This work was supported by the grant from the National Institutes of Health (HL115578 to A. Makino).

The research on mice was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Arizona and was performed according to the National Institute of Health (NIH) guidelines.
NOTE: Sections 1, 2 and 3 should be carried out the day before the experiment as setup.
1. Preparing Solutions
<ol>
	<li>CD31 buffer (50 ml)
	<ol>
		<li>Add 50 ml 1&#215; Phosphate Buffer Saline (PBS) to a 50 ml tube. Weigh 0.05 g Bovine Serum Albumin (BSA) and 0.037 g Ethylenediaminetetraacetic acid (EDTA) and add to the&#160;50 ml tube and put the tube to rotate for about 1 h until powder dissolves. Adjust pH to 7.4, filter through a 0.2 &#181;m filter into a new 50 ml tube, and store at 4 &#730;C until use.</li>
	</ol>
	</li>
	<li>Hank&#39;s Balanced Salt Solution (HBSS) with HEPES (500 ml)
	<ol>
		<li>Add 1.1915 g of&#160;HEPES to make 10 mM HEPES&#160;in&#160;HBSS solution and mix well. Measure the pH and adjust to 7.4. Filter and store at 4 &#730;C.</li>
	</ol>
	</li>
	<li>1x Kreb&#39;s solution (1 L)
	<ol>
		<li>Add 100 ml of 10&#215; Kreb&#39;s solution [Table 1] to double distilled water in a 1 L flask. Weigh 2.1 g Sodium Bicarbonate and 2 g D-Glucose and add to the flask. Mix and adjust the total volume to 1 L.</li>
		<li>Bubble solution with 95% O2/5% CO2 gas for 30 min and filter solution inside the hood. Store at 4 &#730;C and keep on ice during experiment.</li>
	</ol>
	</li>
</ol>
2. Preparing the Magnetic Beads
<ol>
	<li>Add 1 ml CD31 buffer into each 1.5 ml tube (one tube per sample). Mix the sheep anti-rat IgG beads and add the appropriate amount to each sample [Table 1]. Shake the tubes vigorously 30 times and place them in the magnetic plate for 1 min&#160;of shaking. Open the tubes while on the plate and dump the solutions into a bucket.</li>
	<li>Add 1 ml CD31 buffer into each tube. Add 2 &#181;l Rat anti-mouse CD31&#160;antibody to each tube and rotate tubes O/N at 4 &#186;C.</li>
</ol>
3. Autoclaving&#160;
<ol>
	<li>Autoclave all surgery tools and pipette tips. Cut the ends of pipette tips so that there are tips with 4 different diameters, from widest to narrowest. Approximate diameters for the tips are 3 mm, 2.5 mm, 2 mm, and 1.5 mm respectively. Cut the tips and autoclave the day before with 30 min of sterilization at a temperature of 121 &#186;C. 
	NOTE: Sections 4 - 11 should be&#160;carried out on the day of the experiment.</li>
</ol>
4. Preparing Solutions
<ol>
	<li>Prepare washing buffer containing 2% Iron-Fortified Calf Serum (IFCS) in M199. For imaging, prepare&#160;30&#160;ml/mouse.</li>
	<li>Prepare enzyme solution (10 ml/sample).&#160; Weigh 0.6 unit/ml of Dispase and 1 mg/ml of Collagenase Type II into a 50 ml tube. Add M199 and rotate the tube at 4 &#186;C for 15 min. &#160;Filter through 0.2 &#181;m filter into a new 50 ml tube inside the hood and keep at 4 &#730;C until use.</li>
	<li>Prepare HBSS/HEPES with Heparin.&#160; Add 10 ml of HBSS with 10 mM HEPES in 15 ml tube. Add 100 &#956;l of Heparin to the tube and keep at 4 &#730;C until use.</li>
</ol>
5. Washing the Beads
<ol>
	<li>Take out the tubes with beads from the rotator at 4 &#186;C and bring to the hood. Place the tubes in the magnetic plate and shake for 1 min. Dump the solution and add 1 ml CD31 buffer into each tube.</li>
	<li>Take the tubes out of the magnetic plate and shake vigorously 30 times. Put the tubes back in the magnetic plate and repeat for a total of 3 washes. After completion, put the tubes back to 4&#730;C and keep rotating until use.</li>
</ol>
6. Dissection
<ol>
	<li>Inject the mice intraperitoneally with 0.1 ml of Heparin to prevent blood coagulation. Wait 10 mins, then anesthetize the mice using intraperitoneal injection of 0.01 ml of Pentobarbital. Check if the mouse is feeling pain by pinching the foot.</li>
	<li>Place the mouse on a polystyrene board with head up and hold the arms with pins. Use forceps and tough-cut-scissors to cut the skin at the upper thoracic area up to the throat.</li>
	<li>Make a cut in the center of the rib cage just above the diaphragm. Cut the bone sideways and upwards toward the throat to make a triangle exposing the heart and lungs. Cut the aorta just above the diaphragm.</li>
	<li>Hold the thymus with forceps without pulling and use scissors to cut any connective tissue carefully to remove lungs and heart together. Place tissues in a beaker with 1&#215; Kreb&#39;s, shake with forceps and transfer to 6 cm dish with 1&#215; Kreb&#39;s.</li>
