Name: isolation of primary mouse lung endothelial cells
Uploaded: 2021-11-10T13:00:00
Duration: 6 min 41 s
Description: Watch this Scientific Journal Video about Isolation of Primary Mouse Lung Endothelial Cells at JoVE.com

This work was supported by NIH T32GM008440 (JH/EW) and NIH R01AI058106 (JH). We acknowledge the UCSF Parnassus Flow Cytometry Core (RRID:SCR_018206) supported in part by Grant NIH P30 DK063720 and by the NIH S10 Instrumentation Grant S10 1S10OD021822-01.

All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of California San Francisco. Male C57BL/6 neonatal mouse pups (age 5-7 days) were used for the study. The entire protocol takes 7-10 days (Figure 1).
1. Preparation of endothelial cell medium
<ol>
	<li>Prepare the basal medium: Dulbecco&#39;s Modified Eagle Medium with 4,500 mg/L of glucose and 4 mM of L-Glutamine (DMEM) supplemented with 20% (v/v) heat-inactivated fetal bovine serum, 100 U/100 &#956;g/mL of penicillin/streptomycin, and 25 mM of HEPES (see Table of Materials). Sterile filter with 22 &#956;M filter. Store basal medium at 4 &#176;C and use it within a month of preparation.</li>
	<li>Prepare the complete medium: Basal medium supplemented with 100 &#956;g/mL of heparin, 100 &#956;g/mL of endothelial cell growth supplement, 1x non-essential amino acids, and 1 mM sodium pyruvate (see Table of Materials). Sterile filter with 22 &#956;M filter. Store at 4 &#176;C and use within a month of preparation.</li>
</ol>
2. Precoating of anti-rat magnetic beads13
<ol>
	<li>Make 50 mL of bead wash buffer: Phosphate-buffered solution (PBS) supplemented with 0.1% (w/v) bovine serum albumin (BSA). Sterile filter with 22 &#956;M filter and store at 4 &#176;C and use within a month of preparation.</li>
	<li>Vortex the magnetic beads for a few seconds to resuspend the beads and pipette 50 &#956;L of the beads (2 x 107 beads) into two 1.5 mL microcentrifuge tubes, one for CD-31 and one for CD-102 (see Table of Materials). Add 1 mL of bead wash buffer and mix well. 
	NOTE: All the mentioned volumes were used to isolate endothelial cells from 3-4 neonatal mouse pups (5-7 days old). CD-31 and CD-102 are the endothelial cell surface markers used for magnetic immunobead selection.</li>
	<li>Place the tubes on a magnetic separator and remove the supernatant with a glass Pasteur pipet. Repeat wash 3 times with 1 mL of bead wash buffer each time.</li>
	<li>Resuspend beads in original bead volume, 50 &#956;L, with bead wash buffer. Then add 5 &#956;L (2.5 &#956;g) of antibody (CD-31/CD-102) to every 50 &#956;L of beads.</li>
	<li>Incubate bead suspension on gentle rotation on an end-over-end rotator overnight at 4 &#176;C or for 3 h at room temperature. Repeat wash 4 times.</li>
	<li>Resuspend the immunobeads in the original volume, 50 &#956;L, with bead wash buffer and store at 4 &#176;C and use within 1-2 weeks.</li>
</ol>
3. Isolation and plating of the lung cells
<ol>
	<li>Prepare Type 1 collagenase solution on the day of isolation: Add 25 mg of Type 1 collagenase to 25 mL of HBSS (see Table of Materials). Incubate at 37 &#176;C for 1 h with gentle rotation and sterile filter using 22 &#956;M filter. Keep solution warm at 37 &#176;C.</li>
	<li>Inject 5-7 day old mouse pups intramuscularly with heparin (25 &#956;L/mouse, 1,000 U/mL) 10 min before euthanasia to minimize endothelial cell activation and clot formation14,15.</li>
	<li>Euthanize the mouse pup with decapitation using&#160;sharp scissors in accordance with the 2020 AVMA Guidelines for Euthanasia. Then,&#160;pin the mouse to a dissection board with ventral side up.</li>
	<li>Spray the carcass with 70% ethanol, then expose the thoracic cavity by first cutting the diaphragm and then cutting superiorly upwards along the entire lateral walls of the chest. Reflecting the rib cage back then exposes the heart and lungs. 
	NOTE: Use smaller forceps and scissors for this step given the small size of neonatal mice, and ensure not to pierce the heart or lungs as this will lead to inadequate blood removal from the lungs.</li>
