Name: Single Nuclei Isolation from Coronary Endarterectomy Tissue of Coronary Artery Bypass Graft Patients (Video) | JoVE Journal
Uploaded: 2026-04-03T13:08:00.000+00:00
Duration: 9 min 9 s
Description: Learn a protocol for isolating intact nuclei from human coronary endarterectomy tissue to enable single-nuclei RNA sequencing. This method preserves nuclear integrity for transcriptomic profiling of high-risk atherosclerotic plaques.

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This work was supported by the Indian Council of Medical Research (ICMR) (EM/Dev/SG/34/01106/2024) and by collaboration from the U. N. Mehta Institute of Cardiology and Research Centre, Ahmedabad. Khushi Jani acknowledges the University Grant Commission (UGC) and Council of Scientific and Industrial Research (CSIR), Government of India, for awarding the Joint CSIR-UGC NET qualification (NTA Reference No.: 241620143543).

The present study was performed with approval and in accordance with the guidelines of the Human Ethical Committee of U. N. Mehta Institute of Cardiology and Research Centre (EC/Approval/26/Cardio/14/12/2024) and Gujarat Biotechnology University (GBU_IHEC/2023/006). Informed consent was obtained from all participants prior to sample collection. The patients undergoing coronary artery bypass graft surgery with coronary endarterectomy were selected for the current study. Following surgical removal, the endarterectomy tissue was immediately placed into serum-free RPMI medium, maintained at 4 &#176;C, and transported to the laboratory within approximately 12 h in a cryovial. See the Table of Materials for details on all materials used in the following protocol.
1. Nuclei isolation
NOTE: Perform all the steps on ice and use sterile materials. Prepare all buffers freshly.
<ol>
	<li>Wash the tissue once or twice with serum-free RPMI medium to remove blood stains and fragments. Using sterile forceps, transfer the tissue onto a sterile petri dish and record the weight.</li>
	<li>Using a surgical blade (no. 10), mince the tissue into small pieces &#8764;1 mm3 in size.</li>
	<li>Prepare 1 mL lysis buffer by combining 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 (using stock solutions of 1 M for each component), 0.1% (v/v) Nonidet P-40 in nuclease-free water. Add the components sequentially in the following order: nuclease-free water first, then Tris-HCl, NaCl, MgCl&#8322;, and NP-40 last, to avoid foaming. 
	NOTE: The lysis buffer should be prepared fresh on the day of use and kept chilled on ice at all times. Do not freeze.</li>
	<li>Add chilled 1 mL lysis buffer to the tissue in a 5 mL microcentrifuge tube.</li>
	<li>Lyse the tissue on ice using a micro pestle by applying a gentle, consistent force and swirling the micro pestle clockwise and anticlockwise alternately. Continue the lysis for approximately 30 min, ensuring the sample remains cold throughout. 
	NOTE: Lyse the tissue until it turns turbid. Use a 1000 &#181;L tip to mix gently by pipetting.</li>
	<li>Filter the solution using a 100 &#181;m pore-size strainer into a 15 mL centrifuge tube to remove debris and tissue chunks. 
	NOTE: The filtrate should appear less turbid with visible debris retained on the strainer.</li>
	<li>Centrifuge the nuclei at 500 x g for 5 min at 4 &#730;C using a swinging bucket rotor. 
	NOTE: A visible pellet should form at the bottom of the tube.</li>
	<li>Remove the supernatant using a regular-bore pipette tip without disturbing the pellet.</li>
	<li>Prepare nuclei wash and resuspension buffer by combining 1% bovine serum albumin (BSA) (using 10% of stock solution), 0.2 U/&#181;L RNase inhibitor (using 20 U/&#181;L stock solution) in 1X Dulbecco&#39;s phosphate buffer saline (DPBS). Add the components sequentially in the following order: DPBS first, then BSA, and finally RNase inhibitor. 
	NOTE: The buffer should be prepared fresh on the day of use and kept chilled on ice at all times. Do not freeze.</li>
	<li>Resuspend the pellet in nuclei wash and resuspension buffer. Chill the buffer on ice before use.</li>
	<li>Assess lysis efficiency and cell viability by staining with 5 &#181;L trypan blue and counting in a hemocytometer. 
	NOTE: If viable cells are still present, centrifuge at 500 x g for 5 min at 4 &#730;C, then repeat sections 3-10.</li>
	<li>Filter the solution using a 30 &#181;m pore-size strainer into a 15 mL centrifuge tube to remove debris and clumps.</li>
	<li>Centrifuge the nuclei at 500 x g for 5 min at 4 &#730;C using a swinging bucket rotor.</li>