	<li>Remove the lung lobes using scissors and forceps. Insert a 20 G sterile catheter into the aorta. Flush the blood in the coronary circulatory system with HBSS/Heparin. Make a small cut in the ventricle, shake the heart and transfer to a sample tube of M199. Keep tubes on ice.</li>
</ol>
7. Tissue Digestion
<ol>
	<li>Transfer the heart to a 6 cm dish filled with 10 ml of enzyme solution and mince it into small pieces using scissors. Place the dishes in the 37 &#186;C incubator for 15 min. Agitate digested materials by pipetting up and down 30 times through a tip with a cut end to allow the tissue to pass through easier.</li>
	<li>Put it back in the 37 &#186;C incubator for another 15 min. Repeat agitation three times, each time using pipette tips from biggest openings to narrowest&#160;and 15 min incubation between each.</li>
	<li>After last 15 min incubation, take samples out and into the hood. Pipet solution up and down 30 times using pipet tip with the narrowest opening and pass through a 40 &#181;m cell strainer into a new 50 ml tube.</li>
	<li>Wash the dish with 10 ml washing buffer using a&#160;10 ml pipette and transfer onto cell strainer. Put the 50 ml tubes in the centrifuge and spin at 400 g for 10 min at RT.</li>
</ol>
8. Washing and Conjugation with Beads
<ol>
	<li>Remove supernatant without disrupting the pellet. Wash cells with 5 ml washing buffer twice.</li>
	<li>Get one of the previously prepared 1.5 ml tubes with beads and work on one tube at a time. Put one 1.5 ml tube in the magnetic plate, shake 3 times and open it.</li>
	<li>After last centrifuge, remove as much supernatant as possible without disrupting the pellet. Disassociate the cell clumps by flicking vigorously.</li>
	<li>Add 1 ml washing buffer to the 50 ml tube and mix the cells with a 1 ml pipette. Dump the solution from the 1.5 ml tube and add 1 ml of cell suspension from the 50 ml tube to the beads. Flick the tubes 30 times then rotate at 4 &#186;C for 30 min.</li>
</ol>
9. Preparing for Cell Culture/Imaging
<ol>
	<li>Prepare chambers for plating cells. Add gelatin solution (5% w/v, 300 &#181;l) to the chamber well to coat the surface and incubate for 30 min at 37 &#186;C. After incubation, remove gelatin and dry the surface.</li>
	<li>Take out the tubes with beads/cells after 30 min of rotation and put in the magnetic plate inside the hood. Shake for 2 min, dump and add 1 ml washing buffer.</li>
	<li>Take tubes out of the plate, shake vigorously 30 times then flick 30 times. Put them back in magnetic plate and shake for 1 min. Repeat 4 times for a total of 5 washes, but with 1 min shaking on the plate.</li>
	<li>After the last wash, take out one tube and work on each tube one at a time. Shake 30 times then flick 30 times. Put it on the magnetic plate and shake for 1 min. Dump and add 1 ml 20% IFCS EC media (pre-warmed at 37 &#186;C).</li>
	<li>Shake vigorously 30 times then flick 30 times. Adjust pipet to 500 &#181;l and pipet up and down 10 times. Transfer 500 &#181;l to each chamber.</li>
	<li>Put at 37 &#186;C incubator and leave O/N. Next day, wash cells with 10% FBS EC media.</li>
</ol>
10. Preparing for Western Blot (WB) Samples
<ol>
	<li>Take out the tubes with beads/cells after 1 hr of rotation. Put them in the magnetic plate and shake for 2 min. Dump supernatant and add 1 ml cold HBSS. Shake vigorously 30 times then flick 30 times. Repeat twice for 3 washes, but with 1 min shaking on the plate.</li>
	<li>After last wash, put tubes on magnetic plate and shake for 1 min. Remove buffer using pipette and lyse the cells for WB using a method previously described11,12. Collected lysate usually contains around 30 &#181;g of protein.</li>
</ol>
11. Imaging
<ol>
	<li>Wash the cells 3 times with media, with final volume of 250 &#181;l for a chamber. Add 1.5 &#181;l of Dil-acLDL to the cells. From this point, keep the cells in the dark.</li>
	<li>Incubate for 3.5 hr at 37 &#730;C. Add 1.5 &#181;l of Hoechst, mix well and incubate for 30 min at 37 &#730;C.Wash cells with PBS and fix with 4% Paraformaldehyde (PFA) for 20 min at RT.</li>
	<li>Wash twice with PBS and block with 5% BSA-PBS for 30 min at RT. Wash twice with 1% BSA-PBS. Dilute BS-lectin-FITC using 1% BSA-PBS to a concentration of 2 &#181;g/ml and add 300 &#181;l to each chamber.</li>
	<li>Put the chamber on the orbital shaker at RT for 30 min. Wash with PBS 3 times.</li>
	<li>Visualize cells under the fluorescent microscope with exposure time of 200 msec for FITC (Lectin staining, an EC marker, excitation/emission 488 nm/519 nm), 30 msec for DAPI (Hoechst, a nuclear staining, excitation/emission at 358 nm/461 nm), and 300 msec for TRITC (acLDL staining, an EC marker, excitation/emission at 557 nm/576 nm). Acquire images using a 20X objective lens.</li>
</ol>

