This work was supported by an American Heart Association Fellowship Award (16POST29950007) to FL, a training grant from the National Institutes of Health (NIH T32 HL069765) to BLD, an American Heart Association Fellowship Award (14POST20420025) to MA Saleh, and an NIH K08 award (HL121671) to MSM. MSM is also supported by a research grant from Gilead Sciences, Inc.

Vanderbilt University&#39;s Institutional Animal Care and Use Committee has approved the procedures described herein. Mice are housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (National Academies Press. Revised 2010).
1. Isolation of the Aortic Draining Lymph Nodes, Kidney and Aorta from Mice
<ol>
	<li>Euthanize the mice by CO2 inhalation. Spray the chest with 70% ethanol and carefully open the skin and the chest wall with scissors to expose the heart.</li>
	<li>To perfuse the vasculature, perform a small incision in the right atrium and steadily inject at least 10 ml of cold PBS (approximately 1 ml/sec) into the apex of the left ventricle using a 21 or 23 G needle. Perfuse until all organs have blanched. The heart blanches first. Blanching of the liver indicates that the perfusion has been well performed.</li>
	<li>Using small forceps and fine scissors, gently cut away and pull out the intestines, stomach, spleen, pancreas and liver to better visualize the aorta. 
	NOTE: This step has to be done very precisely as damage to the gastrointestinal tract can induce contamination.</li>
	<li>Cut out and remove each lung with scissors. Rinse the chest cavity with phosphate buffered saline (PBS) using a needleless syringe. Remove excess blood and fluid using sterile gauze.</li>
	<li>Remove the abdominal aortic draining lymph nodes using fine dissecting forceps. Make sure not to rupture the capsule. Remove the remaining fat on the surface of each lymph node with the dissecting forceps. Cut out the two kidneys with fine scissors.</li>
	<li>Dissect the entire aorta (thoracic and abdominal aorta) with fine, curved scissors. Start at the level of the heart and carefully dissect away from the esophagus and the vertebrae down to the level of the iliac bifurcation making sure to keep the surrounding perivascular fat attached to the aorta. 
	NOTE: Further dissection of the aorta to remove surrounding structures can be done under a microscope. Specifically, remove arterial branches (carotid arteries, subclavian arteries, celiac, mesenteric, and renal arteries) and lymph nodes. Do keep a consistent layer of perivascular fat around each aorta since this is a site where many inflammatory cells reside in the setting of hypertension. Place each tissue in separate tubes containing cold PBS.</li>
</ol>
2. Generation of Single Cell Suspensions from Each Tissue
<ol>
	<li>The kidneys 
	NOTE: The kidney can be dissociated into a single cell suspension by combining mechanical dissociation plus enzymatic digestion.
	<ol>
		<li>Prepare the kidney digestion solution by adding collagenase D (2 mg/ml) and DNase I (100 &#181;g/ml) to RPMI 1640 medium (supplemented with 5% fetal bovine serum (FBS)). Prepare 10 ml per kidney sample.</li>
		<li>Use forceps to transfer the two kidneys into a dissociation tube containing 10 ml of kidney digestion solution.</li>
		<li>Perform the mechanical dissociation using a semi-automated dissociator device according to the manufacturer&#39;s instructions.</li>
		<li>After mechanical dissociation, detach the tube from the device and perform the enzymatic digestion. Incubate the samples for 20 min at 37 &#186;C under continuous rotation. Immediately stop the enzymatic digestion by adding cold RPMI 1640 medium supplemented with 5% FBS.</li>
		<li>Transfer the solution by pipetting into a 50 ml conical tube after filtering through a 40 &#181;m strainer. Tease the remaining tissue apart by pressing with the plunger of a 1 ml syringe. Pellet the cells (500 x g, 8 min, 4 &#186;C).</li>