	<li>Attach a 25 G needle to a 10 mL syringe, and inject 5 mL of cold DMEM into the heart&#39;s right ventricle to remove blood from the lungs. The lungs will turn white.</li>
	<li>Remove the lungs from the thoracic cavity, one lobe at a time, by cutting the lobes distal to the corresponding bronchi.</li>
	<li>Pool all the lung lobes into a 50 mL conical tube prefilled with 20 mL of ice-cold basal medium (from step 1.1).</li>
	<li>Repeat steps 3.2-3.7 with the other mouse pups, pooling all lung lobes into the same 50-mL conical tube.</li>
	<li>Gently agitate the tube by hand for 10-15 s to wash any excess red blood cells visually appreciated on the surface of the lungs. 
	NOTE: It is not necessary to remove all blood from within or around the lungs; however, this improves purity. Any further tissue handling needs to be done in a sterile hood.</li>
	<li>Use a cell strainer to remove the lungs from the media, and transfer the tissue to a sterile, disposable 100 mm diameter tissue culture dish and mince with sterilized scissors.</li>
	<li>Transfer the minced tissue to the 50 mL conical tube with 25 mL of pre-warmed collagenase solution (step 3.1). Gently agitate on a rotating mixer for 45 min at 37 &#176;C. 
	NOTE: Overdigestion for even 60 min can lead to lower yields.</li>
	<li>Attach a 20 mL syringe to a 15 G blunt cannula (see Table of Materials) and triturate the suspension 10-15 times to break up the tissue to a cellular suspension. Be vigorous, but avoid over-frothing. 
	NOTE: There will no longer be visible pieces of the lung, and instead, the suspension will be cloudy upon completion of this step.</li>
	<li>Filter the suspension through a 70 &#956;m cell strainer into a fresh 50 mL conical tube. Further, rinse the conical tube used for the digestion and the cell strainer with an additional 15 mL of basal medium.</li>
	<li>Centrifuge the cellular suspension at 400 x g for 8 min at 4 &#176;C.</li>
	<li>Remove the supernatant with a glass Pasteur pipet and resuspend the pellet in 2 mL of complete medium. Transfer to a T-75 flask and add 8 mL of complete medium-culture at 37 &#176;C in a humidified, 5% CO2 incubator.</li>
	<li>The following day, wash flasks twice with 10 mL of Hank&#39;s Balanced Salt Solution without calcium and magnesium (HBSS/CMF) (see Table of Materials) and add 10 mL complete medium. 
	&#8203;NOTE: After 2-3 days, the culture will be 90-95% confluent and ready for primary immunobead isolation.</li>
</ol>
4. Primary immunobead isolation and culturing of endothelial cells
<ol>
	<li>Wash adherent endothelial cells twice with 10 mL HBSS/CMF. Add 2 mL of trypsin-free cell detachment solution (see Table of Materials) and incubate at 37 &#176;C for ~5 min. Ensure all the cells have been lifted from the flask. 
	NOTE: A trypsin-free detachment solution needs to be used instead of trypsin as trypsin can disrupt cell surface adhesion marker CD-31.</li>
	<li>Add 8 mL of basal medium to neutralize the cell detachment solution and transfer contents to a 15 mL conical tube. Spin down at 400 x g for 4 min at room temperature.</li>
	<li>Remove the supernatant using a glass Pasteur pipet and resuspend cells in 2 mL of basal medium. Transfer the 2 mL cell suspension to a 5 mL round bottom polystyrene tube (see Table of Materials).</li>
	<li>Vortex anti-CD-31 coated beads for a few seconds to resuspend the beads and add 30 &#956;L of beads for every 2 mL of cell suspension. Secure the lid.</li>
	<li>Incubate tube for 10 min at room temperature on an end-over-end rotator. Then place the tubes on a magnetic separator and leave for 2 min.</li>
	<li>Gently using a glass Pasteur pipet, aspirate the supernatant. Remove the tube from the magnet, resuspend the bead-cell pellet by adding 3 mL of basal medium to the tube, and pipette up and down several times.</li>
	<li>Replace the tube on the magnetic separator for 2 min, and then carefully aspirate the supernatant using a glass Pasteur pipet.</li>
	<li>Repeat washes (step 4.6-4.7) at least 4 times until the supernatant appears clear.</li>
