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>

<ol>
	<li>Kumar, S., Kang, D. W., Rezvan, A., Jo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+atherosclerosis+development+in+C57Bl6+mice+by+overexpressing+AAV-mediated+PCSK9+and+partial+carotid+ligation.">Accelerated atherosclerosis development in C57Bl6 mice by overexpressing AAV-mediated PCSK9 and partial carotid ligation.</a> Laboratory Investigation. 97 (8), 935-945 (2017).</li><li>Nam, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+carotid+ligation+is+a+model+of+acutely+induced+disturbed+flow,+leading+to+rapid+endothelial+dysfunction+and+atherosclerosis.">Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.</a> American Journal of Physiology Heart and Circulation Physiology. 297 (4), 1535-1543 (2009).</li><li>Nam, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+disturbed+flow-induced+atherosclerosis+in+mouse+carotid+artery+by+partial+ligation+and+a+simple+method+of+RNA+isolation+from+carotid+endothelium.">A model of disturbed flow-induced atherosclerosis in mouse carotid artery by partial ligation and a simple method of RNA isolation from carotid endothelium.</a> Journal of Visualized Experiments: JoVE. (40), e1861 (2010).</li><li>Dunn, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+epigenetic+DNA+methylation+regulates+endothelial+gene+expression+and+atherosclerosis.">Flow-dependent epigenetic DNA methylation regulates endothelial gene expression and atherosclerosis.</a> Journal of Clinical Investigation. 124 (7), 3187-3199 (2014).</li><li>Kumar, S., Kim, C. W., Son, D. J., Ni, C. W., Jo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+regulation+of+genome-wide+mRNA+and+microRNA+expression+in+endothelial+cells+in+vivo.">Flow-dependent regulation of genome-wide mRNA and microRNA expression in endothelial cells in vivo.</a> Scientific Data. 1 (1), 140039 (2014).</li><li>Ni, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+novel+mechanosensitive+genes+in+vivo+using+mouse+carotid+artery+endothelium+exposed+to+disturbed+flow.">Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow.</a> Blood. 116 (15), 66-73 (2010).</li><li>Son, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+atypical+mechanosensitive+microRNA-712+derived+from+pre-ribosomal+RNA+induces+endothelial+inflammation+and+atherosclerosis.">The atypical mechanosensitive microRNA-712 derived from pre-ribosomal RNA induces endothelial inflammation and atherosclerosis.</a> Nature Communications. 4, 3000 (2013).</li><li>Andueza, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+reprogramming+by+disturbed+flow+revealed+by+single-cell+RNA+and+chromatin+accessibility+study.">Endothelial reprogramming by disturbed flow revealed by single-cell RNA and chromatin accessibility study.</a> Cell Reports. 33 (11), 108491 (2020).</li><li>Stuart, T., Srivastava, A., Lareau, C., Satija, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+single-cell+chromatin+analysis+with+Signac.">Multimodal single-cell chromatin analysis with Signac.</a> bioRxiv. , (2020).</li><li>Stuart, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+integration+of+single-cell+data.">Comprehensive integration of single-cell data.</a> Cell. 177 (7), 1888-1902 (2019).</li><li>Crowley, L. C., Marfell, B. J., Christensen, M. E., Waterhouse, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+cell+death+by+trypan+blue+uptake+and+light+microscopy.">Measuring cell death by trypan blue uptake and light microscopy.</a> Cold Spring Harbor Protocols. 2016 (7), (2016).</li><li>Li, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA-seq+reveals+cellular+heterogeneity+of+mouse+carotid+artery+under+disturbed+flow.">Single-cell RNA-seq reveals cellular heterogeneity of mouse carotid artery under disturbed flow.</a> Cell Death and Discovery. 7 (1), 180 (2021).</li><li>Wu, Y. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals, Peptides, and Recombinant Proteins</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 &#181;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 &#38; Next GEM Accessory Kit</td>
			<td>10X Genomics</td>
			<td>1000204</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Next GEM Single Cell 3&#39; 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&#38;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 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (100 &#181;m)</td>
			<td>&#160;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>Deposited Data</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>Software and Algorithms</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-...

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

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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3.0K Views.  Georgia Institute of Technology and Emory University. 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.

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

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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 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 Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria&#39;s distribution, fusion, and fission events without staining the cells.

The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAMexcised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive.

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...