Name: isolation of endothelial cells from the lumen of mouse carotid arteries for single-cell multi-omics experiments
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Description: Watch this Scientific Journal Video about Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments at JoVE.com

This work was supported by funding from National Institutes of Health grants HL119798, HL095070, and HL139757 to HJ. HJ is also supported by the Wallace H. Coulter Distinguished Faculty Chair Professorship. The services provided by the Emory Integrated Genomics Core (EIGC) were subsidized by the Emory University School of Medicine and were also partly supported by the Georgia Clinical and Translational Science Alliance of the National Institutes of Health under award no. UL1TR002378. The content provided above is solely the authors&#39; responsibility and does not reflect the official views of the National Institutes of Health.

All animal procedures described below were approved by the Institutional Animal Care and Use Committee at Emory University. Non-hypercholesterolemic, age- and sex-matched C57BL/6 mice were used to mitigate sex-dependent variation and offset any complication of hypercholesterolemic conditions.
1. Partial carotid ligation (PCL) surgery
NOTE: Partial carotid artery ligation of LCA was carried out as previously described and demonstrated3.
<ol>
	<li>Anesthetize the mouse by isoflurane inhalation (5% isofluorane in oxygen for induction and 1.5% after that for maintenance) throughout the procedure using an anesthetic vaporizer. Place the mouse supine on a Deltaphase isothermal pad to prevent loss of body heat during surgery.</li>
	<li>Apply an ocular ointment to prevent exposure keratitis, and confirm the depth of anesthesia by the absence of toe pinch response.</li>
	<li>Inject buprenorphine 0.05 mg/kg subcutaneously to manage pain preemptively. Disinfect the epilated area by applying and cleaning with betadine and isopropanol solution three times. Ensure that betadine is the last step to sterilize the surgery area, starting from the center and finishing at the sides.</li>
	<li>Using aseptic techniques, make a ventral midline incision (~1 cm) in the neck region, and expose the LCA branch point by blunt dissection.</li>
	<li>Ligate the left internal carotid, external carotid, and occipital arteries with 6-0 silk suture; leave the superior thyroid artery untouched.</li>
	<li>Approximate the skin and close the incision with tissue glue and/or sutures.</li>
	<li>Transfer the mouse to a pre-warmed recovery cage (maintained at 37 &#176;C) and place it on a clean towel for up to 1 h to avoid post-surgery hypothermia. 
	NOTE: In our experience, a pre-emptive single dose of Buprenorphine (0.05 mg/kg) is usually sufficient for post-surgery pain management. A repeat dose of Buprenorphine can be administered if the animal is in distress after the first 24 hours. If performed correctly, there is no mortality associated with this procedure, and postoperative stress to the mouse is minimal. The mice are returned to their respective cages and monitored daily following the surgery.</li>
	<li>Prepare the perfusion setup.
	<ol>
		<li>Use 0.9% NaCl (normal saline) containing 10 U/mL heparin in an IV bag. Hook the IV bag 244-274 cm (8-9 feet) from the ground or approximately 122-152 cm (4-5 feet) higher than the dissection board. Attach the butterfly needle to the end of the saline line and flush any air bubbles out of the IV line.</li>
	</ol>
	</li>
</ol>
2. Isolation of carotid arteries post sacrifice
<ol>
	<li>Sacrifice the mice by CO2 inhalation following the institutional IACUC protocol.</li>
	<li>Spray 70% ethanol on the mouse skin and place it in the supine position on the dissection board containing adsorbent paper towels. Secure the paws of the mouse to the adsorbent towels on the dissection board using adhesive tape or a 21 G needle.</li>
	<li>Using a pair of sterile scissors, remove the skin of the mouse starting from the abdomen and all the way to the top of the thorax.</li>
	<li>Use a pair of scissors to open the abdominal wall below the ribcage by blunt dissection.</li>