	<li>After the final wash, resuspend the bead-cell pellet in 3 mL complete growth medium and transfer it to a T-75 flask. Add 7 mL of the complete growth medium, and incubate at 37 &#176;C in a humidified 5% CO2 incubator.</li>
	<li>The next day wash cells with HBSS/CMF, then add 10 mL of fresh complete medium.&#160;Replace media with 10 mL of fresh complete medium every 2-3 days until 90-95% confluent. 
	&#8203;NOTE: Once cells are ~90-95% confluent, which takes ~3-4 days, the culture is ready for secondary immunobead isolation.</li>
</ol>
5. Secondary immunobead isolation and culturing of endothelial cells
<ol>
	<li>Wash adherent endothelial cells twice with 10 mL of HBSS/CMF. Add 2 mL of trypsin-free cell detachment solution and incubate at 37 &#176;C for ~5 min. Ensure all the cells have been lifted from the flask.</li>
	<li>Add 8 mL of basal medium to neutralize cell detachment solution and transfer to a 15 mL conical tube. Spin down at 400 x g for 4 min at room temperature.</li>
	<li>Remove the supernatant using a glass Pasteur pipet and resuspend the cells in 2 mL of basal medium. Transfer the 2 mL cell suspension to a 5 mL round bottom polystyrene tube.</li>
	<li>Vortex anti-CD-102 coated beads for a few seconds to resuspend the beads and add 30 &#956;L of beads for every 2 mL of cell suspension. Secure the lid.</li>
	<li>Incubate the tube for 10 min at room temperature on an end-over-end rotator. Place the tubes on a magnetic separator and leave for 2 min. Then gently aspirate the supernatant using a glass Pasteur pipet.</li>
	<li>Remove the tube from the magnet, resuspend the bead-cell pellet by adding 3 mL of basal medium to the tube, and pipette up and down several times.</li>
	<li>Replace the tube on the magnetic separator for 2 min before carefully aspirating the supernatant using a glass Pasteur pipet.</li>
	<li>Repeat washes (step 5.6-5.7) 4 times or more until the supernatant appears clear.</li>
	<li>After the final wash, resuspend the bead-cell pellet in 3 mL complete growth medium and transfer it to a T-75 flask. Add 7 mL complete growth medium, and incubate at 37 &#176;C in a humidified 5% CO2 incubator.</li>
	<li>The next day wash cells with HBSS/CMF, then add 10 mL of fresh complete medium. Replace media with 10 mL of fresh complete medium every 2-3 days until 90-95% confluent. Once the endothelial cells are fully confluent, they are ready for experimentation. The cells should not be used beyond passage six as senescence may occur, and other cell types may take over.</li>
</ol>
6. Confirmation of the endothelial cell surface markers by flow cytometry
<ol>
	<li>After using trypsin-free cell detachment solution to detach the cells, resuspend cell pellet to 1 x 105 cells/mL and aliquot 100 &#956;L of the cell suspension into three 1.5 mL microcentrifuge tubes, labeled 1, 2, and 3.</li>
	<li>Wash cells with 1 mL of 1x PBS. Centrifuge at 500 x g for 2 min at 4 &#176;C. Remove wash buffer and repeat twice.</li>
	<li>Resuspend the cell pellet in 100 &#956;L of 1x PBS. To tube 1, add 1 &#956;L of conjugated anti-mouse CD-31 (PECAM-1) antibody (ex: phycoerythrin (PE) anti-mouse CD-31 antibody) and 1 &#956;L of conjugated anti-mouse CD-102 (ICAM-2) (ex: fluorescein isothiocyanate (FITC) anti-mouse CD102 antibody) (see Table of Materials). To tube 2, add the appropriate isotype controls. Tube no. 3 will remain unstained.</li>
	<li>Incubate the tubes on ice and in the dark for 30-45 min.</li>
	<li>Add 1 mL of PBS to each tube, vortex gently, and centrifuge at 500 x g for 2 min at 4 &#176;C. Remove wash and repeat three times. Resuspend the final pellet in 500 &#956;L of PBS.</li>
	<li>Keep the tubes covered with aluminum foil at 4 &#176;C until analysis. Analysis needs to be completed within 3-4 h.</li>
	<li>Acquire flow data using a cytometer. Use an unstained sample as a negative control to properly set laser voltage according to cell autofluorescence. Gate total cell population with a forward scattered plot and side scatter plot. 
	NOTE: After excluding doublets, CD31+ CD102+ double-positive cells are defined as endothelial cells.</li>
</ol>