	<li>Remove the supernatant using a regular-bore pipette tip, then resuspend the pellet in 1 mL of nuclei wash and resuspension buffer.</li>
</ol>
2. Characterization of isolated nuclei
<ol>
	<li>Trypan blue staining for assessing the integrity of nuclei.
	<ol>
		<li>Take 15 &#181;L of the isolated nuclei suspension in a 1.5 mL microcentrifuge tube.</li>
		<li>Add 5 &#181;L of trypan blue dye (0.4% w/v) and mix gently by pipetting.</li>
		<li>Incubate at room temperature for 5 min to allow proper staining.</li>
		<li>Load 10 &#181;L of the stained mixture onto a hemocytometer.</li>
		<li>Observe under a compound inverted microscope to examine morphology and count the number of nuclei.</li>
	</ol>
	</li>
	<li>Flow cytometry analysis of single nuclei suspension
	<ol>
		<li>Take 1 mL of isolated nuclei suspension and centrifuge the nuclei suspension at 500 x g for 5 min at 4 &#730;C using a swing bucket rotor.</li>
		<li>Remove the supernatant using a regular-bore pipette tip and resuspend the pellet in 200 &#181;L FACS Buffer (1X PBS and 1% BSA).</li>
		<li>Divide 200 &#181;L of resuspended nuclei into two aliquots: keep 100 &#181;L as the unstained control and use 100 &#181;L as the stained sample.</li>
		<li>Add 1 &#181;L 4&#39;, 6-diamidino-2-phenylindole (DAPI) (1 mg/mL stock) to the stained sample to achieve a final concentration of 10 &#181;g/mL. Mix gently and incubate at room temperature in the dark for 5 min prior to flow cytometric analysis.</li>
		<li>In the flow cytometer used for this study, use the violet laser at 405 nm, and detect using 448/45 nm. Run the unstained control sample first and acquire a minimum of 10,000 events of the total population.</li>
		<li>Set the voltage at 183 V for forward scatter (FSC), 300 V for side scatter (SSC), and 400 V for V450 (DAPI Laser). Set the threshold at 10,000 for FSC. 
		NOTE: All instrument voltage and gain settings are constant across samples to maintain consistency.</li>
		<li>Plot forward scatter area (FSC-A) versus forward scatter height (FSC-H) to distinguish singlets from doublets and aggregates. Draw a gate around events displaying a linear relationship to retain only singlet nuclei.</li>
		<li>Generate a bivariate plot of FSC-A versus side scatter area (SSC-A). Apply a broad gate encompassing the entire scatter population, as isolated nuclei typically exhibit a heterogeneous scatter profile without distinct subpopulations.</li>
		<li>Create a 2D density (pseudo color) plot of SSC-A versus Violet 450 nm-area (V450-A). Use the unstained control to define background fluorescence and guide precise gate placement. Identify and gate the distinct DAPI&#8314; population corresponding to intact nuclei. 
		NOTE: As a single fluorochrome (DAPI) is used, fluorescence compensation is not required.</li>
	</ol>
	</li>
	<li>Imaging the isolated nuclei using confocal microscopy
	<ol>
		<li>Take 50 &#181;L of nuclei suspension and add 1 &#181;L DAPI (1 mg/mL) to the stained sample to achieve a final concentration of 10 &#181;g/mL, mix gently, and incubate at room temperature for 5 min in the dark prior to confocal imaging.</li>
		<li>Mount 5 &#181;L of stained nuclei suspension onto a clean glass slide and cover it with a coverslip. 
		NOTE: Avoid fixation to preserve nuclear morphology.</li>
		<li>Visualize the nuclei using an inverted confocal microscope. Open the confocal microscope software and observe first in 10X bright field, then switch to 40X bright field.</li>
		<li>Acquire confocal images using a laser scanning confocal microscope equipped with a spectral detector and objectives PLAN APO &#955;D 40X (NA 0.95, RI 1.0). Excite DAPI using a 405 nm laser and collect emission between 429-474 nm.</li>
		<li>Imaging parameters were as follows:
		<ol>
			<li>Detector mode: Multi-channel (GaAsP)</li>
			<li>Scanner: Galvano unidirectional</li>
			<li>Scan mode: Band</li>
			<li>Line averaging: 2X</li>
			<li>Resolution: 1024 &#215; 1024 pixels</li>
			<li>Dwell time: 2.0 &#181;s</li>
			<li>Pinhole size: 47.7 &#181;m</li>
			<li>Laser power: 0.1-9.3% (depending on field intensity)</li>
			<li>Detector gain: 37.8-45.9%</li>
			<li>Zoom: 1.0X</li>
			<li>Calibration: 0.43-0.58 &#181;m/pixel</li>
		</ol>
		</li>
		<li>Acquire all images using the AX camera with minimal crosstalk mode under identical optical configurations, and the perfect focus system (PFS) should be active during imaging to maintain z-plane stability.</li>
	</ol>
	</li>
</ol>