	<li>Using the scissors, carefully make an incision along the length of the diaphragm, and continue through the ribs on both sides of the thorax until the sternum can be lifted away. Gently lift away the sternum with a pair of forceps; after that, remove the ribcage to expose the heart.</li>
	<li>Carefully remove the thymus and any connective tissue over the heart to visualize the major vessels.</li>
	<li>Sever the vena cava with scissors to allow blood to exit from closed circulation.</li>
	<li>Insert a 21 G butterfly needle connected to the IV line through the apex of the heart into the left ventricle and allow retrograde perfusion for 2-3 min with normal saline at room temperature. Ensure that a constant flow rate of normal saline is maintained by keeping the saline bag at the height of 8-9 feet from the ground. 
	NOTE: The lungs and liver become pale, indicating optimal perfusion. Approximately 20 mL of perfusion buffer is used for each mouse.</li>
	<li>Remove the skin from the neck region and remove all the fat, muscles, and connective tissues to expose the carotid arteries (Figure 1A). 
	NOTE: The rest of the procedure is carried out under the dissecting microscope.</li>
	<li>Adjust the field of view to locate the ligation sites. Using the 10 mm micro-dissection scissors, make a small incision below the ligation site in the LCA to allow perfusion.</li>
	<li>Perfuse again for an additional 1 min via the left ventricle and ensure that the LCA is well perfused and free from any visible traces of blood.</li>
	<li>Use fine-tip forceps and small spring scissors to carefully remove the peri-adventitial tissues surrounding the carotids while the carotid is attached to the body. 
	NOTE: Be extremely careful not to squeeze or stretch the carotid arteries during this cleaning step, as this can increase the number of non-viable cells. If performed correctly, the cell viability in the final cell suspension is usually &#62;93%.</li>
</ol>
3. Endothelial-cell-enriched single-cell isolation from mice carotids
NOTE: The reagents described below can be prepared in advance and stored at 4 &#176;C until use: 1x and 0.1x single-nuclei lysis buffers with the reagents listed in Table 1; single-nuclei wash buffer with the reagents listed in Table 1; single-nuclei Nuclei Buffer recipe with the reagents listed in Table 1. The working stocks of these buffers are to be prepared following the manufacturer&#39;s protocol. Digestion buffer composition: Collagenase Type II 600 U/mL and DNase I 60 U/mL in 0.5% fetal bovine serum (FBS)-containing phosphate-buffered saline (PBS).
<ol>
	<li>While the carotids are still attached, wash the exterior of the carotid arteries with normal saline solution to flush away any traces of blood.</li>
	<li>Using an insulin syringe fitted with a 29 G needle, inject ~ 50 &#181;L of the digestion buffer into the lumen of the distal end of the left carotid artery. As the digestion buffer starts to fill the carotid artery, clamp the proximal end of the carotid with a micro-clip (Figure 1B). Introduce an additional 15-20 &#181;L of digestion buffer into the lumen and clip the distal end to avoid the release of the digestion buffer.</li>
	<li>Carefully explant the carotid arteries from the mouse (Figure 1B,C) and place them in 35 mm dishes containing warm (at 37 &#176;C) Hepes-buffered saline solution. 
	NOTE: Make sure that both the clamps are securely placed. If the clamp becomes loose, the digestion solution can leak out of the lumen and affect the obtained cell count. If this happens, add additional enzymatic solution using an insulin syringe fitted with a 29 G needle from the open end of the carotid and secure the clamp again (Figure 1D). If the enzyme buffer solution leaks again, it is likely that the carotid is severed during the explantation process. In this case, discard the carotid and exclude the sample from the study. Repeated rough handling of the carotid will decrease the cell viability and the quality of single-cell preparation. Take care to identify and correctly label the left and right carotid arteries.</li>
	<li>Incubate the explanted carotids for 45 min at 37 &#176;C with intermittent rocking.</li>
</ol>
4. Flushing the carotid arteries
<ol>
	<li>After completing the luminal enzymatic digestion, remove the carotid artery together with the clamps from the 35 mm dish. Carefully remove each clamp, taking care that the digestion buffer should not leak.</li>
