composition is listed in Table 2) and hold it at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Isolation of atrial myocytes
	<ol>
		<li>During the final centrifugation for the ventricular myocyte fraction in step 3.2, start isolating the atrial myocytes. Transfer the atria, stored as in step 3.1, to the prewarmed CIB-Ca2+-BSA solution, tear it into pieces and dissociate the cells by pipetting with pipette tip at 10 &#181;L on the heater mat.</li>
		<li>Centrifuge the cell mixture at 14 &#215; g for 3 min and resuspend the pelleted atrial cells with cell resuspension solution.</li>
	</ol>
	</li>
	<li>Isolation and culture of cardiac fibroblasts
	<ol>
		<li>Transfer the supernatant from the first centrifugation in step 3.2 to another 15-mL centrifuge tube, and centrifuge at 190 &#215; g for 5 min. Wash the precipitated cells twice with centrifugation in Dulbecco&#39;s Modified Eagle Medium (DMEM), and then suspend the cells with DMEM supplemented with 10% fetal bovine albumin (FBS) and antibiotics.</li>
		<li>Plate the final cell fraction in a culture flask (25 cm2) and allow the cells to adhere to the bottom of the flask in a humidified atmosphere of 95% air and 5% CO2. After 90 min incubation, discard the unattached cells, and add fresh culture medium. Cells should near confluency after approximately 4 days, at which point they should be amplified by trypsinization and seeded into new culture dishes.</li>
	</ol>
	</li>
</ol>
4. Harvesting proteins from atria and ventricles
<ol>
	<li>After perfusion, separate the atria and ventricles, and homogenize them in lysis buffer at a ratio of 10 mg of tissue weight to 100 &#181;L of buffer using a small grinder in a 1.5-mL sample tube.</li>
	<li>Keep the homogenate in ice for 40 min with vortex mixing every 10 min to extract proteins, and then centrifuge the tube at 15000 &#215; g for 20 min at 4 &#176;C. Store the supernatant fraction at -80 &#176;C as a protein sample.</li>
</ol>
5. Immunostaining of isolated heart cells
NOTE: Immobilization of non-adherent cardiomyocytes to the bottom of the cell imaging dish using biological glue is necessary.
<ol>
	<li>Adhesion of isolated cardiomyocytes to a glass-bottom culture dish
	<ol>
		<li>Before starting cell isolation, coat the glass-bottom culture dish with biological glue (e.g., Cell-Tak) according to the manufacturer&#39;s instruction, rinse with DW, and air-dry at room temperature.</li>
		<li>After resuspension of the isolated cardiomyocytes in CIB-Ca2+-BSA solution, drop the cell suspension on the bottom of the glue-coated dishes, and incubate for 20 min at room temperature without agitation.</li>
	</ol>
	</li>
	<li>Immunostaining
	<ol>
		<li>Plate the isolated cardiomyocytes on a glass-bottom dish precoated with biological glue and keep it at room temperature for 40 min to allow the cells to adhere to the dish. Culture cardiac fibroblasts in bottom-glass culture dishes.</li>
		<li>Rinse cells with phosphate-buffered saline (PBS), and fix them with 4% paraformaldehyde (PFA) for 5 min with shaking. Wash the fixed cells with PBS for 10 min three times, and incubate them in blocking-permeabilization solution for 60 min at room temperature with shaking.</li>
		<li>Probe cells with primary antibody diluted in blocking-permeabilization solution for 60 min at room temperature or overnight at 4 &#176;C. Wash the cells with PBS for 10 min three times, and incubate then with fluorescence-labeled secondary antibody for 60 min at room temperature.</li>
		<li>After washing them three times with PBS for 10 min, stain the nuclei with DAPI (1:10000 dilution in PBS). Analyze the fluorescent signals using a confocal laser scanning microscope.</li>
	</ol>
	</li>
</ol>
6. Whole-cell patch clamp recordings
<ol>
	<li>Fabricate the patch electrodes from a glass capillaries using a horizontal microelectrode puller. The resistance of the electrode ranged from 2 to 4 M&#937; when filled with a K+-rich pipette solution (Table 2). Transfer an aliquot of isolated cardiomyocytes to a recording chamber mounted on the stage of an inverted microscope superfused with Tyrode at a rate of 1 mL/min&#160;at 36-37 &#176;C.</li>
	<li>Record action potentials using the perforated patch-clamp method with K+-rich pipette solution containing 30 mg/mL amphotericin B by applying current pulses of 5-10 ms in duration at a rate of 1 Hz via the patch electrode.</li>