<ol><li>Roth, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+burden+of+cardiovascular+diseases+and+risk+factors-+1990-2019%5BTitle%5D)+AND+%22J.+Am.+Coll.+Cardiol%22%5BJournal%5D)">Global burden of cardiovascular diseases and risk factors- 1990-2019.</a> J. Am. Coll. Cardiol. 76 (25), 2982-3021 (2020).</li>
<li>de Winther, M. P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translational+opportunities+of+single-cell+biology+in+atherosclerosis%5BTitle%5D)+AND+%22Eur.+Heart+J%22%5BJournal%5D)">Translational opportunities of single-cell biology in atherosclerosis.</a> Eur. Heart J. 44 (14), 1216-1230 (2023).</li>
<li>Mohebbati, R., Momeni-Moghaddam, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+red+blood+cells+in+cholesterol+accumulation+and+atherosclerotic+plaque+instability%3A+a+perspective+on+atherosclerosis%5BTitle%5D)+AND+%22Curr.+Cardiol.+Rev%22%5BJournal%5D)">The role of red blood cells in cholesterol accumulation and atherosclerotic plaque instability: a perspective on atherosclerosis.</a> Curr. Cardiol. Rev. 21 (5), (2025).</li>
<li>Ajoolabady, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammation+in+atherosclerosis%3A+pathophysiology+and+mechanisms%5BTitle%5D)+AND+%22Cell+Death+Dis%22%5BJournal%5D)">Inflammation in atherosclerosis: pathophysiology and mechanisms.</a> Cell Death Dis. 15 (11), 817(2024).</li>
<li>Dawson, L. P., Layland, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-risk+coronary+plaque+features%3A+a+narrative+review%5BTitle%5D)+AND+%22Cardiol.+Ther%22%5BJournal%5D)">High-risk coronary plaque features: a narrative review.</a> Cardiol. Ther. 11 (3), 319-335 (2022).</li>
<li>Tong, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+sensitive+magnetic+particle+imaging+of+vulnerable+atherosclerotic+plaque+with+active+myeloperoxidase-targeted+nanoparticles%5BTitle%5D)+AND+%22Theranostics%22%5BJournal%5D)">Highly sensitive magnetic particle imaging of vulnerable atherosclerotic plaque with active myeloperoxidase-targeted nanoparticles.</a> Theranostics. 12 (17), 7641-7641 (2022).</li>
<li>Lu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coronary+computed+tomography+angiography+assessment+of+high-risk+plaques+in+predicting+acute+coronary+syndrome%5BTitle%5D)+AND+%22Front.+Cardiovasc.+Med%22%5BJournal%5D)">Coronary computed tomography angiography assessment of high-risk plaques in predicting acute coronary syndrome.</a> Front. Cardiovasc. Med. 8, (2021).</li>
<li>Kinoshita, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-risk+plaques+on+coronary+computed+tomography+angiography%5BTitle%5D)+AND+%22JACC+Cardiovasc.+Imaging%22%5BJournal%5D)">High-risk plaques on coronary computed tomography angiography.</a> JACC Cardiovasc. Imaging. 17 (4), 382-391 (2024).</li>
<li>Canu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-invasive+multimodality+imaging+of+coronary+vulnerable+patient%5BTitle%5D)+AND+%22Front.+Cardiovasc.+Med%22%5BJournal%5D)">Non-invasive multimodality imaging of coronary vulnerable patient.</a> Front. Cardiovasc. Med. 9, (2022).</li>
<li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+coronary+computed+tomography+angiography-derived+plaque+features+in+the+diagnosis+of+optical+coherence+tomography-defined+vulnerable+plaques%5BTitle%5D)+AND+%22Quant.+Imaging+Med.+Surg%22%5BJournal%5D)">Assessment of coronary computed tomography angiography-derived plaque features in the diagnosis of optical coherence tomography-defined vulnerable plaques.</a> Quant. Imaging Med. Surg. 15 (3), 2029-2041 (2025).</li>
<li>Chiorescu, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vulnerable+atherosclerotic+plaque%3A+is+there+a+molecular+signature%5BTitle%5D)+AND+%22Int.+J.+Mol.+Sci%22%5BJournal%5D)">Vulnerable atherosclerotic plaque: is there a molecular signature.</a> Int. J. Mol. Sci. 23 (21), 13638(2022).</li>
<li>The applications of single-cell RNA sequencing in atherosclerotic disease. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Front.+Cardiovasc.+Med%22">Front. Cardiovasc. Med</a>. Slenders, L., Tessels, D. E., van der Laan, S. W., Pasterkamp, G., Mokry, M. 9, (2022).</li>
<li>Adam, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+biomarkers+of+atherosclerotic+vascular+disease-latest+insights+in+the+research+field%5BTitle%5D)+AND+%22Int.+J.+Mol.+Sci%22%5BJournal%5D)">Novel biomarkers of atherosclerotic vascular disease-latest insights in the research field.</a> Int. J. Mol. Sci. 23 (9), 4998(2022).</li>