	<li>Gently hold one end of the carotid artery with fine forceps on top of a 1.5 mL microcentrifuge tube. Insert a 29 G needle fitted with an insulin syringe containing 100 &#181;L of warm digestion buffer (37 &#176;C) into the lumen with the other hand (Figure 1D).</li>
	<li>Quickly flush the lumen of the carotid into the microcentrifuge tube. Block the enzymatic reaction by adding 0.3 mL of FBS into the 1.5 mL tube. Place the tube on ice. 
	NOTE: The flushing contains the cells from the luminal enzymatic digestion. Adding FBS and reducing the temperature stops the enzymatic digestion process. If the number of cells in the preparation is high and contains debris or fat tissue, the use of a 50 or 70 &#181;m cell strainer is highly recommended to filter out unwanted tissues debris. To increase the single-cell count, flushing multiple carotids is recommended. Here, to obtain at least 5,000 cells, 10-12 carotids were flushed and pooled as one sample.</li>
	<li>Centrifuge the cells at 500 &#215; g for 5 min at 4 &#176;C using a centrifuge equipped with a fixed-angle rotor (see the Table of Materials).</li>
	<li>Discard the supernatant and resuspend the cell pellet in digestion buffer containing cell dissociation reagent (see the Table of Materials) for 5 min at 37 &#176;C to separate all the cells into single cells. 
	NOTE: If the number of cells in the preparation is low, increase the centrifuge speed up to 2,000-3,000 &#215; g to spin down the cells. Moreover, using 0.5 mL centrifuge tubes facilitates better visualization of the cell pellet at the bottom of the tube.</li>
	<li>Block the enzymatic reaction by adding 0.15 mL of FBS into the 0.5 mL tube.</li>
	<li>Centrifuge the single-cell suspension at 500 &#215; g for 5 min at 4 &#176;C using a centrifuge equipped with a fixed-angle rotor, as in step 4.4.</li>
	<li>Discard the supernatant and resuspend the cells in 100 &#181;L of ice-cold 1% bovine serum albumin (BSA) solution in PBS in a 0.2 mL microcentrifuge tube. 
	NOTE: Using 0.2 mL centrifuge tubes enhances the visualization of the single-cell-pellet at the bottom of the tube.</li>
</ol>
5. Single-cell and single-nucleus analyses
<ol>
	<li>Resuspend and submit the single-cell preparation for scRNAseq.
	<ol>
		<li>Resuspend the single-cell pellet with 100 &#181;L of ice-cold 1% BSA in PBS in a 0.2 mL tube.</li>
		<li>After resuspending the cells for single-cell encapsulation, immediately proceed for single-cell encapsulation and barcoding using a microfluidics-based single-cell partitioning and barcoding system (see the Table of Materials).</li>
		<li>Before submitting the samples to the Genomics Core, count and inspect the single-cell preparations; ensure the absence of cell aggregates. 
		&#8203;NOTE: The aggregation of single cells can be further minimized by increasing the BSA amount up to 2%.</li>
	</ol>
	</li>
	<li>Resuspend and submit the single-cell preparation for scATACseq.
	<ol>
		<li>Resuspend the cell pellet in 100 &#181;L of 0.04% BSA in ice-cold 1x PBS and centrifuge the single-cell suspension at 500 &#215; g at 4 &#176;C.</li>
		<li>Lyse the single-cell suspension with ice-cold 0.1x lysis buffer (Table 1) and incubate for 5 min on ice.</li>
		<li>Mix the lysate 10 times with a P-20 pipette and incubate for an additional 10 min.</li>
		<li>Add 500 &#181;L of chilled wash buffer (Table 1) to the lysed cells and mix 5 times using a pipette. 
		NOTE: Use a 70 &#181;m cell strainer to filter any debris from the cell suspension and transfer them into a 2 mL tube.</li>
		<li>Centrifuge the lysate at 500 &#215; g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the nuclei-pellet in the diluted nuclei buffer (150 &#181;L) (see the Table of Materials and Table 1). Count and inspect the single-nuclei preparations using a hemocytometer11.</li>
		<li>Submit the single-nuclei sample to the Genomics Core for single-nuclei transposition, nuclei partitioning, library preparation, and sequencing. 
		NOTE: If the initial cell number is low, it is common to observe debris along the nuclei. Therefore, it is recommended to start with a high cell number.</li>
	</ol>
	</li>
</ol>