<ol><li>Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+cell+functions%5BTitle%5D)+AND+%22J+Cell+Physiol%22%5BJournal%5D)">Endothelial cell functions.</a> J Cell Physiol. 196, 430-443 (2003).</li>
<li>Sumpio, B. E., Riley, J. T., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cells+in+focus%3A+endothelial+cell%5BTitle%5D)+AND+%22Int+J+Biochem+Cell+Biol%22%5BJournal%5D)">Cells in focus: endothelial cell.</a> Int J Biochem Cell Biol. 34, 1508-1512 (2002).</li>
<li>Cines, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+cells+in+physiology+and+in+the+pathophysiology+of+vascular+disorders%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Endothelial cells in physiology and in the pathophysiology of vascular disorders.</a> Blood. 91, 3527-3561 (1998).</li>
<li>Deanfield, J. E., Halcox, J. P., Rabelink, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+function+and+dysfunction%3A+testing+and+clinical+relevance%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Endothelial function and dysfunction: testing and clinical relevance.</a> Circulation. 115, 1285-1295 (2007).</li>
<li>Jin, Y., Liu, Y., Antonyak, M., Peng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+vascular+endothelial+cells+from+murine+heart+and+lung%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Isolation and characterization of vascular endothelial cells from murine heart and lung.</a> Methods Mol Biol. 843, 147-154 (2012).</li>
<li>Makino, A., Platoshyn, O., Suarez, J., Yuan, J. X., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Downregulation+of+connexin40+is+associated+with+coronary+endothelial+cell+dysfunction+in+streptozotocin-induced+diabetic+mice%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice.</a> Am J Physiol Cell Physiol. 295, C221-C230 (2008).</li>
<li>Marelli-Berg, F. M., Peek, E., Lidington, E. A., Stauss, H. J., Lechler, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+endothelial+cells+from+murine+tissue%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Isolation of endothelial cells from murine tissue.</a> J Immunol Methods. 244, 205-215 (2000).</li>
<li>van Beijnum, J. R., Rousch, M., Castermans, K., vander Linden, E., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+endothelial+cells+from+fresh+tissues%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Isolation of endothelial cells from fresh tissues.</a> Nat Protoc. 3, 1085-1091 (2008).</li>
<li>Nolan, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev Cell. 26, 204-219 (2013).</li>
<li>Sobczak, M., Dargatz, J., Chrzanowska-Wodnicka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+pulmonary+endothelial+cells+from+neonatal+mice%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation and culture of pulmonary endothelial cells from neonatal mice.</a> J Vis Exp. , (2010).</li>
<li>Makino, A., Dai, A., Han, Y., Youssef, K. D., Wang, W., Donthamsetty, R., Scott, B. T., Wang, H., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((O-GlcNAcase+overexpression+reverses+coronary+endothelial+cell+dysfunction+in+type+1+diabetic+mice%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.</a> Am J Physiol Cell Physiol. 309, C593-C599 (2015).</li>
<li>Sasaki, K., Donthamsetty, R., Heldak, M., Cho, Y., Scott, B. T., Makino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((VDAC%3A+old+protein+with+new+roles+in+diabetes%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">VDAC: old protein with new roles in diabetes.</a> Am J Physiol Cell Physiol. 303, C1055-C1060 (2012).</li>
</ol>

The authors have nothing to disclose.