<li>Ye, W., Chen, Z., Xia, Z., Li, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+RNA+sequencing+in+human+atherosclerotic+plaques+reveals+a+novel+smooth+muscle+cell+subtype+that+possesses+multi+differentiation+potential+and+shapes+the+microenvironment%5BTitle%5D)+AND+%22Clin.+Exp.+Med%22%5BJournal%5D)">Single-cell RNA sequencing in human atherosclerotic plaques reveals a novel smooth muscle cell subtype that possesses multi differentiation potential and shapes the microenvironment.</a> Clin. Exp. Med. 25 (1), 251(2025).</li>
<li>Kelly, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coronary+endarterectomy%3A+analysis+of+the+society+of+thoracic+surgeons+adult+cardiac+surgery+database%5BTitle%5D)+AND+%22Ann.+Thorac.+Surg%22%5BJournal%5D)">Coronary endarterectomy: analysis of the society of thoracic surgeons adult cardiac surgery database.</a> Ann. Thorac. Surg. 114 (3), 667-674 (2022).</li>
<li>Ghatanatti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coronary+endarterectomy%3A+recent+trends%5BTitle%5D)+AND+%22J.+Clin.+DIAGNOSTIC+Res%22%5BJournal%5D)">Coronary endarterectomy: recent trends.</a> J. Clin. DIAGNOSTIC Res. , (2017).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+transcriptome+analysis+reveals+dynamic+cell+populations+and+differential+gene+expression+patterns+in+control+and+aneurysmal+human+aortic+tissue%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Single-cell transcriptome analysis reveals dynamic cell populations and differential gene expression patterns in control and aneurysmal human aortic tissue.</a> Circulation. 142 (14), 1374-1388 (2020).</li>
<li>Alsaigh, T., Evans, D., Frankel, D., Torkamani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decoding+the+transcriptome+of+calcified+atherosclerotic+plaque+at+single-cell+resolution%5BTitle%5D)+AND+%22Commun.+Biol%22%5BJournal%5D)">Decoding the transcriptome of calcified atherosclerotic plaque at single-cell resolution.</a> Commun. Biol. 5 (1), 1084(2022).</li>
<li>Iqbal, F., Lupieri, A., Aikawa, M., Aikawa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Harnessing+single-cell+RNA+sequencing+to+better+understand+how+diseased+cells+behave+the+way+they+do+in+cardiovascular+disease%5BTitle%5D)+AND+%22Arterioscler.+Thromb.+Vasc.+Biol%22%5BJournal%5D)">Harnessing single-cell RNA sequencing to better understand how diseased cells behave the way they do in cardiovascular disease.</a> Arterioscler. Thromb. Vasc. Biol. 41 (2), 585-600 (2021).</li>
<li>Ahmad, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+cell+RNA+sequencing+of+haematopoietic+cells+in+fresh+and+frozen+human+atheroma+tissue%5BTitle%5D)+AND+%22Cardiovasc.+Res%22%5BJournal%5D)">Single cell RNA sequencing of haematopoietic cells in fresh and frozen human atheroma tissue.</a> Cardiovasc. Res. 121 (3), 396-404 (2025).</li>
<li>Depuydt, M. A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microanatomy+of+the+human+atherosclerotic+plaque+by+single-cell+transcriptomics%5BTitle%5D)+AND+%22Circ.+Res%22%5BJournal%5D)">Microanatomy of the human atherosclerotic plaque by single-cell transcriptomics.</a> Circ. Res. 127 (11), 1437-1455 (2020).</li>
<li>Williams, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+cell+RNA+sequencing+in+atherosclerosis+research%5BTitle%5D)+AND+%22Circ.+Res%22%5BJournal%5D)">Single cell RNA sequencing in atherosclerosis research.</a> Circ. Res. 126 (9), 1112-1126 (2020).</li>
<li>Kim, N., Kang, H., Jo, A., Yoo, S. -A., Lee, H. -O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perspectives+on+single-nucleus+RNA+sequencing+in+different+cell+types+and+tissues%5BTitle%5D)+AND+%22J.+Pathol.+Transl.+Med%22%5BJournal%5D)">Perspectives on single-nucleus RNA sequencing in different cell types and tissues.</a> J. Pathol. Transl. Med. 57 (1), 52-59 (2023).</li>
<li>Gruenbaum, Y., Foisner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lamins%3A+Nuclear+intermediate+filament+proteins+with+fundamental+functions+in+nuclear+mechanics+and+genome+regulation%5BTitle%5D)+AND+%22Annu.+Rev.+Biochem%22%5BJournal%5D)">Lamins: Nuclear intermediate filament proteins with fundamental functions in nuclear mechanics and genome regulation.</a> Annu. Rev. Biochem. 84 (1), 131-164 (2015).</li>
<li>Zwerger, M., Ho, C. Y., Lammerding, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+mechanics+in+disease%5BTitle%5D)+AND+%22Annu.+Rev.+Biomed.+Eng%22%5BJournal%5D)">Nuclear mechanics in disease.</a> Annu. Rev. Biomed. Eng. 13 (1), 397-428 (2011).</li>
<li>Dechat, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+lamins%3A+major+factors+in+the+structural+organization+and+function+of+the+nucleus+and+chromatin%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin.</a> Genes Dev. 22 (7), 832-853 (2008).</li>