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The authors have nothing to disclose.

This simple protocol lends itself to high-purity endothelial cells from murine lungs. Although the total time from start to finish is 7-10 days, the hands-on time is around 3-4 h. Compared to other methods13,14,15,16, the current protocol is streamlined, keeping essential steps without compromising yield and purity.
It is worth noting some critical aspects of this protocol. First, it is important to use neonatal pups rather than adult mice. In our experience, endothelial cells from adult mice do not proliferate as easily as cells from neonatal pups, and they are easily overrun by cells with spindle-like morphology consistent with fibroblasts or mesenchymal stem cells15,16. One possible explanation for the difference is that lung development is in the alveolar stage in neonatal mice, characterized by the more rapid proliferation of endothelial cells20. There may also be some degree of senescence with aging21. Enzymatic digestion time also needs to be precise, as underdigestion can lead to a low yield single-cell suspension. In contrast, over digestion can lead to a substantial amount of cell death. We have determined that the optimal time for collagenase digestion is determined to be 45 minutes. This is then followed by mechanical dissociation with a blunt cannula. Instilling intratracheal collagenase during enzymatic digestion has also been reported14,15; however, this can be time-consuming and leads to incomplete digestion during the current work. Lastly, some protocols proceed directly to immunobead isolation immediately following enzymatic digestion13,16. We have&#160;found that culturing the cells from the dissociated lung in the medium for a day or two before making the immunobead selection substantially increases the yield of viable and highly pure endothelial cells. This may be related to the speed of getting isolated cells into the tissue culture medium, which leads to less cell death in the early stages after removal from the mice, higher initial seeding density, and time for the endothelial cells to adhere and proliferate before the first immunobead isolation.
Limitations of the preparation and analyses of mouse endothelial cells are as follows. Each mouse only provides a limited number of cells that must be used within a few passages. This issue can be overcome by pooling lung digest from several mice. Unfortunately, we are unsuccessful in our efforts to cryopreserve and thaw the cells for future use. Despite these limitations, mouse endothelial cells have some advantages. They can be helpful, particularly in situations where human endothelial cells either cannot be used (e.g., gene knockout), when complementary studies are required to corroborate results with human cells, or when the heterogeneity of responses of endothelial cells from different human donors makes reproducibility between experiments problematic6. The homogenous endothelial cell population from genetically identical mice allows for reproducibility between experiments and the study of specific genes and pathways under stable background conditions.
Mouse endothelial cells can be used for multiple applications to provide insights into endothelial activation and dysfunction in different disease processes. For example, endothelial cells play an integral role in sepsis, contributing to both beneficial and pathological responses through their roles in activating localized and systemic inflammation, promoting leukocyte trafficking and activation in tissues, regulating coagulation, and modulating endothelial permeability2,22,23. This simple protocol provides viable, functional endothelial cells that can be used in assays for each of these endothelial processes.