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HJ is the founder of FloKines Pharma. Other authors have no conflicts of interest to declare.

This paper provides a detailed protocol to isolate single-cell preparations from the mouse carotid arteries. The influence of d-flow on the endothelial cells can be accurately studied if the PCL surgery is performed correctly. It is crucial to correctly identify the branches of the common carotid, such as the external carotid, internal carotid, occipital artery, and superior thyroid artery. Validation of flow patterns by ultrasonography further validates the successful onset of d-flow conditions. Althou...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63128">Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-cell Multi-omics Experiments</a>. The Authors section was&#160;updated.
The Authors section was updated from:
Sandeep Kumar*1 
Aitor Andueza*1 
Juyoung Kim1 
Dong-Won Kang1 
Hanjoong Jo1,2 
1Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 2Division of Cardiology, Georgia Institute of Technology and Emory University 
* These authors contributed equally
to:
Sandeep Kumar*1 
Aitor Andueza*1 
Nicolas Villa-Roel1 
Juyoung Kim1 
Dong-Won Kang1 
Hanjoong Jo1,2 
1Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 2Division of Cardiology, Georgia Institute of Technology and Emory University 
* These authors contributed equally

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63128">Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-cell Multi-omics Experiments</a>. The Authors section was&#160;updated.

Erratum: Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-cell Multi-omics Experiments

This laboratory previously demonstrated that induction of d-flow leads to quick and rugged atherosclerosis development in hyperlipidemic mice1,2. The novel mouse model of d-flow-induced atherosclerosis was possible using partial carotid ligation (PCL) surgery 3. PCL surgery induces low and oscillatory blood flow condition or d-flow in the ligated left carotid artery (LCA). In contrast, the contralateral right carotid artery (RCA) continues to face stable laminar flow (s-flow). Previously, to understand the effect of d-flow on endothelial cells, the carotid arteries were dissected out after partial ligation surgery and flushed with a phenol and guanidine isothiocyanate-based lysing agent (luminal RNA/DNA flushing method)2,4, which provided endothelial-enriched &#34;pooled bulk&#34; RNAs or DNAs. These pooled bulk RNAs or DNAs were then processed for transcriptomic studies or epigenomic DNA methylome studies, respectively4,5,6. These studies helped discover multiple flow-sensitive genes and microRNAs whose roles in endothelial biology and atherosclerosis were extensively investigated4,6,7.
However, despite endothelial enrichment, these bulk RNA/DNA studies could not distinguish the specific role of each cell type in the artery wall in d-flow-induced atherosclerosis. Endothelial-enriched single-cell (sc) isolation and&#160;scRNA and scATAC sequencing studies were performed to overcome this limitation8. For this, C57Bl6 mice were subjected to the PCL surgery to induce d-flow in the LCA while using the s-flow-exposed RCA as control. Two days or two weeks after the PCL surgery, the mice were sacrificed, and the carotids were dissected and cleaned up. The lumen of both LCAs and RCAs were infused with collagenase, and the luminal collagenase digests containing ECs as a significant fraction and other arterial cells were collected. The single-cell suspension (scRNAseq) or single-nuclei suspension (scATACseq) were prepared and barcoded with unique identifiers for each cell or nucleus using a 10x Genomics setup. The RNAs were subjected to cDNA library preparation and sequenced.
The scRNAseq and scATACseq datasets were processed using the Cell Ranger Single-Cell Software and further analyzed by Seurat and Signac R packages9,10. Each cell and nucleus was assigned a cell type from these analyses and clustered into the cell type based on the marker genes and unique gene expression patterns. The results of the scRNAseq and scATACseq demonstrated that these single-cell preparations are enriched with ECs and also contain smooth muscle cells (SMCs), fibroblasts, and immune cells.
Further analysis revealed that the EC population in the luminal digestion is highly divergent and plastic (8 different EC clusters) and responsive to blood flow. Most importantly, these results demonstrated that d-flow reprograms ECs from an athero-protected anti-inflammatory phenotype to pro-atherogenic phenotypes, including pro-inflammatory, endothelial-to-mesenchymal transition, endothelial stem/progenitor cell transition, and most surprisingly, endothelial-to-immune cell-like transition. In addition, scATACseq data reveal novel flow-dependent chromatin accessibility changes and transcription factor binding sites in a genome-wide manner, which form the basis of several new hypotheses. The methodology and protocol for preparing single endothelial cells for single-cell multi-omics studies from the mouse carotid arteries are detailed below.