<li>Oh, J. -M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+cell+type+distribution+between+single-cell+and+single-nucleus+RNA+sequencing%3A+enrichment+of+adherent+cell+types+in+single-nucleus+RNA+sequencing%5BTitle%5D)+AND+%22Exp.+Mol.+Med%22%5BJournal%5D)">Comparison of cell type distribution between single-cell and single-nucleus RNA sequencing: enrichment of adherent cell types in single-nucleus RNA sequencing.</a> Exp. Mol. Med. 54 (12), 2128-2134 (2022).</li>
<li>Nott, A., Schlachetzki, J. C. M., Fixsen, B. R., Glass, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclei+isolation+of+multiple+brain+cell+types+for+omics+interrogation%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Nuclei isolation of multiple brain cell types for omics interrogation.</a> Nat. Protoc. 16 (3), 1629-1646 (2021).</li>
<li>Gulko, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+flow+cytometry-assisted+single-nucleus+RNA+sequencing+of+human+and+mouse+adipose+tissue+with+sample+multiplexing%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing.</a> STAR Protoc. 5 (1), 102893(2024).</li>
<li>Fernandez, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+immune+landscape+of+human+atherosclerotic+plaques%5BTitle%5D)+AND+%22Nat.+Med%22%5BJournal%5D)">Single-cell immune landscape of human atherosclerotic plaques.</a> Nat. Med. 25 (10), 1576-1588 (2019).</li>
<li>Galvis, A. E., Fisher, H., Camerini, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NP-40+fractionation+and+nucleic+acid+extraction+in+mammalian+cells%5BTitle%5D)+AND+%22BIO-PROTOCOL%22%5BJournal%5D)">NP-40 fractionation and nucleic acid extraction in mammalian cells.</a> BIO-PROTOCOL. 7 (20), (2017).</li>
<li>Wirka, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atheroprotective+roles+of+smooth+muscle+cell+phenotypic+modulation+and+the+TCF21+disease+gene+as+revealed+by+single-cell+analysis%5BTitle%5D)+AND+%22Nat.+Med%22%5BJournal%5D)">Atheroprotective roles of smooth muscle cell phenotypic modulation and the TCF21 disease gene as revealed by single-cell analysis.</a> Nat. Med. 25 (8), 1280-1289 (2019).</li>
<li>Pan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+genomics+reveals+a+novel+cell+state+during+smooth+muscle+cell+phenotypic+switching+and+potential+therapeutic+targets+for+atherosclerosis+in+mouse+and+human%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Single-cell genomics reveals a novel cell state during smooth muscle cell phenotypic switching and potential therapeutic targets for atherosclerosis in mouse and human.</a> Circulation. 142 (21), 2060-2075 (2020).</li>
<li>Bakken, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-nucleus+and+single-cell+transcriptomes+compared+in+matched+cortical+cell+types%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Single-nucleus and single-cell transcriptomes compared in matched cortical cell types.</a> PLoS One. 13 (12), e0209648(2018).</li>
<li>Litviňuková, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cells+of+the+adult+human+heart%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cells of the adult human heart.</a> Nature. 588 (7838), 466-472 (2020).</li>
<li>Hu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-nucleus+transcriptomic+survey+of+cell+diversity+and+functional+maturation+in+postnatal+mammalian+hearts%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">Single-nucleus transcriptomic survey of cell diversity and functional maturation in postnatal mammalian hearts.</a> Genes Dev. 32 (19-20), 1344-1357 (2018).</li>
<li>Rani, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+use+of+RPMI+Medium+to+preserve+cell+morphology+for+pleural%2Fperitoneal+fluid+cytology%5BTitle%5D)+AND+%22J.+Cytol%22%5BJournal%5D)">Evaluation of use of RPMI Medium to preserve cell morphology for pleural/peritoneal fluid cytology.</a> J. Cytol. 39 (1), 26-29 (2022).</li>
<li>Machado, L., Relaix, F., Mourikis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stress+relief%3A+emerging+methods+to+mitigate+dissociation-induced+artefacts%5BTitle%5D)+AND+%22Trends+Cell+Biol%22%5BJournal%5D)">Stress relief: emerging methods to mitigate dissociation-induced artefacts.</a> Trends Cell Biol. 31 (11), 888-897 (2021).</li>
<li>Slyper, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single-cell+and+single-nucleus+RNA-seq+toolbox+for+fresh+and+frozen+human+tumors%5BTitle%5D)+AND+%22Nat.+Med%22%5BJournal%5D)">A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors.</a> Nat. Med. 26 (5), 792-802 (2020).</li>
<li>Krishnaswami, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+single+nuclei+for+RNA-seq+to+capture+the+transcriptome+of+postmortem+neurons%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons.</a> Nat. Protoc. 11 (3), 499-524 (2016).</li>
</ol>