		<li>Resuspend the pellet into 3 ml of 36% density gradient centrifugation media. Transfer the suspension into a 15 ml conical tube. Gently add 3 ml of 72% density gradient centrifugation media beneath the 3 ml 36% cell suspension without any disruption. Centrifuge (1,000 x g without brake, 20 min, 4 &#176;C).</li>
		<li>The immune cells will be located at the interface. Remove the topmost yellowish layer containing cellular debris and then bring the volume up to 15 ml using cold PBS. Shake well to breakdown the gradient. Spin the cells (300 x g, 8 min, 4 &#176;C) and resuspend the pellet in 3 ml of RPMI with 5% FBS. Count the number of live cells on a hemocytometer using Trypan blue exclusion.</li>
	</ol>
	</li>
	<li>The aorta
	<ol>
		<li>Prepare the aorta digestion solution by adding collagenase A (1 mg/ml), collagenase B (1 mg/ml), and DNase I (100 &#181;g/ml) to RPMI 1640 medium (supplemented with 5% FBS).</li>
		<li>Using fine forceps, transfer the aorta into a 2 ml tube containing 1 ml of aorta digestion solution. Mince the whole aorta into very small pieces with fine scissors in the 1 ml digestion solution. Keep on ice. Incubate the samples for 30 min at 37 &#176;C under continuous rotation.</li>
		<li>Transfer the solution to a tissue culture dish and stop the enzymatic digestion by adding 5 times the volume of cold RPMI with 5% FBS.</li>
		<li>Transfer the solution by pipetting into a 50 ml conical tube after filtering through a 40 &#181;m strainer. Tease the remaining tissue apart into a single cell suspension by pressing with the plunger of a 1 ml syringe. Flush the strainer and pellet the cells (300 x g, 8 min, 4 &#186;C).</li>
		<li>Wash the pellet by adding 10 ml of RPMI with 5% FBS. Spin the cells (300 x g, 8 min, 4 &#176;C) and resuspend the pellet in 3 ml of RPMI with 5% FBS. Count the number of live cells on a hemocytometer using Trypan blue exclusion.</li>
	</ol>
	</li>
	<li>The aortic draining lymph nodes
	<ol>
		<li>Place a 40 &#181;m strainer in a tissue culture dish (60 mm x 15 mm). Place the lymph nodes on the strainer and tease them apart into a single cell suspension by pressing with the plunger of a 1 ml syringe. Flush the strainer with 2 ml of RPMI with 5% FBS. Collect the cells in the dish.</li>
		<li>Transfer the cells into a 15 ml falcon tube and pellet the cells (300 x g, 8 min, 4 &#186;C). Resuspend the pellet in 500 &#181;l of RPMI with 5% FBS. Count the number of live cells on a hemocytometer using Trypan blue exclusion.</li>
	</ol>
	</li>
</ol>
3. Ex Vivo Stimulation for Cytokine Detection by Flow Cytometry
<ol>
	<li>Prepare the cell culture media: RPMI with 5% FBS, 0.1 mM of non-essential amino acids, 50 &#181;M &#946;-mercaptoethanol and 1% penicillin/streptomycin.</li>
	<li>Centrifuge each cell suspension obtained from the kidney, aorta and lymph nodes (300 x g, 8 min, 4 &#186;C). Resuspend each pellet with the cell culture media at a concentration of 1 x 106 cells per ml.</li>
	<li>Stimulate cells by adding 2 &#181;l of the cell activation cocktail (containing Phorbol 12-Myristate 13-Acetate (PMA), the calcium ionophore ionomycin, and the Golgi inhibitor Brefeldin A) for every 1 ml of cell culture. 
	NOTE: The final concentrations of each component are: 81 nM of PMA, 1.33 &#181;M of ionomycin and 5 &#181;g/ml of Brefeldin A. Treatment with PMA and ionomycin activates cells to produce cytokines. Cytokines are secreted proteins that must be trapped in the cells to be detected. Brefeldin A causes the accumulation of normally secreted proteins in the endoplasmic reticulum and Golgi apparatus. Thus, the technique described here enhances the detectability of cytokine-producing lymphocytes by flow cytometry.</li>
	<li>Plate 1 to 2 million cells in a 12-well (aorta or kidney) or 24-well (lymph node) low-adherence plates. Place the plate in a 37 &#176;C humidified CO2 incubator for 3 hr. 