<table><tbody><tr><td>&lt;strong&gt;Chemicals, Peptides, and Recombinant Proteins&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1x PBS (Cell Culture Grade)</td><td>Corning</td><td>21040CMX12</td><td></td></tr><tr><td>1.5 mL Protein LoBind Microcentrifuge Tubes</td><td>Eppendorf</td><td>022-43-108-1</td><td></td></tr><tr><td>15 mL Centrifuge Tube - Foam Rack, Sterile</td><td>Fablab</td><td>FL4022</td><td></td></tr><tr><td>50 mL SuperClear Centrifuge Tubes</td><td>Labcon</td><td>3191-335-028</td><td></td></tr><tr><td>6-0 Silk Suture Sterile</td><td>Covidien</td><td>s-1172 c2</td><td></td></tr><tr><td>70 &amp;micro;m Cell Strainer, White, Sterile, Individually Packaged</td><td>Thermo Fisher Scientic</td><td>08-771-2</td><td></td></tr><tr><td>Accutase solution,sterile-filtered</td><td>Sigma-Aldrich</td><td>A6964-100ML</td><td>or equivalent</td></tr><tr><td>ATAC Buffer (Component I of Transposition Mix)</td><td>10x Genomics</td><td>2000122</td><td></td></tr><tr><td>ATAC Enzyme (Component II of Transposition Mix)</td><td>10x Genomics</td><td>2000123/ 2000138</td><td></td></tr><tr><td>Bovine Serum albumin</td><td>Sigma-Aldrich</td><td>A7906-500G</td><td></td></tr><tr><td>Buprenorphine</td><td>Med-Vet International</td><td>RXBUPRENOR5-V</td><td></td></tr><tr><td>Chromium Controller &amp;amp; Next GEM Accessory Kit</td><td>10X Genomics</td><td>1000204</td><td></td></tr><tr><td>Chromium Next GEM Single Cell 3&#039; Reagent Kits v3.1</td><td>10X Genomics</td><td>1000121</td><td></td></tr><tr><td>Chromium Next GEM Single Cell ATAC Reagent Kits v1.1</td><td>10X Genomics</td><td>1000175</td><td></td></tr><tr><td>Collagenase II</td><td>MP Biomedicals</td><td>2100502.5</td><td></td></tr><tr><td>Digitonin</td><td>Sigma-Aldrich</td><td>D141-100MG</td><td></td></tr><tr><td>Dissecting Forceps</td><td>Roboz Surgical Instruments Co</td><td>RS-5005</td><td></td></tr><tr><td>Dnase1</td><td>New England Biolabs Inc</td><td>M0303S</td><td></td></tr><tr><td>Centrifuge (Benchtop-Model # 5425)</td><td>Eppendorf</td><td>22620444230VR</td><td></td></tr><tr><td>Fetal Bovine Serum - Premium Select</td><td>R&amp;amp;D systems</td><td>S11550</td><td></td></tr><tr><td>Fixed Angle Rotor</td><td>Eppendorf</td><td>FA-45-24-11-Kit Rotor</td><td></td></tr><tr><td>HEPES buffered saline</td><td>Millipore Sigma</td><td>51558</td><td></td></tr><tr><td>Insulin syringe (3/10 mL 29 G syringe)</td><td>BD</td><td>305932</td><td></td></tr><tr><td>Isoflurane</td><td>Patterson vet</td><td>789 313 89</td><td></td></tr><tr><td>MACs Smart Strainers (30 &amp;micro;m)</td><td>Miltenyi Biotec</td><td>130-098-458</td><td></td></tr><tr><td>MACS SmartStrainers (100 &amp;micro;m)</td><td>&amp;nbsp;Miltenyi Biotec</td><td>130-098-463</td><td></td></tr><tr><td>Normal Saline (0.9% sodium chloride)</td><td>Baxter International Inc</td><td>2B1323</td><td></td></tr><tr><td>Nuclei Buffer (20x)</td><td>10x Genomics</td><td>PN 2000153/2000207</td><td></td></tr><tr><td>PBS (10x), pH 7.4</td><td>Thermo Fisher Scientic</td><td>70011-044</td><td></td></tr><tr><td>Small scissors</td><td>Roboz Surgical Instruments Co</td><td>RS-5675</td><td></td></tr><tr><td>Stainless Steel Micro Clip Applying Forceps With Lock</td><td>Roboz Surgical Instruments Co.</td><td>RS-5480</td><td>or similar</td></tr><tr><td>Tissue Mend II</td><td>Webster Veteinanry</td><td>07-856-7946</td><td></td></tr><tr><td>Type II Collagenase</td><td>MP biomedicals</td><td>2100502.1</td><td></td></tr><tr><td>&lt;strong&gt;Deposited Data&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>scATACseq FastQ files</td><td>NCBI</td><td>www.ncbi.nlm.nih.gov/bioproject Accession # PRJNA646233</td><td></td></tr><tr><td>scRNAseq FastQ files</td><td>NCBI</td><td>www.ncbi.nlm.nih.gov/bioproject Accession # PRJNA646233</td><td></td></tr><tr><td>&lt;strong&gt;Software and Algorithms&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Ranger 3.1.0</td><td>10X Genomics</td><td>https://support.10xgenomics.com/ single-cell-gene-exp</td><td></td></tr><tr><td>Cicero</td><td>Pliner et al., 2018</td><td>https://cole-trapnell-lab.github.io/cicero-release/</td><td></td></tr><tr><td>Ggplot2 v3.2.1</td><td>Hadley Wickham</td><td>https://cran.r-project.org</td><td></td></tr><tr><td>Harmony</td><td>Korsunsky et al., 2019</td><td>https://github.com/immunogenomics/harmony</td><td></td></tr><tr><td>ImageJ</td><td>Schneider et al., 2012</td><td>https://imagej.nih.gov</td><td></td></tr><tr><td>Monocle 2.8.0</td><td>Qiu et al., 2017</td><td>https://github.com/cole-trapnell-lab/ monocle-release</td><td></td></tr><tr><td>R version 3.6.2</td><td>R Foundation</td><td>https://www.r-project.org</td><td></td></tr><tr><td>Seurat 3.1.3</td><td>Stuart et al., 2019</td><td>https://github.com/satijalab/seurat</td><td></td></tr><tr><td>Signac 0.2.5</td><td>Stuart et al., 2019</td><td>https://github.com/timoast/signac</td><td></td></tr></tbody></table>