Interest in studying the vascular endothelium has grown recently, as dysfunction of microvascular endothelial cells occurs in multiple human diseases, such as stroke, cardiovascular disease, diabetes, acute lung injury, sepsis, and non-infectious injuries1,2,3,4,5. To define the pathogenesis of these conditions and understand the roles of specific genes in protective and dysregulated endothelial cell responses, there is a need for reliable methods to isolate and culture high purity microvascular endothelial cells from mice. While many studies utilize human endothelial cells such as human umbilical vein endothelial cells (HUVEC) or human lung microvascular endothelial cells (HMVEC), there are multiple reasons to use mouse endothelial cells to study endothelial function and dysfunction. First, there is considerable heterogeneity in responses of endothelial cells from different human donors, which leads to variability in results between individual experiments6,7,8. Second, silencing gene targets in primary human cell lines can also lead to variable protein expression levels9,10. In contrast, there is minimal heterogeneity in the responses of mouse endothelial cells11, assuming the genetic background is the same between study groups. Furthermore, large numbers of mouse endothelial cells can be prepared by pooling lungs from several neonatal mice. These factors allow for more consistent and reproducible results between experiments. Finally, the availability of knockout or transgenic mice also provides for the isolation of cell types lacking proteins of interest.
The considerable challenges with isolating healthy and pure populations of mouse lung endothelial cells using published methods12,13,14,15,16 led to developing a more reliable and straightforward method to isolate high-quality mouse lung microvascular endothelial cells. Advantages of the current protocol include using neonatal mice, which are readily available, limiting animal husbandry and housing costs. Additionally, in our experience, the viability of endothelial cells from neonatal mice is substantially higher than endothelial cells prepared from adult mice. Because neonatal mice have small lung tissue, endothelial cells can be harvested by&#160;digesting&#160;excised lungs into collagenase&#160;rather than using tracheal instillation of collagenase, which is recommended for collection from adult mice16. This also reduces the time between euthanizing mice and getting their endothelial cells into cell culture media and the incubator without affecting the purity or yield or the reproducibility of endothelial responses. Lastly, pure cell populations were isolated without flow cytometry, which can damage or lead to lower yields of the endothelial cells14,15,17.
The current modified protocol consistently leads to high purity and viable endothelial cells in 7-10 days that can be used immediately for in vitro experiments. Isolating cells directly from knockout animals also minimizes the manipulation of cells before experimentation. These methods could be used to investigate the role of various proteins in endothelial function to discover endothelial-based therapeutic targets for multiple diseases.