</ol>
7. Western blot analyses
<ol>
	<li>In this study, perform a Western blot analysis of small-molecular-weight proteins, such as atrial marker atrial natriuretic peptide (ANP).</li>
	<li>Dissolve the protein sample in the final concentration of 1X Sample buffer, 2% 2&#39; mercaptoethanol, and denature the proteins for 60 min at 37 &#176;C. Load 20 &#181;g of protein into each well, and perform electrophoresis in running buffer with 20 mA per gel for 120 min.</li>
	<li>Transfer the protein to a PVDF membrane in transfer buffer at 10 V for 40 min. Wash the transferred membrane twice with TBST for 5 min, then block with 5 % skim milk in TBST for 60 min at room temperature, and probe with the primary antibody dissolved in TBST overnight at 4 &#176;C.</li>
	<li>Wash the membrane 5 times with TBST for 7 min, and incubate it with the secondary antibody diluted in TBST for 120 min at room temperature.</li>
	<li>After washing the membrane 5 times with TBST for 7 min, visualize the signals with a chemi-luminescence assay and analyze them with a lumino-image analyzer.</li>
</ol>

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The authors have nothing to disclose.

Since the heart is highly susceptible to ischemia, the time taken to excise the heart and immerse it in ice-cold CIB-EGTA to stop contraction should be kept short as possible (&#60;1 min). This is the first critical step of this method. The second critical step concerns the direction of the heart. The particular orientation of the excised heart in step 2.1.2 makes it easier to see and remove the fat and connective tissues around the aorta. After cleaning around the aorta, place the clamped heart with anterior surface side up on the perfusion plate. The final critical step involves the insertion of the injection needle. When advancing the needle towards the heart, the injection needle should not be detached from the perfusion plate in order to maintain a constant distance from the plate. The position of the insertion is near the apex of the left ventricle. Insert the needle carefully without twisting, since such twisting may enlarge the hole. The depth of the insertion of the needle can be estimated by watching the red mark. If the needle is inserted too deep, the tip may pierce through the ventricular septum and enter the right ventricle or though the mitral valve and enter the left atrium. After confirming the disappearance of the blood from the coronary artery, the needle should be fixed with tape to the perfusion plate.
A longer aorta length makes it difficult to clamp the aorta at the right position. If the clamp is too distant from the atria, the heart may rotate after perfusate infusion. To prevent this, cut off the aorta just under the brachiocephalic artery to shorten the aorta before clamping.
If the blood does not begin to discharge after perfusion at an initial speed of 0.5 mL/min, increase the speed to 1 mL/min. If that does not help, the injection needle may be positioned incorrectly, such as in the right ventricle, ventricular septum or left myocardial wall. In such a case, remove the needle immediately and try to re-insert it near the apex of the left ventricle. When inserting the needle several times, digested cells may flow out from the opened holes. Note that this does not usually seriously affect the cell isolation.
The operators can monitor the entire process of antegrade perfusion of the heart using a stereoscopic microscope to observe the changes in color and transparency and restarting of the beating of the atria along with the digestion. A total of 10 mL of enzyme mix should be the maximum required, even for an old heart. In younger hearts (5-7 weeks old), we reduce the volume to 9 mL, which is similar to the approach via retrograde perfusion with the same enzyme mix.
The supernatant at the final centrifugation contains debris, blood cells, and non-myocytes whereas, the pellet contains mainly cardiomyocytes and contaminating non-myocytes, such as fibroblasts and endothelial cells. To purify the cardiomyocytes, more steps are needed. In general, the pellet should be resuspended in the appropriate cell culture medium and preplated for 2 h at 37&#176;C on a tissue cell culture dish, and then gently remove the cardiomyocytes by pipetting and preplating for culture.