	NOTE: The number of plated cells may be lower in lymph nodes from control mice. Also, the time of stimulation has to be empirically determined for each cell population and cytokine studied. In the case of T cells isolated from aorta, kidney or lymph nodes, we recommend stimulating for 3 to 4 hr. A longer stimulation will not increase the cytokine secretion and will induce a further downregulation of several T cell surface markers like CD3, CD4 and CD8. (Note that even a 3-4 hr stimulation will induce some downregulation of these markers.)</li>
</ol>
4. Surface Staining
<ol>
	<li>Transfer the cells into a polystyrene FACS tube and centrifuge (300 x g, 8 min, 4 &#176;C). Wash the cells twice with FACS buffer (Ca2+ and Mg+ free PBS containing 4% FBS and 2mM EDTA) and divide the cell suspension equally into different FACS tubes based on the number of antibody panels desired and the number of control tubes needed (see below).</li>
	<li>To perform compensation for each panel, prepare unstained control tubes and single stained tubes for each tissue type and antibody panel. In addition, prepare fluorescence minus one (FMO) control tubes for each antibody panel by adding all but one of the antibodies.</li>
	<li>Adjust the volume of each tube to 2 ml with FACS Buffer. Centrifuge the cell suspension (300 x g, 8 min, 4 &#176;C), remove the supernatant and block Fc receptors by incubating the cells with 0.5 &#181;g of anti-CD16/CD32 antibodies per million cells for 10 min on ice. 
	NOTE: CD16 is low affinity IgG Fc receptor III (FcR III) and CD32 is FcR II. CD16/CD32 are expressed on granulocytes, mast cells, monocytes/macrophages, natural killer cells, B cells, dendritic cells and some activated mature T cells.</li>
	<li>Perform viability staining: Wash the cells with 1x PBS, centrifuge (300 x g, 8 min, 4 &#176;C) and resuspend in 1,000 &#181;l of 1x PBS. Add 1 &#181;l of a cell-impermeant amine-reactive dye (viability stain). Incubate for 15 min at 4 &#176;C protected from light.</li>
	<li>During the incubation, prepare the cocktail of antibodies (for surface staining) in an appropriate volume of FACS buffer. Wash the cells twice with 1-2 ml of FACS buffer, centrifuge (300 x g, 8 min, 4 &#176;C) and resuspend each pellet with 100 &#181;l of the antibody mixture. Incubate for 30 min at 4 &#176;C protected from light. 
	NOTE: For each flow cytometry antibody, it is useful to determine the optimal amount of antibody needed by performing a dose titration curve.</li>
	<li>Wash the cells twice with 2 ml of FACS buffer and pellet the cells (300 x g, 8 min, 4 &#176;C).</li>
</ol>
5. Fixation, Permeabilization and Intracellular Staining
<ol>
	<li>Perform the intracellular staining with a fixation and permeabilization kit containing appropriate buffers by following the manufacturer&#39;s instructions. Fix and permeabilize the cells by resuspending them in the appropriate buffer. Incubate samples for 40 min at 4 &#176;C protected from light.</li>
	<li>Add 1 ml of 1x permeabilization and wash buffer directly to the fixed and permeabilized cells. Centrifuge (500 x g, 8 min, 4 &#176;C) and remove the supernatant. Resuspend the pellet with 2 ml of 1x wash buffer.</li>
	<li>Prepare the mixtures of antibodies (for intracellular staining only) in an appropriate volume of 1x wash buffer (usually 100 &#181;l per tube). 
	NOTE: As described above, the optimal antibody concentration needs to be determined for each antibody in the panel. For example, for antibodies to IL-17A or IL-17F, we use a dilution factor of 1:30, but for antibodies to IFN&#947; and T-bet, we use a dilution factor of 1:100.</li>
	<li>Centrifuge the cells (500 x g, 8 min, 4 &#176;C) and resuspend each pellet with 100 &#181;l of the antibody mixture. Incubate for 40 min at 4 &#176;C protected from light.</li>