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	<tbody>
		<tr>
			<td>50 mL conicals</td>
			<td>Corning Life Sciences</td>
			<td>430829</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase Cell Detachment Solution</td>
			<td>Innovative Cell Technologies, Inc</td>
			<td>AT-104</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle 25 G x 5/8&#34;</td>
			<td>Becton Disckinson</td>
			<td>305122</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioRender.com</td>
			<td>BioRender</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt Cannula 15 G x 1-1/2&#34;</td>
			<td>Covidien</td>
			<td>8881202314</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD-102 Rat anti-mouse (3C4 (MIC2/4))</td>
			<td>BD Biosciences</td>
			<td>553326</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD-31 Rat anti-mouse (MEC 13.3)</td>
			<td>BD Biosciences</td>
			<td>557355</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type 1</td>
			<td>Worthington Biochemical Corp</td>
			<td>LS004194</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Sheep Anti-Rat IgG</td>
			<td>Invitrogen</td>
			<td>11035</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Supplment</td>
			<td>EMD Millipore Corp</td>
			<td>02-102</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon cell strainers (70 &#181;m)</td>
			<td>Corning Life Sciences</td>
			<td>352350</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, heat inactivated</td>
			<td>Gibco</td>
			<td>10082-147</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Disposable Borosilicate Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20B</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Rat Anti-mouse CD102 (3C4)</td>
			<td>BD Biosciences</td>
			<td>557444</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Rat IgG2a,&#160;&#954; Isotype Control (R35-95)</td>
			<td>BD Biosciences</td>
			<td>553929</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11050-10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium Injections (1,000 Units/mL)</td>
			<td>Medline</td>
			<td>0409-2720-02</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630106</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids (100x)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PE Rat Anti-mouse CD31 (MEC 13.3)</td>
			<td>BD Biosciences</td>
			<td>553373</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PE Rat IgG2a,&#160; &#954;&#160;Isotype Control R35-95)</td>
			<td>BD Biosciences</td>
			<td>553930</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (10,000 U/mL, 10,000 &#181;g/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine IFN-&#947;</td>
			<td>Peprotech</td>
			<td>315-05</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine IL-1&#946;</td>
			<td>Peprotech</td>
			<td>211-11B</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine TNF-&#945;</td>
			<td>Peprotech</td>
			<td>315-01A</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom polystyrene test tubes</td>
			<td>Corning Life Sciences</td>
			<td>352058</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Semken Forceps</td>
			<td>Fine Science Tools</td>
			<td>11008-13</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100 mM)</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Quick Release-GP Sterile Vacuum Filtration System, 250 mL</td>
			<td>Millipore</td>
			<td>S2GPU02RE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit</td>
			<td>Milipore</td>
			<td>SCGP00525</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe (10 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-955-459</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe (20 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-955-460</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 cell culture flask with vent cap, CellBIND treated</td>
			<td>Corning Life Sciences</td>