The authors have no conflict of interest to disclose.

An optimized workflow for the isolation of nuclei from human coronary endarterectomy tissue is described in the current protocol. The nuclear isolation protocol described here was specifically designed to overcome the critical limitations of obtaining viable, intact cells from advanced coronary atherosclerotic plaques. Unlike conventional scRNA-seq approaches that rely on harsh enzymatic digestion and mechanical dissociation21,30, our optimized protocol utilizes ...

Atherosclerotic cardiovascular disease (ASCVD) is the world&#39;s leading cause of death and disability, and despite major therapeutic advances, its burden remains undiminished1. Atherosclerosis is a chronic inflammatory disease of the arterial wall, driven by complex and dynamic interactions among diverse cell populations that collectively orchestrate the silent progression from early subclinical disease to life-altering cardiovascular (CV) events2.
In medium and large arteries, the buildup of cholesterol deposits within macrophages is a hallmark of atherosclerosis3. The atherosclerotic plaque consists of fatty acids, cholesterol, inflammatory cells, calcium, fibrin, smooth muscle cells, endothelial cells, and cellular debris within the subendothelium4. Atherosclerotic plaques are classified into two types: low-risk and high-risk. High-risk plaques are identified by their tendency to trigger a subsequent cardiovascular event, typically through mechanisms such as plaque erosion or rupture5.
The characteristics of high-risk plaques include large lipid cores, several inflammatory cells, fewer smooth muscle cells, a thin fibrous cap, microcalcification, intraplaque haemorrhage, neo vessels, and a necrotic core6. The detection of high-risk plaques is clinically performed using non-invasive and invasive imaging modalities, namely, coronary computed tomography angiography (CCTA) and optical coherence tomography (OCT), respectively7,8. Despite numerous advancements in cardiovascular imaging, it is still challenging to predict the rupture of atherosclerotic plaque that causes myocardial infarction (MI), and it is not always useful in clinical settings9.
Despite decades of research, we still lack reliable circulating biomarkers that can specifically identify high-risk plaques phenotypes, leaving a critical gap in our clinical management of atherosclerosis. This underscores the need for molecular strategies that can characterize these plaques at a more fundamental biological level7,10. In this context, RNA-based markers have gained considerable attention, as they can capture the intricate transcriptional activity occurring within the plaque microenvironment11. Recent developments in single-cell RNA sequencing (scRNA-seq) have proven particularly powerful, offering an unprecedented resolution to profile individual cells within atherosclerotic plaques and uncover distinct cell populations, gene expression patterns, and molecular pathways that underlie plaque vulnerability12,13. Despite these developments, little knowledge exists on the regulatory processes, interactions, and functional importance of the various cell types implicated in atherosclerosis development14.
When atherosclerotic burden is severe and diffuse, surgical revascularization becomes the definitive treatment strategy. Coronary endarterectomy (CE) is a surgical adjunct to coronary artery bypass graft (CABG) surgery in which atherosclerotic plaque is mechanically removed from diffusely diseased coronary arteries to enable bypass grafting and achieve complete myocardial revascularization15,16. Atherosclerotic plaques and endarterectomy specimens are particularly challenging to work with because they contain abundant necrotic tissue, lipid deposits, fibrin, and calcifications. When these tissues are broken down for analysis, they generate substantial debris that clogs filters and microfluidic systems, making the isolation of viable single cells extremely difficult17,18. In fact, one carotid plaque study found that viable cells represented less than 1% of all particles after tissue processing; the rest was simply plaque debris18.
Further compounding this challenge, atherosclerotic plaques exist in a state of chronic metabolic and oxidative stress, where cells undergoing apoptosis, senescence, and other degenerative processes render cell membranes increasingly fragile and susceptible to disruption, making the isolation of viable single cells extremely difficult18,19,20. The mechanical and enzymatic digestion required to prepare samples for scRNA-seq often damages these already compromised cells, further reducing viability and resulting in poor cell capture rates. This process also tends to selectively lose fragile or rare cell populations, which introduces significant bias into both the transcriptomic data and the cell types represented in the final results. This problem becomes even more pronounced in calcified or heavily diseased plaques, where the harsh processing conditions needed to break down the tissue cause additional damage to cell viability18,21,22.
In contrast, the nucleus remains structurally intact even under these adverse conditions23. This is primarily due to the nuclear envelope, a double lipid bilayer reinforced by nuclear lamins (Lamin A/C and Lamin B), which provides mechanical rigidity and resistance to the osmotic, enzymatic, and physical stresses that readily disrupt the plasma membrane24,25. The nuclear lamina also preserves nuclear morphology even when the surrounding cytoplasm is severely compromised26. This makes single-nucleus RNA sequencing (snRNA-seq) a more practical and reliable alternative to single-cell RNA sequencing (scRNA-seq) for profiling atherosclerotic tissue27. By capturing transcriptional information from intact nuclei rather than whole cells, snRNA-seq circumvents the limitations imposed by poor cell viability and high debris content, enabling more accurate and comprehensive transcriptomic profiling of the plaque microenvironment18.
Therefore, in this study, we present an optimized nuclei isolation protocol specifically developed for coronary endarterectomy tissue, incorporating non-ionic detergent-based lysis and empirically adjusted lysis duration based on the degree of tissue calcification, to consistently yield high-quality nuclei with preserved structural and RNA integrity suitable for downstream snRNA-seq analysis.