The most widely used endothelial cells in research are human umbilical vein endothelial cells (HUVECs) because of their convenience and ease of culturing. However, a lot of research requires the availability of a physiological model attained by the isolation of endothelial cells from other specific organs10. Endothelial cells make up a very minor population of cells in the heart tissue; their isolation and culturing can prove to be very difficult.
There are three critical steps within this protocol. The first is to flush blood well. This is necessary, as leukocytes within blood also express CD31 as a cell surface marker, and will compete against ECs for the binding of the CD31 antibody. As other endothelial cells may also be present in the blood, flushing is essential to prevent contamination by such cells. The second critical step is to maintain a sterile environment throughout the isolation process. Any contamination results in a dramatic decrease in the number of cells attained. Finally, the third critical step is to adjust the pH of the CD31 buffer, as failure to do so will decrease the efficiency of the isolation.
Cell quality is most important during this isolation. Smooth muscle cell growth can be inhibited by the addition of an appropriate amount of heparin to the culture media. Fibroblast proliferation could be slowed down by adding D-Valine to the media. Endothelial cells can be characterized by the uptake of Dil-AcLDL and co-staining with BS-Lectin in order to test for the purity of the population.The endothelial cell phenotype is unstable and their behavior can change rapidly once taken out of the original tissue and cultured. A limitation of the technique is that cells should not be used after 5 days of culturing to ensure that they still retain their phenotype. It is strongly recommended not to pass the cells. The primary cells will provide a consistent&#160;result.
Overall, the technique results in a good number of cells of outstanding purity. With the required skills, the isolation process may be completed within 6 hr. It provides a great method for obtaining mouse coronary endothelial cells for either cell culture or molecular biological experiment.

Mouse models of various cardiovascular diseases and metabolic disorders bear physiological and molecular changes which are similar to those found in patients. Furthermore, genetic alteration of mice is a powerful tool that allows us to investigate the pathogenic role of specific genes in the development and progression of diseases. Nowadays, cell-type-specific gene-knock-in or -out mice are easily generated in many laboratories and the measurement of mRNA and protein levels in specific cell types is the first step in determining whether the mice were truly genetically modified.
Endothelium is a thin single layer of endothelial cells (ECs) that lines the inner vascular wall. Endothelial cells play a critical role in regulating vascular tension, vascular permeability, and new vascular formation1,2. Endothelial dysfunction is a hallmark of many pathological conditions and changes in endothelial function can lead to various cardiovascular diseases3,4. It is thus significant to study the function of endothelial cells under physiological and pathophysiological conditions.
There are several ways to isolate ECs5-8 and the isolation method has to be optimized depending on which tissues and species will be used. This is because ECs from different tissues display a high level of heterogeneity with respect to their surface markers and protein expression9. Successful isolation of endothelial cells can often be challenging and requires some degree of training and practice. Once achieved, the method of isolation proposed in this protocol proves to be stable and efficient.
The overall goal of this method is to obtain mouse coronary ECs (MCECs) of high quality and quantity. Even though the quantity of cells collected from a heart is less than cells collected from other tissues using other techniques, this technique still provides better quality. The purity of endothelial cells in the resulting population is greater than 90%. Thus, this technique would be ideal for applications that rely on purely isolated endothelial cells such as cell imaging.