The enzyme mix contains a low concentration of Ca2+ (0.3 mM). We therefore incubate digested cells in CIB-Ca2+-BSA (1.2 mM Ca2+) before the final resuspension with the cell resuspension solution (1.8 mM Ca2+), and the gradual increase in Ca2+ avoids causing cell damage7. As long as the isolated cardiomyocytes are intact (quiescent cells with no contraction) this Ca2+-adapting procedure does not affect cell viability in mice. As the damaged cells are dying during this incubation, we consequently obtain a healthy cell group. Similarly, isolated intact atrial myocytes (quiescent cells without irregular contraction) can be stored in the same cell resuspension solution. However, the atrial myocytes tend to be more delicate to be stored compare to the ventricular myocytes.
In the laboratory, this isolation method is almost always successful unless the needle insertion into the left ventricle fails. We have also succeeded in isolating cells from the hypertrophied heart prepared by surgical transverse aortic constriction. However, in aged mice, which often have small myocardial infarctions, perfusion ceases in some places, resulting in incomplete digestion and thus a low yield (Figure 1C), similar to the Langendorff-based retrograde method. In such cases, the distorted shape of the heart can be observed even at the start of perfusion.
This antegrade perfusion method is useful for isolating heart cells from mice of various ages but not larger animals, such as rabbits and guinea pigs. It may be possible to apply this method to neonatal or juvenile rats before weaning.
One of the advantages of this antegrade perfusion method is that it decreases the technical obstacles associated with using the Langendorff-based retrograde perfusion method for small mouse hearts. The time required for perfusion is approximately 7 min with 10 mL of the enzymes, this short digestion period increases the viability of the cells. In addition, it enables perfusion to be performed through the coronary circulation of the heart, even after the aortic valves have been digested. Isolation of atrial myocytes usually requires Langendorff-based retrograde perfusion and further incubation with enzymes17. This antegrade perfusion approach, however, can deeply perfuse the tissue with the enzyme to isolate atrial myocytes.
In experiments using multiple mice, the Langendorff apparatus should be cleaned before perfusing the next heart. However, in the present antegrade method, as long as desired number of instrument sets (e.g., syringes needles and perfusion plates) are prepared in advance, perfusion can be performed continuously.
We herein report the basic methodology of the antegrade perfusion of the mouse heart using the same solutions as the Langendorff-based retrograde perfusion method with no additional chemicals. The composition of the perfusate can be changed to suit the purpose of the experiment, such as using a detergent containing EGTA instead of the enzymes to make a decellularized heart18.

Generally, the first step of the single cell isolation of dissected tissue involves mincing the tissue into small pieces, followed by the digestion of the connective tissue and extracellular matrix with enzymes. However, cardiomyocytes cannot be isolated with such a chopping method, as enrichment with extracellular matrix components, including collagen and elastin fibers, makes the myocardium too tough to mince, and the cardiomyocytes are highly sensitive to hypoxia and other changes in the microenvironment. Thus, using the Langendorff-based retrograde perfusion system1, a method of digesting the extracellular matrix with enzymes has been developed to isolate individual cardiomyocytes from the heart2,3,4.
In mouse models, Langendorff-based retrograde perfusion of the heart with enzymes is also used for the isolation of individual cardiomyocytes5,6,7,8.&#160;However, the cannulation of the small and thin mouse aorta and its mounting on the Langendorff apparatus to perform retrograde perfusion requires a high degree of skill, since the diameter of the aorta in the adult heart is approximately 1.2 mm. Furthermore, it takes time to perform multiple experiments as the Langendorff apparatus should be cleaned before perfusing the next heart.