isolation of endothelial cells from the lumen of mouse carotid arteries for single-cell multi-omics experiments

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

Partial carotid ligation surgeries were performed on 44 mice, and the onset of d-flow in the LCA was validated by performing ultrasonography one day post partial ligation surgery. Successful partial ligation surgery causes reduced blood flow velocity and reverses blood flow (disturbed flow) in the LCA3. The carotid arteries were dissected out either at two days or at two weeks post ligation. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single-...

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

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

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

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Research

JoVE Journal

Biology

3.1K Views.  Georgia Institute of Technology and Emory University. In een bloedvat is het aantal endotheelcellen erg laag, waardoor het moeilijk is om ze te bestuderen. Ons protocol elimineert deze beperking en stelt ons in staat om nieuwe moleculaire routes en therapeutica te bestuderen. Ons protocol stelt ons in staat om endotheelverrijkte monsters te verkrijgen en te bestuderen hoe acute en chronisch verstoorde stroom dit celtype beïnvloedt.Het vermogen om endotheelcellen in meer detail te bestuderen, maakt het mogelijk om doelgenen te definiëren...

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

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Medicine

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Developmental Biology

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

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

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

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

Isolation of Primary Mouse Lung Endothelial Cells

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

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

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

Immunology and Infection

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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

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

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

Isolation of 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 Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the transcriptome and epigenome of ECs is paramount to understanding their roles in development, health, and disease, but is limited in the availability of isolated primary cells. Recent technologies have enabled the high-throughput profiling of EC transcriptome and epigenome, leading to the identification of previously unknown EC cell subpopulations and developmental trajectories. While EC cultures are a useful tool in the exploration of EC function and dysfunction, the culture conditions and multiple passages can introduce external variables that alter the properties of native EC, including morphology, epigenetic state, and gene expression program. To overcome this limitation, the present paper demonstrates a method of isolating human primary ECs from donor mesenteric arteries aiming to capture their native state. ECs in the intimal layer are dissociated mechanically and biochemically with the use of particular enzymes. The resultant cells can be directly used for bulk RNA or single-cell RNA-sequencing or plated for culture. In addition, a workflow is described for the preparation of human arterial tissue for spatial transcriptomics, specifically for a commercially available platform, although this method is also suitable for other spatial transcriptome profiling techniques. This methodology can be applied to different vessels collected from a variety of donors in health or disease states to gain insights into EC transcriptional and epigenetic regulation, a pivotal aspect of endothelial cell biology.

The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Author Spotlight: Magnetic Bead-Based Isolation of Murine Dermal Lymphatic Endothelial Cells

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

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

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