<table><tbody><tr><td>BSA</td><td>Sigma</td><td>A3311-10G</td><td></td></tr><tr><td>EDTA</td><td>Fisher</td><td>BP120-500</td><td></td></tr><tr><td>HBSS</td><td>Fisher</td><td>SH3026801</td><td></td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H4034-500G</td><td></td></tr><tr><td>Sodium Bicarbonate&amp;nbsp;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>D-(+)-Glucose&amp;nbsp;</td><td>Sigma</td><td>G7021-1KG</td><td></td></tr><tr><td>DynaBeads pan mouse IgG beads&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11035</td><td></td></tr><tr><td>Rat anti-mouse CD31 Antibody</td><td>BD Biosciences</td><td>550274</td><td></td></tr><tr><td>IFCS&amp;nbsp;</td><td>Fisher</td><td>SH30072.04</td><td></td></tr><tr><td>M199</td><td>Fisher</td><td>MT10060-CV</td><td></td></tr><tr><td>Dispase (Neutral Protease)</td><td>Worthington Biochemical Corp./Fisher</td><td>LS02109</td><td></td></tr><tr><td>Collagenase Type II&amp;nbsp;</td><td>Worthington Biochemical Corp./Fisher</td><td>LS004176</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Fisher</td><td>BP381-1</td><td></td></tr><tr><td>Igepal</td><td>Sigma</td><td>I3021-50 ml</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail</td><td>Sigma</td><td>P0044-1ml</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>P8340-1 ml</td><td></td></tr><tr><td>Heparin&amp;nbsp;</td><td>Sigma</td><td>H3149-100KU</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Fisher/Mediatech</td><td>MT35010CV</td><td></td></tr><tr><td>D-Valine</td><td>Sigma</td><td>V1255-5G</td><td></td></tr><tr><td>EGS</td><td>Corning</td><td>356006</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Fisher/Mediatech</td><td>MT-30-002-CI</td><td></td></tr></tbody></table>

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.

The successful isolation of coronary endothelial cells from a mouse heart typically yields around 104 cells with over 90% purity with a cobblestone shape. For a pure population of endothelial cells, caution should be taken throughout the protocol to ensure a sterile environment free from any contamination. The appearance of elongated cells or enlarged cells within the population indicates contamination with other cell types, smooth muscle cells and fibroblasts. Endothelial cell purity test is performed by staining cells with an endothelial cell surface marker, BS-Lectin&#160;and acLDL (Figure 1). The percentage of positive cells which exhibit both colors was over 90% in MCEC. Such a result indicates that the technique was performed successfully.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">PBS (10x) (1 L)</td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>MW</td>
			<td>Amount (g)</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.44</td>
			<td>80</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.55</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>141.96</td>
			<td>6.1</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2&#173;PO4</td>
			<td>136.08</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Krebs (10x) (1 L)</td>
		</tr>
		<tr>
			<td>Material</td>
			<td>MW</td>
			<td>Conc (mM)</td>
			<td>Amount (g)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.44</td>
			<td>118</td>
			<td>69</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>74.56</td>
			<td>4.7</td>
			<td>3.5</td>
		</tr>
		<tr>
			<td>CaCl2&#8226;2H2O</td>
			<td>147</td>
			<td>1.8</td>
			<td>2.6</td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H2O</td>
			<td>246.5</td>
			<td>1.2</td>
			<td>2.96</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>120</td>
			<td>1.2</td>
			<td>1.44</td>
		</tr>
		<tr>
			<td colspan="2">EC Media (20%) (500 ml)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>Amount</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M199</td>
			<td>444.5 ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 - 20 U/ml Heparin</td>
			<td>500 &#181;l</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-Valine</td>
			<td>25 mg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IFCS</td>
			<td>100 ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EGS</td>
			<td>10 mg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>5 ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Amount of beads to be used for one mouse</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>For imaging</td>
			<td>5 &#181;l</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>For WB/PCR</td>
			<td>10 &#181;l</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Chemical Compositions of Solutions Used. 
<img alt="Figure 1" src="/files/ftp_upload/53985/53985fig1.jpg" /> 
Figure 1. Representative Images of MCECs. Fluorescence images show that mouse coronary ECs (MCEC) isolated from hearts exhibit high purity of the EC population. Purity was tested by both acLDL uptake of red color (A) and lectin staining in green color (B) in MCEC. The nucleus was stained by Hoechst in blue color (C). The percentage of positive cells which exhibit both colors was over 90% in MCEC. Bar is representative of 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/53985/53985fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Mouse Coronary Endothelial Cells at JoVE.com

Isolation of Mouse Coronary Endothelial Cells

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 ...

isolation-of-mouse-coronary-endothelial-cells

Nanyang Technological University

Research

JoVE Journal

Biology

11.1K Views.  University of Arizona, Tucson. 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.

Video: Isolation of Mouse Coronary Endothelial Cells

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Developmental Biology

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 ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Neuroscience

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Immunology and Infection

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 Murine Coronary Vascular Smooth Muscle Cells

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

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

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

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...