	<li>Wash the cells twice with 2 ml of 1x wash buffer and pellet the cells (500 x g, 8 min, 4 &#176;C). Resuspend the pellet with 300 &#181;l of 1x wash buffer and analyze on a flow cytometer with appropriate filters.</li>
	<li>For quantification of the total number of cells per tissue sample, use counting beads. Prior to acquisition, add the recommended volume/number of counting beads to each tube.</li>
</ol>
6. Compensation, Gating, Normalization, and Tips
<ol>
	<li>Compensation and gating
	<ol>
		<li>Run unstained and single stained control tubes for compensation to adjust for spectral overlap. 
		NOTE: The use of compensation beads rather than cell-based compensation controls is necessary in cases where the antigen of interest or cell population in a sample is present at such low levels that compensation using cells will be difficult. Also of note, when using tandem dyes, it is advisable to prepare compensations for each experiment since tandem dyes vary lot-to-lot and with each day.</li>
		<li>Run fluorescence minus one controls (FMOs) for all the fluorophores in the panel when starting a new multicolor experiment. 
		NOTE: The FMO controls are samples that contain all the antibodies in the panel except for one. These controls permit accurate discrimination of positive versus negative signals for the omitted antibody to properly adjust the gates. These controls are particularly important when the expression levels are low or when the separation of the target population is poor (for example, when the target is a cytokine or transcription factor). Although not always necessary, in some cases, isotype controls may be preferred in place of FMO controls as they provide proper compensation for the antibody isotype and fluorophore conjugate without the specificity of the antigen.</li>
		<li>Set up the primary gate: adjust Forward Scatter Area (FSC-A) and Side Scatter Area (SSC-A) voltage in order to detect the leukocyte population.</li>
		<li>Exclude the dead cells. 
		NOTE: The viability stain reacts with free amines present on both the surface and the interior of cells. In contrast to live cells, dead cells (with compromised membranes) allow the dye to enter into the cytoplasm, increasing the amount of protein labeling. Thus, dead cells will be brighter than live cells allowing easy discrimination. Gate on the live cells. 
		NOTE: The viability of the cell suspensions from the aorta and the kidney may be lower than the viability of the cell suspensions from the lymph nodes due to the additional digestion step.</li>
		<li>Plot Forward Scatter Area (FSC-A) [y-axis] and Forward Scatter Height (FSC-H) [x-axis]. Singlets appear as a diagonal on this dot plot. Gate on this diagonal to exclude doublets. Then plot SSC-A [y-axis] and CD45 [x-axis].</li>
		<li>The CD45+ leukocyte population can be easily identified. Gate on this population. Subsequent populations can be identified from this population.</li>
	</ol>
	</li>
	<li>Normalization of cell counts
	<ol>
		<li>Counting beads have characteristics that result in low FSC and high SSC. For example, if 50,000 beads are added into 300 &#181;l of the single cell suspension, the equation for the calculation of the absolute number of any give cell type per tube is as follows: 
		&#160;&#160;&#160;&#160; Number of cells per tube = (50,000 / number of beads counted by the flow cytometer) x number of cells counted. 
		Using this calculation, the absolute number of any given cell type per tube can be determined. Based on how many tubes were generated from each organ, the number of cells per organ can be calculated. 
		NOTE: Keep all cells and reagents at 4 &#176;C during the protocol. Avoid vigorous vortexing because it can damage cells. Use minimal forces to pellet the cells. Avoid making bubbles in the FACS tube since it can precipitate cell death. Don&#39;t let the pellets dry out at any time.</li>
	</ol>
	</li>
</ol>

