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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 ...

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

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Immunology and Infection

4.7K 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 Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

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

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

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

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

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

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

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

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

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

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

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

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

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

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

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

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

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

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

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 quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

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

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

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

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

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