			<td>3290</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This simple protocol allows one to isolate, culture, and characterize mouse microvascular endothelial cells over 7-10 days (Figure 1). Briefly, lungs are excised and enzymatically digested before plating. Immediately after plating, endothelial cells and other cells will float in cell culture media. By the following day, endothelial cells and the contaminating cells will be adhered to the plate, and a vigorous washing technique is needed to remove debris and the floating cells. Once the cells reach confluence, the first immunobead selection is performed. The CD-31-positive cells then begin to grow in a cobblestone formation (Figure 2). Cells that do not have cobblestone morphology or overgrow are contaminants and not endothelial cells13,14,15,16.
A second immunobead selection is then performed using anti-CD-102 coated beads to increase the purity of endothelial cells, similar to other protocols13,16. Cells that have grown to confluence after second immunobead selection can then be used for experimentation. Fluorescence-activated cell sorting (FACS) is used to confirm that the cell population is CD-31- and CD-102-positive (Figure 3).
These endothelial cells can then be used for many applications, such as studying endothelial inflammatory pathways and endothelial barrier function. For example, it was observed that treatment with murine cytomix (IFNg, TNFa, and IL1b, each at 10 ng/mL) induced IL-6 production by endothelial cells (Figure 4A). Then Electric Cell-substrate Impedance Sensing (ECIS)18 was used to measure transendothelial resistance (TER) to the flow of electrical current across murine lung endothelial cell monolayers. It was observed that the cells treated with cytomix led to a decrease in TER (Figure 4B), which is consistent with increased endothelial permeability. These examples illustrate how the endothelial cells prepared using this protocol can study various endothelial processes. This allows investigators to more easily study genes of interest in endothelial cells, given the ever-growing numbers of available knockout and transgenic mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63253/63253fig01.jpg" /> 
Figure 1: Schematic for isolation of murine lung endothelial cells. Lungs from 5-7 day old neonatal pups are harvested and minced. Minced tissue was enzymatically digested with collagenase I. Cellular suspension is plated and cultured for 2-3 days. Cells then undergo primary immunobead isolation with CD-31-coated beads and are cultured for another 3-4 days. Second immunobead isolation with CD-102-coated beads before culturing another 2-3 days. Cells are now ready for functional experimentation. The image was created by a web-based illustration tool, Biorender. <a href="https://www.jove.com/files/ftp_upload/63253/63253fig01large.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/63253/63253fig02.jpg" /> 
Figure 2: Morphology of the murine lung endothelial cells. Cultured murine lung endothelial cells display a cobblestone morphology. <a href="https://www.jove.com/files/ftp_upload/63253/63253fig02large.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/63253/63253fig03v2.jpg" /> 
Figure 3: Analysis of endothelial cells by flow cytometry. (A) All events are visualized on a dot plot of the Forward Scatter (FSC) and Side Scatter (SSC). (B) Scatter plot confirmation of murine lung endothelial cells being positive for both endothelial-specific cell surface antigens CD-31 and CD-102.&#160;(C) Histogram of the isotype sample and sample stained with CD-31/CD-102 specific antibody. <a href="https://www.jove.com/files/ftp_upload/63253/63253fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63253/63253fig04.jpg" /> 
Figure 4: Cytomix induces murine lung endothelial cell IL-6 production and endothelial cell permeability. Murine lung microvascular endothelial cells from C57BL/6 mice were treated with cytomix (IFNg, TNFa, and IL1b, each at 10 ng/mL). (A) IL-6 levels were then quantified in culture supernatants at 15 h (**p&#60;0.01, n = 4 wells/condition). (B) Electric Cell-Substrate Impedance Sensing was used to measure the transendothelial resistance (TER) repeatedly over 15 h. Data were normalized to the resistance immediately before the addition of the cytomix as designated and was analyzed by two-way analysis of variance (ANOVA) followed by the Tukey post-test19 (**p&#60;0.01, n = 4 wells/condition). <a href="https://www.jove.com/files/ftp_upload/63253/63253fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Isolation of Primary Mouse Lung Endothelial Cells

