This work was supported by American Heart Association grants POST290900 to N.R.B., 17SDG33670829 to L.X. and National Institutes of Health grant K01HL130497 to A.K.

Vanderbilt University&#39;s Institutional Animal Care and Use Committee have 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 Spleens from Mice
<ol>
	<li>Prepare 1640 RPMI: 10% FBS, 0.10 mM HEPES, 1 mM sodium pyruvate, 50 &#181;M &#946;-mercaptoethanol and 1% penicillin/streptomycin.</li>
	<li>Euthanize 10&#8211;12 week-old C57bl/6 male mice...

<ol>
	<li>Kearney, P. M., 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=Global+burden+of+hypertension:+analysis+of+worldwide+data.">Global burden of hypertension: analysis of worldwide data.</a> Lancet. 365, 217-223 (2005).</li><li>Murray, C. J., Lopez, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+global+burden+of+disease.">Measuring the global burden of disease.</a> N Engl J Med. 369, 448-457 (2013).</li><li>Lev-Ran, A., Porta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+and+hypertension:+a+phylogenetic+perspective.">Salt and hypertension: a phylogenetic perspective.</a> Diabetes/metabolism research and reviews. 21, 118-131 (2005).</li><li>Frisoli, T. M., Schmieder, R. E., Grodzicki, T., Messerli, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+and+hypertension:+is+salt+dietary+reduction+worth+the+effort.">Salt and hypertension: is salt dietary reduction worth the effort.</a> The American journal of medicine. 125, 433-439 (2012).</li><li>He, F. J., Li, J., Macgregor, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+longer-term+modest+salt+reduction+on+blood+pressure.">Effect of longer-term modest salt reduction on blood pressure.</a> Cochrane Database Syst Rev. 4, 004937 (2013).</li><li>Weinberger, M. H., Miller, J. Z., Luft, F. C., Grim, C. E., Fineberg, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitions+and+characteristics+of+sodium+sensitivity+and+blood+pressure+resistance.">Definitions and characteristics of sodium sensitivity and blood pressure resistance.</a> Hypertension. 8, 127-134 (1986).</li><li>Morimoto, 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=Sodium+sensitivity+and+cardiovascular+events+in+patients+with+essential+hypertension.">Sodium sensitivity and cardiovascular events in patients with essential hypertension.</a> Lancet. 350, 1734-1737 (1997).</li><li>Weinberger, M. H., Fineberg, N. S., Fineberg, S. E., Weinberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+sensitivity,+pulse+pressure,+and+death+in+normal+and+hypertensive+humans.">Salt sensitivity, pulse pressure, and death in normal and hypertensive humans.</a> Hypertension. 37, 429-432 (2001).</li><li>Barbaro, N. R., 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=Dendritic+Cell+Amiloride-Sensitive+Channels+Mediate+Sodium-Induced+Inflammation+and+Hypertension.">Dendritic Cell Amiloride-Sensitive Channels Mediate Sodium-Induced Inflammation and Hypertension.</a> Cell Rep. 21, 1009-1020 (2017).</li><li>Kirabo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+paradigm+of+sodium+regulation+in+inflammation+and+hypertension.">A new paradigm of sodium regulation in inflammation and hypertension.</a> American journal of physiology. Regulatory, integrative and comparative physiology. 313, 706-710 (2017).</li><li>Machnik, 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=Macrophages+regulate+salt-dependent+volume+and+blood+pressure+by+a+vascular+endothelial+growth+factor-C-dependent+buffering+mechanism.">Macrophages regulate salt-dependent volume and blood pressure by a vascular endothelial growth factor-C-dependent buffering mechanism.</a> Nat Med. 15, 545-552 (2009).</li><li>Kopp, C., 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=23Na+magnetic+resonance+imaging-determined+tissue+sodium+in+healthy+subjects+and+hypertensive+patients.">23Na magnetic resonance imaging-determined tissue sodium in healthy subjects and hypertensive patients.</a> Hypertension. 61, 635-640 (2013).</li><li>Dixon, K. B., Davies, S. S., Kirabo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+isolevuglandins+in+immunity,+inflammation,+and+hypertension.">Dendritic cells and isolevuglandins in immunity, inflammation, and hypertension.</a> American journal of physiology. Heart and circulatory physiology. 312, 368-374 (2017).</li><li>Kirabo, 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=DC+isoketal-modified+proteins+activate+T+cells+and+promote+hypertension.">DC isoketal-modified proteins activate T cells and promote hypertension.</a> J Clin Invest. 124, 4642-4656 (2014).</li><li>McMaster, W. G., Kirabo, A., Madhur, M. S., Harrison, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation,+immunity,+and+hypertensive+end-organ+damage.">Inflammation, immunity, and hypertensive end-organ damage.</a> Circ Res. 116, 1022-1033 (2015).</li><li>Harrison, D. G., Vinh, A., Lob, H., Madhur, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+adaptive+immune+system+in+hypertension.">Role of the adaptive immune system in hypertension.</a> Curr Opin Pharmacol. 10, 203-207 (2010).</li><li>Madhur, M. S., 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=Interleukin+17+promotes+angiotensin+II-induced+hypertension+and+vascular+dysfunction.">Interleukin 17 promotes angiotensin II-induced hypertension and vascular dysfunction.</a> Hypertension. 55, 500-507 (2010).</li><li>Harrison, D. G., 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=Inflammation,+immunity,+and+hypertension.">Inflammation, immunity, and hypertension.</a> Hypertension. 57, 132-140 (2011).</li><li>Crowley, S. 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=Stimulation+of+lymphocyte+responses+by+angiotensin+II+promotes+kidney+injury+in+hypertension.">Stimulation of lymphocyte responses by angiotensin II promotes kidney injury in hypertension.</a> American journal of physiology. Renal physiology. 295, 515-524 (2008).</li><li>Zhang, J. 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+novel+role+for+type+1+angiotensin+receptors+on+T+lymphocytes+to+limit+target+organ+damage+in+hypertension.">A novel role for type 1 angiotensin receptors on T lymphocytes to limit target organ damage in hypertension.</a> Circ Res. 110, 1604-1617 (2012).</li></ol>