As an alternative to retrograde perfusion, a novel method for isolating cardiomyocytes from an adult mouse heart without a Langendorff apparatus was developed. This epoch-making method was based on the antegrade perfusion of the coronary arteries9. We recently improved each step of this antegrade protocol, such as the clamping of the aorta, needle insertion, and temperature control, and monitored all perfusion procedures with a microscope10. We herein report in detail the refinement of this antegrade perfusion method to shorten the time for isolation and provide a supplemental video. In this method, the perfusion of the heart takes approximately 7 min with 10 mL of the enzymes, and this short digestion period increases the viability of the cells. This is a simple method for isolating single heart cells at a high quality without requiring the addition of chemicals, such as 2,3-butanedione monoxime (BDM)6,11 or taurine5,8. We believe that this method will lower the skill threshold of the technique and enhance the utility of mouse cardiomyocytes in basic research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amphotericin B</td>
			<td>Wako Pure Chemical Industries, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-mouse IgG antibody</td>
			<td>Molecular Probes, USA</td>
			<td>A11001</td>
			<td>Fluorescent-labeled secondary antibody. (1:400 dilution for immunostaining)</td>
		</tr>
		<tr>
			<td>Anti-&#945;-actinin (ACTN)</td>
			<td>Sigma-Aldrich, USA</td>
			<td>A7811</td>
			<td>Mouse monoclonal antibody (clone EA-53). (1:400 dilution for immunostaining)</td>
		</tr>
		<tr>
			<td>Anti-atrial natriuretic peptide (ANP)</td>
			<td>Merck-Millipore, USA</td>
			<td>AB5490-I</td>
			<td>Rabbit polyclonal antibody (1:2000 dilution for Western blots)</td>
		</tr>
		<tr>
			<td>Anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)</td>
			<td>Cell Signaling Technology, USA</td>
			<td>2118</td>
			<td>Mouse monoclonal antibody (1:10000 dilution for Western blots)</td>
		</tr>
		<tr>
			<td>Anti-smooth muscle actin (SMA)</td>
			<td>Dako, Denmark</td>
			<td>M0851</td>
			<td>Mouse monoclonal antibody (clone 1A4) (1:400 for immunostaining)</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG antibody</td>
			<td>Amersham, GE Healthcare, USA</td>
			<td>NA934</td>
			<td>Secondary antibody (1:10000 dilution for Western blots)</td>
		</tr>
		<tr>
			<td>ATP disodium salt</td>
			<td>Sigma-Aldrich, USA</td>
			<td>A26209</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich, USA</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Tak</td>
			<td>Corning</td>
			<td>354240</td>
			<td>Biological material for adhesion of the cell or tissues</td>
		</tr>
		<tr>
			<td>Chemi-Lumi One Super&#160;</td>
			<td>Nacalai Tesque, Japan</td>
			<td>02230-14</td>
			<td>Chemiluminescent reagent used for western blotting.</td>
		</tr>
		<tr>
			<td>Collagenase Type 2</td>
			<td>Worthington Biochemicals, USA</td>
			<td>LS004176</td>
			<td>Choose the activity guaranteed to be greater than 300 unit/mg.</td>
		</tr>
		<tr>
			<td>Complete Mini</td>
			<td>Roche, Germany</td>
			<td>11836153001</td>
			<td>A mixture of several protease inhibitors.</td>
		</tr>
		<tr>
			<td>4&#39;6&#39;diamidino-2-phenylindole (DAPI)</td>
			<td>Nacalai Tesque, Japan</td>
			<td>11034-56</td>
			<td>Used for cell-impermeant nuclear stainig&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Nacalai Tesque, Japan</td>
			<td>08458-45</td>
			<td>including 4.5 g/L gluose</td>
		</tr>
		<tr>
			<td>Extension tube</td>
			<td>Top, Japan</td>
			<td>X1-50</td>
			<td>Connect with syringe and injection needle for antegrade perfusion.&#160;</td>
		</tr>
		<tr>
			<td>EPC-8 patch-clamp amplifier</td>
			<td>HEKA, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich, USA</td>
			<td>F7524-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Narishige Scientific Instrument Lab., Japan</td>
			<td></td>
			<td>outside diameter 1.5 mm, inside diameter 0.9 mm</td>
		</tr>
		<tr>