<table><tbody><tr><td>100 &amp;mu;m strainer</td><td>Miltenyi Biotech&amp;nbsp;</td><td>130-098-463</td><td></td></tr><tr><td>30 &amp;mu;m strainer</td><td>Miltenyi Biotech&amp;nbsp;</td><td>130-098-458</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>HiMedia</td><td>TC546-25G</td><td></td></tr><tr><td>Centrifuge</td><td>MPW</td><td>NA</td><td>https://mpw.pl/en/offer/mpw-352r</td></tr><tr><td>Compound Microscope</td><td>Nikon</td><td>NA&amp;nbsp;</td><td>Nikon Eclipse Ts2 inverted microscope (https://www.microscope.healthcare.nikon.com/en_AOM/products/inverted-microscopes/eclipse-ts2)</td></tr><tr><td>Confocal Microscope&amp;nbsp;</td><td>Nikon</td><td>NA</td><td>Nikon Ti2-E inverted confocal microscope (https://www.microscope.healthcare.nikon.com/en_AOM/products/inverted-microscopes/eclipse-ti2-series)</td></tr><tr><td>Coverslips</td><td>HiMedia</td><td>BG011C</td><td></td></tr><tr><td>CRYOCHIL Wide Mouth Specimen Vial</td><td>Tarsons</td><td>883191</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542-5MG</td><td></td></tr><tr><td>DPBS</td><td>Gibco (Thermofischer)</td><td>10010-023</td><td></td></tr><tr><td>Centrifuge Tube (15 mL)</td><td>Tarsons</td><td>546021</td><td></td></tr><tr><td>Flow cytometer&amp;nbsp;</td><td>BD Biosciences</td><td>663029</td><td>BD FACSLyric (3L12C Instrument CEIVD)</td></tr><tr><td>Hemocytometer</td><td>Axibio</td><td>CCB 100</td><td></td></tr><tr><td>Microcentrifuge Tube (1.5 mL)</td><td>Tarsons</td><td>500010C</td><td></td></tr><tr><td>Microcentrifuge Tube (5 mL)</td><td>Tarsons</td><td>500050</td><td></td></tr><tr><td>MgCl2</td><td>Sigma</td><td>M1028-100ML</td><td></td></tr><tr><td>Micro pestle</td><td>Tarsons</td><td>160020</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888-500G</td><td></td></tr><tr><td>NP-40</td><td>HiMedia</td><td>MB143-100ML</td><td></td></tr><tr><td>Nuclease free water</td><td>Gibco (Thermofischer)</td><td>W4502-1L</td><td></td></tr><tr><td>RNase inhibitor</td><td>Sigma</td><td>333540201</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco (Thermofischer)</td><td>11875093</td><td></td></tr><tr><td>Slide&amp;nbsp;</td><td>Riviera</td><td>72910135</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma</td><td>T2194-100ML</td><td></td></tr><tr><td>Trypan blue&amp;nbsp;</td><td>Sigma</td><td>302643-25G</td><td></td></tr></tbody></table>

