Name: Isolation of Mouse Coronary Endothelial Cells (Video) | JoVE Journal
Uploaded: 2016-07-03T13:00:00.000+00:00
Duration: 7 min 36 s
Description: Learn to isolate mouse coronary endothelial cells using CD31 buffer and magnetic bead purification for cardiovascular research. This protocol yields high-quality cells for imaging and molecular studies.

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5 minutes

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 with...

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

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...

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 ...

The overall goal of this experiment is to obtain large numbers of highly purified high quality mouse coronary endothelial cells. This method can help answer key questions in the field of cardiovascular research. The main advantage of this technique is that it facilitates the acquisition of a high yield of good quality endothelial cells.Demonstrating this procedure will be Shizhen Luo, a research specialist, and Dr.Ayako Makino, our principal investigator. Up to one week before the procedure, add one milliliter of CD31 buffer to one 1.5 milliliter microcentrifuge tube per sample. Next, mix sheep antirad IGG beads with gentle pipetting, and add the appropriate volume of beads to each tube.After all of the beads have been added, shake the tubes vigorously 30 times and place them on a magnetic plate for one minute of shaking. Then, with the tubes still attached to the plate, discard the buffer and add one milliliter of fresh CD31 buffer to each tube. Add two microliters of rad anti-mouse CD31 antibody and place the tubes on a rotator at four degrees Celsius overnight.The next day, transfer the tubes under a laminar flow hood and place them back onto the magnetic plate with shaking for one minute. Then replace the supernatant with one milliliter of fresh CD31 buffer and shake the tubes vigorously by hand 30 times. After the third wash, return the tubes to the rotator at four degrees Celsius.On the day of the procedure, begin by injecting the experimental animal intraperitoneally with 0.1 milliliters of heparin to prevent blood coagulation. After ten minutes, anesthetize the mouse with an injection of 0.01 milliliters pentobarbital. Then, confirm a lack of response to toe pinch.After removing the heart and lung tissue together, place them into a beaker of Krebs buffer and rinse the tissues with gentle shaking. Then transfer the tissues into a six centimeter dish containing fresh Krebs, and use scissors and foreceps to remove the lung lobes. Now insert a 20 gauge catheter into the aorta and flush the coronary circulatory system with HBSS supplemented with heparin.When all of the blood has been removed, make a small cut in the ventricle, rinse the heart with gentle shaking, and place the cardiac tissue into a sample tube on ice. After digesting the tissue to release the coronary endothelial cells, pellet and wash the cells two times with five milliliters of washing buffer. Then place a tube of the previously prepared beads onto the magnetic plate and shake the tube three times to evenly distribute the beads.After the second wash, remove as much supernatant as possible without disrupting the pellet, and associate the cells with vigorous flicking. Next, resuspend the cells in one milliliter of washing buffer with gentle mixing and discard the CD31 buffer from the beads. Combine the cells with the beads, and flick the tube 30 times.Then place the cell and bead solution onto the rotator at four degrees Celsius for 30 minutes. While the cells are incubating, coat the surface of one plating chamber well per sample with gelatin solution, and place the chambers at 37 degrees Celsius for 30 minutes. When the gelatin has solidified, remove any excess gel solution and dry the chamber well surface.Then transfer the bead conjugated cells to the magnetic plate under the hood for two minutes of shaking, and discard the washing buffer. Add one milliliter of fresh washing buffer to the cells and remove the tube from the plate for 30 vigorous shakes, followed by 30 flicks. Return the tube to the plate for another minute of shaking.After the last agitation, return the tube to the magnetic plate for a final minute of shaking, and replace the washing buffer with one milliliter of 37 degree Celsius endothelial cell medium, supplemented with 20 percent iron fortified calf serum. Shake and flick the tubes 30 final times. Then use a one milliliter pipetter set to 500 microliters to agitate the conjugated cells ten times, and transfer 500 microliter aliquots of coronary endothelial cells to each plating chamber well for overnight incubation at 37 degrees Celsius.The next day, wash the cells in endothelial cell medium, supplemented with ten percent FBS. The purity of the coronary endothelial cells isolated by this method can be confirmed by immunohistochemical staining against the endothelial cell surface markers, BS lectin, and AC LDL, with over 90 percent of the cells demonstrating a double positive expression of both markers in a typical mouse coronary endothelial cell culture. Following this procedure, as a method, various blood, immunoreception, and fluorescent imaging can be performed to answer additional questions, such as what protein can be modified during different disease states.After its development, this technique provides the tools necessary for researchers to expand their research into the field of coronary endothelial cells. After watching this video, you should have a good understanding of how to isolate mouse coronary endothelial cells of high quantity and quality. There are several key points during this procedure.The most important thing is to maintain a sterile environment throughout the isolation. Second is to flush the blood completely and as soon as possible to avoid blood coagulation in the capillary network. Third is to measure the pH of all the buffer, especially CD31 buffer.

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JoVE Journal

Biology

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Learn to isolate mouse coronary endothelial cells using CD31 buffer and magnetic bead purification for cardiovascular research. This protocol yields high-quality cells for imaging and molecular studies.

Isolation of Mouse Coronary Endothelial Cells

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