			<td>GTP lithium salt</td>
			<td>Sigma-Aldrich, USA</td>
			<td>G5884</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal microelectrode puller&#160;</td>
			<td>Germany)</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Heater mat</td>
			<td>Natsume Seisakusho, Japan</td>
			<td>KN-475-3-40</td>
			<td>Equipment to warm the perfusion plate.</td>
		</tr>
		<tr>
			<td>Infusion pump</td>
			<td>TERUMO, Japan</td>
			<td>TE-311</td>
			<td>Infusion syringe pump for antegrade perfusion.</td>
		</tr>
		<tr>
			<td>Injeciton needle (27 gauge)</td>
			<td>TERUMO, Japan</td>
			<td>NN-2719S</td>
			<td>Needle for insertion into the left ventricle.</td>
		</tr>
		<tr>
			<td>Insulin (from bovine pancrease)</td>
			<td>Sigma-Aldrich, USA</td>
			<td>I5500</td>
			<td>Dissolve in 0.1 M HCl.</td>
		</tr>
		<tr>
			<td>Mini cordless grinder</td>
			<td>Funakoshi, Japan</td>
			<td>cG-4A</td>
			<td>Small grinder for homogenizing tissue in 1.5 mL sample tube.</td>
		</tr>
		<tr>
			<td>4%-Paraformaldehyde Phosphate Buffer solution (4% PFA)</td>
			<td>Nacalai Tesque, Japan</td>
			<td>09154-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G potassium</td>
			<td>Nacalai Tesque, Japan</td>
			<td>26239-84</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Nacalai Tesque, Japan</td>
			<td>26807-21</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Phosphate Buffered Saline (pH7.4) (10X PBS)</td>
			<td>Nacalai Tesque, Japan</td>
			<td>27575-31</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic multi-well culture plate</td>
			<td>Falcon, USA</td>
			<td>353226</td>
			<td>Use the lid of the multi-well culture plate as the perfusion plate.</td>
		</tr>
		<tr>
			<td>Plastic syringe (20 mL)</td>
			<td>TERUMO, Japan</td>
			<td>SS-20ES</td>
			<td>Use for infusion of CIB-EGTA.</td>
		</tr>
		<tr>
			<td>Plastic syringe (30 mL)</td>
			<td>TERUMO, Japan</td>
			<td>SS-30ES</td>
			<td>Use for infusion of Enzyme-mix</td>
		</tr>
		<tr>
			<td>Plastic transfer pipette</td>
			<td>Sarstedt, Germany</td>
			<td>86.1171</td>
			<td>Cut the tip just before sucking mouse heart into the pipette.</td>
		</tr>
		<tr>
			<td>Polyvinylidene difluoride (PVDF) membrane</td>
			<td>Merck-Millipore, USA</td>
			<td>IPVH00010</td>
			<td>Immobilin-P membrane (Transfer membrane for protein blotting)</td>
		</tr>
		<tr>
			<td>Protease</td>
			<td>Sigma-Aldrich, USA</td>
			<td>P5147</td>
			<td>A mixture of three or more proteases including extracellular serine protease.</td>
		</tr>
		<tr>
			<td>4X Sample buffer solution</td>
			<td>Fuji Film, Japan</td>
			<td>198-13282</td>
			<td>Contains 0.25 M Tris-HCl (pH 6.8), 8 w/v% SDS,40 w/v% Glyceroland 0.02 w/v% BPB</td>
		</tr>
		<tr>
			<td>SDS polyacrylamide gel&#160; (15%)</td>
			<td>Fuji Film, Japan</td>
			<td>193-14991</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate</td>
			<td>Nacalai Tesque, Japan</td>
			<td>32237-14</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris-Glycine buffer solution (10X TG)</td>
			<td>Nacalai Tesque, Japan</td>
			<td>09422-81</td>
			<td>Contains 0.25 M-Tris and 1.92 M-Glycine, (pH 8.3)&#160;</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich, USA</td>
			<td>T8003</td>
			<td>Trypsin from bovine Type 1.&#160;</td>
		</tr>
		<tr>
			<td>Vascular clamp</td>
			<td>Karl Hammacher GmbH, Germany</td>
			<td>HSE 004-35</td>
			<td>Small straight vascular clamp used for clamping aorta.</td>
		</tr>
		<tr>
			<td>All other reagents</td>
			<td>Nacalai Tesque, Japan</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an antegrade perfusion method for cardiomyocyte isolation from mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

The principle of this method is simple: the perfusate flows from the left chamber, the aortic valve is opened, and the perfusate runs into the coronary artery in the same direction as the blood run, since the aorta is closed by clamping, which enables the deep perfusion of the myocardium in order to digest the extracellular matrix.