single nuclei isolation from coronary endarterectomy tissue of coronary artery bypass graft patients

Single Nuclei Isolation from Coronary Endarterectomy Tissue of Coronary Artery Bypass Graft Patients

Atherosclerosis is a chronic, progressive inflammatory condition characterized by the accumulation of plaque in the walls of arteries. During the progression of plaque formation, certain lesions become unstable, referred to as high-risk plaques, and have the potential to cause myocardial infarction (MI). An atherosclerotic plaque comprises smooth muscle cells, endothelial cells, immune cells, fat cells, and necrotic cells, any of which can cause plaque rupture. It is crucial to study cellular heterogeneity within atherosclerotic plaques to better understand the mechanisms underlying plaque instability. We present an easily reproducible protocol for isolating and validating nuclei from human coronary endarterectomy tissue obtained from patients undergoing coronary artery bypass graft (CABG) surgery. All tissues are kept on wet ice in Roswell Park Memorial Institute (RPMI) medium to preserve cellular integrity before harvesting. The tissue samples are finely minced and then gently disrupted with a micro pestle in lysis buffer to lyse cells and release nuclei. The isolated nuclei are then assessed for integrity and yield using the trypan blue exclusion assay in a compound inverted microscope. DAPI staining enables visualization of intact nuclei by confocal microscopy, and flow cytometry provides fluorescence-based quantification to assess nuclear isolation yield and validate the nuclear population. In contrast to traditional methods of isolation via single-cell dissociation, nuclear isolation reduces cellular stress and maintains the native transcriptomic environment, thereby preventing changes in gene expression. The high-quality nuclear preparation obtained through this method is compatible with downstream applications such as single-nuclei RNA sequencing (snRNA-seq).

The human coronary endarterectomy tissue was collected from patients undergoing CABG surgery. The tissue was weighed and processed for nuclei isolation, and further isolated nuclei were confirmed by flow cytometry and confocal microscopy (Figure 1).
Trypan blue assessment of nuclei isolation quality 
The tissue kept in RPMI medium was weighed, and it measured 247.8 mg. The tissue was minced, then homogenized in lysis buffer using a micropestle ...

Coronary endarterectomy tissue samples were processed to isolate single nuclei using an optimized dissociation protocol. The integrity of the isolated nuclei was validated through three complementary approaches: trypan blue exclusion staining to assess membrane integrity, DAPI-based confocal microscopy to confirm nuclear morphology, and flow cytometry to quantify nuclear yield and purity.

Atherosclerosis is a chronic, progressive inflammatory condition characterized by the accumulation of plaque in the walls of arteries. During the ...

Our major research focus is to map the cellular diversity in unstable atherosclerotic plaques to understand cell-to-cell interactions, and then develop RNA-based prognostic markers. Membrane fragility hinders single cell isolation from the tissue obtained during endarterectomy. Therefore, this protocol involves isolating of preserved nuclei, enabling transcriptomic profiling of high-risk plaques.To begin, gather all the reagents and icebox to perform the procedure. Wash the tissue once or twice with serum-free RPMI medium to remove bloodstains and fragments. Using sterile forceps, transfer the tissue onto a sterile Petri dish and record the weight.Using a surgical blade number 10, mince the tissue into small pieces of approximately one cubic millimeter in size. Now add one milliliter chilled lysis buffer, and the tissue to a 1.5 milliliter micro centrifuge tube. Lice the tissue on ice using a micro pestle by applying a gentle, consistent force and swirling the micro pestle clockwise and anti-clockwise alternately.Continue lysis for approximately 30 minutes while keeping the sample on ice. Filter the solution through a 100 micrometer pore size strainer into a 15 milliliter centrifug

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Learn a protocol for isolating intact nuclei from human coronary endarterectomy tissue to enable single-nuclei RNA sequencing. This method preserves nuclear integrity for transcriptomic profiling of high-risk atherosclerotic plaques.

Single Nuclei Isolation from Coronary Endarterectomy Tissue of Coronary Artery Bypass Graft Patients

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