<ol><li>Saleh, M. A., McMaster, W. G., Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphocyte+adaptor+protein+LNK+deficiency+exacerbates+hypertension+and+end-organ+inflammation%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Lymphocyte adaptor protein LNK deficiency exacerbates hypertension and end-organ inflammation.</a> J Clin Invest. 125 (3), 1189-1202 (2015).</li>
<li>Madhur, M. S., Lob, H. E., McCann, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin+17+promotes+angiotensin+II-induced+hypertension+and+vascular+dysfunction%5BTitle%5D)+AND+%22Hypertension%22%5BJournal%5D)">Interleukin 17 promotes angiotensin II-induced hypertension and vascular dysfunction.</a> Hypertension. 55 (2), 500-507 (2010).</li>
<li>Laroumanie, F., Douin-Echinard, V., Pozzo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD4%2B+T+Cells+Promote+the+Transition+From+Hypertrophy+to+Heart+Failure+During+Chronic+Pressure+Overload%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">CD4+ T Cells Promote the Transition From Hypertrophy to Heart Failure During Chronic Pressure Overload.</a> Circulation. 129 (21), 2111-2124 (2014).</li>
<li>Guzik, T. J., Hoch, N. E., Brown, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+the+T+cell+in+the+genesis+of+angiotensin+II+induced+hypertension+and+vascular+dysfunction%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Role of the T cell in the genesis of angiotensin II induced hypertension and vascular dysfunction.</a> J Exp Med. 204 (10), 2449-2460 (2007).</li>
<li>Ketelhuth, D. F. J., Hansson, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adaptive+Response+of+T+and+B+Cells+in+Atherosclerosis%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Adaptive Response of T and B Cells in Atherosclerosis.</a> Circ Res. 118 (4), 668-678 (2016).</li>
<li>McMaster, W. G., Kirabo, A., Madhur, M. S., Harrison, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammation%2C+Immunity%2C+and+Hypertensive+End-Organ+Damage%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Inflammation, Immunity, and Hypertensive End-Organ Damage.</a> Circ Res. (6), 1022-1033 (2015).</li>
<li>Luckheeram, R. V., Zhou, R., Verma, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD4%2BT+Cells%3A+Differentiation+and+Functions%5BTitle%5D)+AND+%22Clin+Dev+Immunol%22%5BJournal%5D)">CD4+T Cells: Differentiation and Functions.</a> Clin Dev Immunol. 2012, 1-12 (2012).</li>
<li>Mittrücker, H. -W., Visekruna, A., Huber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterogeneity+in+the+differentiation+and+function+of+CD8%2B+T+cells%5BTitle%5D)+AND+%22Arch+Immunol+Ther+Exp+%28Warsz%29%22%5BJournal%5D)">Heterogeneity in the differentiation and function of CD8+ T cells.</a> Arch Immunol Ther Exp (Warsz). 62 (6), 449-458 (2014).</li>
<li>Autengruber, A., Gereke, M., Hansen, G., Hennig, C., Bruder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+enzymatic+tissue+disintegration+on+the+level+of+surface+molecule+expression+and+immune+cell+function%5BTitle%5D)+AND+%22Eur+J+Microbiol+Immunol+%28Bp%29%22%5BJournal%5D)">Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function.</a> Eur J Microbiol Immunol (Bp). 2 (2), 112-120 (2012).</li>
<li>Finak, G., Langweiler, M., Jaimes, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standardizing+Flow+Cytometry+Immunophenotyping+Analysis+from+the+Human+ImmunoPhenotyping+Consortium%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.</a> Sci Rep. 6, 20686(2016).</li>
<li>Herzenberg, L. A., Parks, D., Sahaf, B., Perez, O., Roederer, M., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+history+and+future+of+the+fluorescence+activated+cell+sorter+and+flow+cytometry%3A+a+view+from+Stanford%5BTitle%5D)+AND+%22Clin+Chem%22%5BJournal%5D)">The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford.</a> Clin Chem. 48 (10), 1819-1827 (2002).</li>
<li>Hrvatin, S., Deng, F., O'Donnell, C. W., Gifford, D. K., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MARIS%3A+method+for+analyzing+RNA+following+intracellular+sorting%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">MARIS: method for analyzing RNA following intracellular sorting.</a> PLoS One. 9 (3), 89459(2014).</li>
<li>Kalisky, T., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+genomics%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Single-cell genomics.</a> Nat Methods. 8 (4), 311-314 (2011).</li>
<li>Chattopadhyay, P. K., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytometry%3A+Today%27s+technology+and+tomorrow%27s+horizons%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Cytometry: Today's technology and tomorrow's horizons.</a> Methods. 57 (3), 251-258 (2012).</li>
<li>Jiang, L., Tixeira, R., Caruso, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+the+progression+of+cell+death+and+the+disassembly+of+dying+cells+by+flow+cytometry%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry.</a> Nat Protoc. 11 (4), 655-663 (2016).</li>
<li>Lemoine, S., Jaron, B., Tabka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dectin-1+activation+unlocks+IL12A+expression+and+reveals+the+TH1+potency+of+neonatal+dendritic+cells%5BTitle%5D)+AND+%22J+Allergy+Clin+Immunol%22%5BJournal%5D)">Dectin-1 activation unlocks IL12A expression and reveals the TH1 potency of neonatal dendritic cells.</a> J Allergy Clin Immunol. 136 (5), 1355-1368 (2015).</li>
<li>Gaublomme, J. T., Yosef, N., Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-Cell+Genomics+Unveils+Critical+Regulators+of+Th17+Cell+Pathogenicity%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Single-Cell Genomics Unveils Critical Regulators of Th17 Cell Pathogenicity.</a> Cell. 163 (6), 1400-1412 (2015).</li>
</ol>