Ventricular myocytes freshly isolated with the present method are shown in Figure 1A. Figure 1B shows enlarged images of the ventricular and atrial myocytes. This isolation procedure resulted in a high yield (70%-80%) of rod-shaped quiescent ventricular myocytes from adult mice (8-10 weeks), which were available within roughly 5 h after isolation (Figure 1C), a similar interval to that when using the traditional Langendorff-based procedure7. However, the ratio of freshly isolated viable cells was lower in aged mice of &#62;2 years old (Figure 1C). The total number of ventricular myocytes obtained per adult heart using this protocol was approximately 3 x 106 cells, which was similar to the value previously reported7,12. The action potentials recorded in the ventricular and atrial myocytes (Figure 1D) were similar to those in cells obtained by the Langendorff-based method10. An immunostaining analysis confirmed that the sarcomeric structure of the ventricular myocytes was well-organized with a clearly visible cell membrane (Figure 2A). The individual cardiomyocytes isolated with this method can be directly used in experiments, such as an electrophysiological analysis10 or immunostaining experiment.
Cardiac fibroblasts exist in interstitial spaces. Sufficient digestion of the extracellular matrix results in the isolation of those cells. The isolated cardiac fibroblasts proliferate under culture conditions and can be passaged several times or stored in liquid nitrogen in an appropriate cell reservoir solution. Figure 2B shows that most of the cultured cardiac fibroblasts had transformed into myofibroblasts during subculture, as confirmed by the increased expression of &#945;-smooth muscle actin13,14. Also, the cardiac progenitors can be isolated with the present method and cultured in appropriate culture medium, which start beating automatically10,15.
Homogenization of the robust myocardium is not easy, especially for cardiac tissue from aged mice, which possesses a large amount of extracellular fibers. After antegrade perfusion, protein from the atria and ventricles can be easily homogenized in the lysis buffer with light force to extract proteins. A Western blot analysis demonstrated the specific expression of ANP in atria but not in ventricles from adult (20 weeks old) and aged (108 weeks old) mice (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61866/61866fig1.jpg" /> 
Figure&#160;1. Isolated cardiomyocytes from mice.&#160;A. Ventricular myocytes freshly isolated with the antegrade perfusion, with images acquired with low magnification. After the final washing, the cardiomyocytes were resuspended with 2 mL of cell resuspension solution, 100 &#181;L of which was dropped onto the glass-bottom culture dish and cell settlement awaited. Bar, 100 &#181;m. B. Enlarged images of isolated ventricular myocytes (upper) and atrial myocytes (lower). Bar, 100 &#181;m. C. Isolated cells were suspended in the cell resuspension solution and stored at 37&#176;C for the desired period, and the number of live ventricular myocytes was counted in 10-15 fields under a microscope. Rounded cells were considered to have been irreversibly injured or dead16. Green, blue and red symbols were obtained from 3 mice at 8-10 weeks old, and black symbols were from 106 weeks old mouse. Yellow symbol indicates the mean of each group. D. Representative action potentials recorded from ventricular (black) and atrial (red) myocytes of 8-10 mice. The data were obtained from the cells approximately 3 h after isolation. <a href="https://www.jove.com/files/ftp_upload/61866/61866fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61866/61866fig2.jpg" /> 
Figure&#160;2. Immunostaining for &#945;-actinin in isolated mouse ventricular myocytes and &#945;-smooth muscle actin in cultured mouse cardiac fibroblasts.&#160;A. Confocal laser scanning microscopy of immunostaining for &#945;-actinin (green), DAPI staining for nuclei (blue) and a DIC image of ventricular myocytes isolated from mouse heart with antegrade perfusion. Bar, 50 &#181;m. B. Immunostaining for &#945;-smooth muscle actin (green), DAPI staining for nuclei (blue) and a DIC image of cardiac fibroblasts isolated from mouse heart with antegrade perfusion. Cardiac fibroblasts were cultured for four days. Bar, 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61866/61866fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61866/61866fig3.jpg" /> 