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 ...

このプロトコルは、複数の用途を持つ可能性のあるマウスから高純度の内皮細胞を単離および培養するための信頼性が高く再現性のある方法を実証しています。この技術は、内皮細胞の収量と純度を維持するより簡単な方法です。手順を実演するのは、私の研究室のスタッフ研究員であるニーナ・グエンです。まず、磁気ビーズを数秒間ボルテックスします。50マイクロリットルのビーズをCD31およびCD102用の2本の1.5ミリリットルマイクロ遠心チューブにピペットで入れます。1ミリリットルのビーズ洗浄バッファーを加え、よく混ぜます。チューブを磁気分離器の上に置き、ガラスパスツールピペットで上清を除去します。ビーズを50マイクロリットルのビーズ洗浄バッファーに再懸濁します。次に、ビーズ50マイクロリットルごとに5マイクロリットルまたは2.5マイクログラムのCD31またはCD102抗体を追加します。ビーズ懸濁液を、端部ローテーター上で穏やかに回転させて、摂氏4度で一晩、または室温で3時間インキュベートします。イムノビーズを4回洗浄した後、イムノビーズを50マイクロリットルのビーズ洗浄バッファーに再懸濁します。分離当日、25ミリリットルのHBSSを25ミリグラムの1型コラゲナーゼに加え、穏やかに回転させながらインキュベートします。次に、22マイクロメートルのフィルターを使用してろ過します。生後5〜7日のマウスの子犬を安楽死させた後、枝肉に70%エタノールをスプレーします。次に、胸部の側壁全体に沿って横隔膜を上向きに切り、胸腔を露出させます。肺が白くなるまで、5ミリリットルの冷たいDMEMを心臓の右心室に注射します。次に、対応する気管支の遠位葉を1つずつ切断して、肺を取り除きます。20ミリリットルの氷冷基礎培地があらかじめ充填された50ミリリットルの円錐形のチューブで肺葉を引っ張ります。チューブを手で10〜15秒間静かに攪拌して、余分な赤血球を洗い流します。セルストレーナーで培地から肺を取り除き、滅菌ハサミでミンチします。ミンチ組織を、25ミリリットルの予熱したコラゲナーゼ溶液と共に50ミリリットルの円錐管に移します。回転ミキサーで静かに攪拌します。20ミリリットルの注射器を15ゲージの鈍いカニューレに取り付け、細胞懸濁液に10〜15回泡立てずに懸濁液を激しく粉砕します。懸濁液を70マイクロメートルのセルストレーナーを通して50ミリリットルの円錐管に濾過する。次に、消化に使用した円錐形のチューブとセルストレーナーを15ミリリットルの基礎培地ですすいでください。細胞懸濁液を摂氏4度で400倍Gで8分間遠心分離する。ペレットを2ミリリットルの完全培地に再懸濁します。8ミリリットルの完全培地を加えてインキュベートします。翌日、カルシウムとマグネシウムを含まない10ミリリットルのHBSSでフラスコを2回洗浄し、10ミリリットルの完全培地を加えます。細胞が90〜95%コンフルエントになると、一次イムノビーズ単離の準備が整います。付着した内皮細胞をカルシウムとマグネシウムを含まない10ミリリットルのHBSSで2回洗浄します。2ミリリットルのトリプシンフリー細胞剥離溶液を加え、それをインキュベートして、フラスコからすべての細胞を確実に持ち上げます。8ミリリットルの基礎培地を追加します。次に、細胞を室温で4分間400倍Gでスピンダウンし、2ミリリットルの基礎培地に再懸濁します。ボルテックス抗CD31コーティングビーズを数秒間、ビーズを再懸濁させた。細胞懸濁液2ミリリットルごとに30マイクロリットルのビーズを加え、蓋をしっかり閉めます。チューブをエンドオーバーエンドローテーターで室温で10分間インキュベートします。次に、チューブを磁気分離器に2分間置きます。上澄み液を吸引し、磁石からチューブを取り外します。3ミリリットルの基礎培地をチューブに加え、数回上下にピペッティングして、ビーズ細胞ペレットを再懸濁します。磁気分離器のチューブを2分間交換します。その後、ガラス製パスツールピペットを用いて上清を注意深く吸引する。最終洗浄後、上清が透明になったら、ビーズ細胞ペレットを3ミリリットルの完全増殖培地に再懸濁し、T75フラスコに移します。7ミリリットルの完全増殖培地を加えてインキュベートします。翌日、カルシウムとマグネシウムを含まないHBSSで細胞を再洗浄します。次に、10ミリリットルの完全培地を追加します。細胞が90〜95%コンフルエントになると、二次イムノビーズ単離の準備が整います。最初のイムノビーズの選択は、石畳の形成で成長するCD31陽性細胞を識別します。抗CD102コーティングビーズを用いた2回目のイムノビーズ選択は、内皮細胞の純度を高め、蛍光活性化セルソーティングを使用して、細胞集団がCD31陽性およびCD102陽性であることを確認する。内皮炎症経路と内皮バリア機能を研究するために、マウスCytomixによる処理を使用して、P値が0.01未満の内皮細胞による有意なIL-6産生を誘導しました。さらに、経内皮抵抗は減少し、内皮透過性の増加を示した。単離された内皮細胞は、内皮細胞刺激またはバリア機能アッセイにおいて、内皮機能障害に対する内皮ベースの治療標的を発見するために使用することができる。

Research

JoVE Journal

Immunology and Infection

Isolation of Mouse Peritoneal Cavity Cells

マウス腹腔細胞の分離

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Isolation of Mouse Lung Dendritic Cells

マウス肺樹状細胞の分離

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

フローの状況下で好中球移住研究におけるヒト臍帯静脈内皮細胞とそれらの使用の分離

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

マウスの腸からの樹状細胞およびマクロファージの単離とキャラクタリゼーション

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

機能と分子生物学的研究のためのプライマリマウスII型肺胞上皮細胞のフローサイトメトリ分離

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

定量的な<em&gt;インビトロ</em静的な条件の下で活性化された初代ヒト微小血管内皮細胞への好中球の接着を測定するために&gt;アッセイ

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

単離とマウス骨髄由来の単球の静脈注射

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

レジオネラ・ニューモ外膜小胞：マクロファージ上における炎症誘発ポテンシャルの単離と解析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

精密カットライブ肺樹状細胞を可視化するために、マウスの肺スライスを

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

検出細胞毒性アミロイド次感染症の肺による内皮細胞緑膿菌法

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...