MSM is supported by a grant from Gilead Cardiovascular Sciences.

The protocol described herein has been optimized to properly identify T cell subsets present within murine kidneys, aorta and lymph nodes. This protocol can be easily adapted to examine other immune cell subsets such as B lymphocytes and innate immune cells and can be modified to include other tissue types. The digestion step is critical and has to be modified and optimized for each tissue9. A prolonged digestion step or the use of an inappropriate enzyme can affect the stability of antigen expression. Similar...

Recent evidence demonstrates that the adaptive immune system, particularly T lymphocytes, play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, in the model of angiotensin II induced hypertension, an accumulation of T cells in the vessels and kidneys of mice has been described6. The vascular accumulation is predominantly in the adventitia and the perivascular fat. In the kidney, T cells accumulate in both the medulla and renal cortex. Depending on which subset is involved, these T cells give rise to different cytokines that can affect vascular and renal function and lead to the development of pathology (reviewed by McMaster et al.6).
CD4+ T helper lymphocytes can be divided into several subsets: T helper 1 (Th1), Th2, Th9, Th17, Th22, T regulatory (Treg) cells, and T follicular helper (Tfh) cells based on their functions and signature cytokines7. Similarly, CD8+ cytotoxic T cells can be classified as Tc1, Tc2, Tc17 or Tc98. There are also double negative T cells (i.e. cells that do not express the CD4 or CD8 T cell markers). A subset of these cells possess an alternate gamma delta T cell receptor (instead of the classical alpha and beta receptors) and are therefore referred to as gamma delta T cells. The multi-parameter analysis by flow cytometry of surface marker, cytokine and transcription factor constitutes the best approach to identify these cells. Although this method is extensively used in the field of immunology, it is less well described in solid organs and in the setting of cardiovascular diseases.
Historically, the identification of lymphocytes in tissues was limited to immunohistochemistry or RT-PCR approaches. Although immunohistochemistry and immunofluorescence are powerful methods to determine the tissue distribution of an antigen of interest, they are inadequate to phenotypically identify the subsets involved. In addition, while RT-PCR analysis is useful to detect mRNA expression of antigens, cytokines or transcription factors, it doesn&#39;t allow the detection of multiple proteins simultaneously at the level of individual cells.
The advent of flow cytometry, especially when combined with intracellular staining to detect cytokines and transcription factors, provides investigators with a powerful technique that allows identification and quantification at the single cell level of immune cell subsets in solid organs. We have optimized an intracellular staining assay to identify by flow cytometry the major T cell subsets present within murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The optimization of each step: tissue digestion, ex vivo activation, permeabilization, and surface and intracellular staining results in a highly reproducible assay that can be applied to other cardiovascular and renal disease models.