Figure&#160;3. Western blot analyses of ANP in atria and ventricles.&#160;Western blot analyses for the atrial marker atrial natriuretic peptide (ANP) in atria (A) and ventricles (V) prepared from adult (20 weeks) and aged (108 weeks) hearts. ANP is present in the atria but absent in the ventricles. Use glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control house-keeping protein. <a href="https://www.jove.com/files/ftp_upload/61866/61866fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Stock solutions for isolating heart cells</td>
		</tr>
		<tr>
			<td colspan="2">10X CIB (500 mL)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>37.99 g</td>
		</tr>
		<tr>
			<td>KCI</td>
			<td>2.01 g</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>2.5 mL</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>0.23 g</td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>29.79 g</td>
		</tr>
		<tr>
			<td>DW</td>
			<td>Fill up to 500 mL</td>
		</tr>
		<tr>
			<td colspan="2">100 mM CaCl2 stock soution</td>
		</tr>
		<tr>
			<td>CaCl2&#160;</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td colspan="2">400 mM EGTA stock solution</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>400 mM</td>
		</tr>
		<tr>
			<td colspan="2">Insulin solution</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>1 unit/mL in 0.1 M HCl</td>
		</tr>
		<tr>
			<td colspan="2">50X Antibiotics stock solution (20 mL)</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>1.5 g</td>
		</tr>
		<tr>
			<td>DW</td>
			<td>20 mL and sterilize with filtering</td>
		</tr>
		<tr>
			<td colspan="2">10X Tyrode solution (1000 mL)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>81.82 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>4.03 g</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>0.47 g</td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>11.92 g</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>0.8 g</td>
		</tr>
		<tr>
			<td>DW</td>
			<td>Fill up to 1000 mL</td>
		</tr>
		<tr>
			<td colspan="2">Stock solutin for immunostaining</td>
		</tr>
		<tr>
			<td colspan="2">DAPI stock</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>2 mg/mL in methanol</td>
		</tr>
		<tr>
			<td colspan="2">Stock solutions for Western blots</td>
		</tr>
		<tr>
			<td colspan="2">&#160;HEPES buffer (100 mL)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>0.88 g</td>
		</tr>
		<tr>
			<td>400 mM EGTA</td>
			<td>0.25 mL</td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>0.24 g</td>
		</tr>
		<tr>
			<td>1M NaOH</td>
			<td>adjust pH to 7.4</td>
		</tr>
		<tr>
			<td>DW</td>
			<td>Fill up to 500 mL</td>
		</tr>
		<tr>
			<td colspan="2">Protease inhibitors cocktail&#160;</td>
		</tr>
		<tr>
			<td>Complete mini</td>
			<td>1 tablet</td>
		</tr>
		<tr>
			<td>DW</td>
			<td>0.4 mL</td>
		</tr>
	</tbody>
</table>
Table 1. Description of the stock solutions.&#160;Keep stock solutions at 4 &#176;C. Aliquot protease inhibitors cocktail for storage at -20 &#176;C.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Solutions for isoltaing heart cells</td>
		</tr>
		<tr>
			<td colspan="2">CIB (200 mL)</td>
		</tr>
		<tr>
			<td>10X CIB</td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>Insulin solution</td>
			<td>0.01 mL</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>0.79 g</td>
		</tr>
		<tr>
			<td>1M NaOH</td>
			<td>pH adjust to 7.4</td>
		</tr>
		<tr>
			<td>DW</td>
			<td>Fill up to 200 mL</td>
		</tr>
		<tr>
			<td colspan="2">Enzyme-mix solution (30 mL)</td>
		</tr>
		<tr>
			<td>Collagenase type2</td>
			<td>30 mg</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>1.8 mg</td>
		</tr>
		<tr>
			<td>Protease</td>
			<td>1.8 mg</td>
		</tr>
		<tr>
			<td>100 mM CaCl2 stock solution</td>
