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.

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

isolation-of-primary-mouse-lung-endothelial-cells

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JoVE Journal

Immunology and Infection

5.2K Views.  UCSF Benioff Children's Hospital. In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Video: Isolation of Primary Mouse Lung Endothelial Cells

Isolation of Mouse Lung Dendritic Cells

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 respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

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 Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Neuroscience

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 ...

Biology

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

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 ...

Isolation of Immune and Non&mdash;Immune Cells from Mouse Lungs for Flow Cytometry

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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

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

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

Isolation of Mouse Coronary Endothelial Cells

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

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

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

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. ...

Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although biobanks can provide a wide collection of primary and immortalized cells for biomedical research, these do not cover all experimental needs, particularly those related to specific diseases or genotypes. Vascular endothelial cells (ECs) are key components of the immune inflammatory reaction and, thus, play a central role in the pathogenesis of a variety of disorders. Notably, ECs from different sites display different biochemical and functional properties, making the availability of specific EC types (i.e., macrovascular, microvascular, arterial, and venous) essential for designing reliable experiments. Here, simple procedures to obtain high-yield, virtually pure human macrovascular and microvascular endothelial cells from the pulmonary artery and lung parenchyma are illustrated in detail. This methodology can be easily reproduced at a relatively low cost by any laboratory to achieve independence from commercial sources and obtain EC phenotypes/genotypes that are not yet available.

Although challenging, the isolation of pulmonary endothelial cells is essential for studies on lung inflammation. The present protocol describes a procedure for the high-yield, high-purity isolation of macrovascular and microvascular endothelial cells.

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although ...