<table><tbody><tr><td>Collagenase D</td><td>ROCHE</td><td>11088882001</td><td></td></tr><tr><td>Collagenase A</td><td>ROCHE</td><td>10103586001</td><td></td></tr><tr><td>Collagenase B</td><td>ROCHE</td><td>11088815001</td><td></td></tr><tr><td>Dnase</td><td>ROCHE</td><td>10104159001</td><td></td></tr><tr><td>1x Red blood cell lysis buffer</td><td>eBioscience</td><td>00-4333-57</td><td></td></tr><tr><td>RPMI Medium 1614 1x</td><td>Gibco</td><td>11835-030</td><td></td></tr><tr><td>DPBS without calcium and magnesium</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-5445-02</td><td>For density gradient centrifugation</td></tr><tr><td>GentleMACS&amp;trade; C tube</td><td>Miltenyi Biotec</td><td>130-096-334</td><td></td></tr><tr><td>GentleMACS dissociator device</td><td>Miltenyi Biotec</td><td>130-093-235</td><td>Use the program SPLEEN_04</td></tr><tr><td>Cell activation cocktail (with Brefeldin A)</td><td>Biolegend</td><td>423303</td><td></td></tr><tr><td>anti-CD16/32</td><td>eBioscience</td><td>14-0161-81</td><td>dilute 1:100</td></tr><tr><td>LIVE/DEAD fixable violet dead cell stain kit</td><td>Life Technologies</td><td>L34955</td><td></td></tr><tr><td>Transcription factor buffer set</td><td>BD Pharmingen</td><td>562725</td><td></td></tr><tr><td>OneComp eBeads&amp;nbsp;</td><td>eBioscience</td><td>01-1111-42</td><td></td></tr><tr><td>123 count eBeads</td><td>eBioscience</td><td>01-1234-42</td><td></td></tr><tr><td>CD45 AmCyan (clone 30-F11)</td><td>BioLegend</td><td>103138</td><td></td></tr><tr><td>CD3 PerCP-Cy5.5 (clone 17A2)</td><td>BioLegend</td><td>100218</td><td></td></tr><tr><td>IL-17A FITC (clone TC11-18H10.1)</td><td>BioLegend</td><td>506910</td><td></td></tr><tr><td>IL-17F APC (clone 9D3.1C8)</td><td>BioLegend</td><td>517004</td><td></td></tr><tr><td>CD4 APC-Cy7 (clone GK1.5)</td><td>BD Biosciences</td><td>560181</td><td></td></tr><tr><td>CD8 APC (clone 53-67)</td><td>eBioscience</td><td>17-0081-82</td><td></td></tr><tr><td>T-bet PE-Cy7 (clone 4B10)</td><td>BioLegend</td><td>644823</td><td></td></tr><tr><td>IFN&amp;gamma; FITC (clone XMG1.2)</td><td>BD Biosciences</td><td>557724</td><td></td></tr></tbody></table>

intracellular staining and flow cytometry to identify lymphocyte subsets within murine aorta, kidney and lymph nodes in a model of hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

The protocol described permits the identification of surface and intracellular markers in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. Representative results are presented below.
Figure 1 demonstrates the gating strategy used to identify the T cell population in a single cell suspension prepared from the aorta of a WT mouse infused ...

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Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

intracellular-staining-flow-cytometry-to-identify-lymphocyte-subsets

Research

JoVE Journal

Immunology and Infection

18.2K Views.  Vanderbilt University Medical Center.  Dit artikel bevat gedetailleerde methodologie voor het identificeren en kwantificeren van functionele T-lymfocyten subsets in muizen nieren, aorta en de lymfeklieren aanwezig door intracellulaire kleuring en flowcytometrie. Het model van angiotensine II geïnduceerde hypertensie werd gekozen uitleggen, stap voor stap, de procedures en de grondbeginselen van flowcytometrie en intracellulaire kleuring. 

Video: Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Medicine

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

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