			<td>0.09 mL</td>
		</tr>
		<tr>
			<td>CIB</td>
			<td>30 mL</td>
		</tr>
		<tr>
			<td colspan="2">CIB-Ca2+-BSA (15 mL)</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>30 mg</td>
		</tr>
		<tr>
			<td>100 mM CaCl2 stock solution</td>
			<td>0.18 mL</td>
		</tr>
		<tr>
			<td>CIB</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td colspan="2">CIB-EGTA (150 mL)</td>
		</tr>
		<tr>
			<td>400 mM EGTA stock solution</td>
			<td>0.150 mL</td>
		</tr>
		<tr>
			<td>CIB</td>
			<td>150 mL</td>
		</tr>
		<tr>
			<td colspan="2">Tyrode solution (1000 mL)</td>
		</tr>
		<tr>
			<td>10X Tyrode stock solution</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>0.99 g</td>
		</tr>
		<tr>
			<td>1M CaCl2</td>
			<td>1.8 mL</td>
		</tr>
		<tr>
			<td>1M NaOH</td>
			<td>pH adjust to 7.4</td>
		</tr>
		<tr>
			<td>DW</td>
			<td>Fill up to 1000&#160; mL</td>
		</tr>
		<tr>
			<td colspan="2">Cell resuspension solution (15 mL)</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>30 mg</td>
		</tr>
		<tr>
			<td>50X Antibiotics stock solution</td>
			<td>0.3 mL</td>
		</tr>
		<tr>
			<td>Tyrode solution</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td colspan="2">Solutions for immunostaining</td>
		</tr>
		<tr>
			<td colspan="2">Cell adherent solution (0.3 mL)</td>
		</tr>
		<tr>
			<td>Cell-Tak</td>
			<td>0.01 mL</td>
		</tr>
		<tr>
			<td>0.1 M NaHCO3 (pH8.0)</td>
			<td>0.285 mL</td>
		</tr>
		<tr>
			<td>0.1 M NaOH</td>
			<td>0.005 mL</td>
		</tr>
		<tr>
			<td colspan="2">Blocking-permeabilizatin solution (10 mL)</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>DW</td>
			<td>7 mL</td>
		</tr>
		<tr>
			<td colspan="2">K+ rich pipette solution</td>
		</tr>
		<tr>
			<td>Potassium aspartate</td>
			<td>70 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>ATP disodium salt</td>
			<td>3 mM</td>
		</tr>
		<tr>
			<td>GTP lithium salt</td>
			<td>0.1 mM</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>pH adjust to 7.2</td>
		</tr>
		<tr>
			<td colspan="2">Solutions for Western blots</td>
		</tr>
		<tr>
			<td colspan="2">Lysis buffer (1 mL)</td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>0.86 mL</td>
		</tr>
		<tr>
			<td>Nonidet-P40</td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td>Protease inhibitors cocktail</td>
			<td>0.04 mL</td>
		</tr>
		<tr>
			<td colspan="2">Runnning buffer (1000 mL)</td>
		</tr>
		<tr>
			<td>10X TG (0.25 M Tris and 1.92 M Glycine)</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>DW</td>
			<td>900 mL</td>
		</tr>
		<tr>
			<td colspan="2">Transfer buffer (1000 mL)</td>
		</tr>
		<tr>
			<td>10X TG</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>200 mL</td>
		</tr>
		<tr>
			<td>DW</td>
			<td>700 mL</td>
		</tr>
		<tr>
			<td colspan="2">Blotting buffer (TBST)&#160; (1000 mL)</td>
		</tr>
		<tr>
			<td>5M NaCl</td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>2M Tris-HCl (pH 7.5)</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>10% Tween 20</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>DW</td>
			<td>965 mL</td>
		</tr>
	</tbody>
</table>
Table 2. Description of the working solutions for isolating heart cells, immunostaining and Western blotting.&#160;Prepare all working solutions just before the experiments.
Supplementary Figure&#160;1. Outline of the cell isolation.&#160;Flow diagram of the isolation of ventricular and atrial myocytes and cardiac fibroblasts from a single heart.&#160;<a href="https://www.jove.com/files/ftp_upload/61866/SupplementaryFig1R.pdf" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice at JoVE.com

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating ...

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10.2K Views.  Shiga University of Medical Science. We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

Video: An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

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 ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

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 ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.