In the current protocol, we have optimized procedures to isolate CD11c+ DCs from spleens of mice and adoptively transfer them into na&#239;ve animals to study the role of DCs in salt-induced hypertension. This protocol can be adapted to isolate and adoptively transfer other immune cell subsets including macrophages, monocytes, and adaptive immune cells including T and B lymphocytes. We have optimized the splenic digestion process to achieve adequate cell survival and stability of DC surface expression markers....

Excess dietary salt is a major risk factor for hypertension.1,2 The American Heart Association recommends a maximum of 2,300 milligrams (mg) of sodium (Na+) intake per day, however; less than 10% of the U.S. population observes this recommendation.3,4 Modest reductions in Na+ intake lower blood pressure and reduce the annual new cases of coronary heart disease and stroke in the U.S. by 20%.5 A major problem with excess salt consumption is that 50% of the hypertensive population exhibits salt-sensitivity, defined as a 10 mmHg increase in the blood pressure following Na+ loading or a similar drop in blood pressure after Na+ restriction and diuresis.6 Salt-sensitivity also occurs in 25% of normotensive individuals, and is an independent predictor of death and cardiovascular events.7,8 Salt-sensing mechanisms in hypertension involving the kidney have been well studied; however, recent studies suggest that immune cells can sense Na+.9,10
Recent evidence suggests that changes in extra-renal Na+ handling can cause accumulation of Na+ in the interstitium and promote inflammation.11,12 Our laboratory and others have shown that cells of both the innate and adaptive immune system contribute to the exacerbation of hypertension.9,13,14,15 Various hypertensive stimuli, including angiotensin II, norepinephrine, and salt cause macrophages, monocytes and T lymphocytes to infiltrate the kidney and vasculature and promote Na+ retention, vasoconstriction, blood pressure elevation and end-organ damage.9,16,17,18,19,20 In prior studies, we found that DCs accumulate isolevuglandin (IsoLG)-protein adducts in response to various hypertensive stimuli including angiotensin II and DOCA-salt hypertension.14 IsoLGs are highly reactive products of lipid peroxidation that rapidly and covalently adduct to lysines on proteins and their accumulation is associated with DC activation.14 We have recently established that elevated Na+ is a potent stimulus for IsoLG-protein adduct formation in murine DCs.9 Na+ entry into DCs is mediated through amiloride sensitive transporters. Na+ is then exchanged for calcium (Ca2+) via the Na+/Ca2+ exchanger. Ca2+ activates protein kinase C (PKC) which activates the NADPH oxidase leading to increased superoxide (O2&#183;-) and IsoLG-protein adduct formation.9 Adoptive transfer of salt-exposed DCs primes hypertension in response to a sub-pressor dose of angiotensin II.9
Identification of CD11c+ DCs from tissues has been previously limited to immunohistochemistry and RT-PCR, and isolation of DCs has been limited to cell sorting by flow cytometry. Although flow cytometry cell sorting is a powerful method for the isolation of immune cells, it is costly, time-consuming, and leads to a low yield of viable cells. Therefore, we have optimized a step by step protocol for tissue digestion, in vitro stimulation, and adoptive transfer of CD11c+ DCs to study hypertension.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">APC/Cy7 anti-mouse CD11c</td>
			<td style="width:160px;">Biolegend</td>
			<td align="right" style="width:140px;">117324</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">autoMACS Running Buffer&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD11c MicroBeads Ultrapure&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-108-338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase D</td>
			<td>Roche</td>
			<td align="right">11088866001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS without calcium and magnesium</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FcR Blocking Reagent</td>
			<td>Miltenyi Biotec</td>
			<td>&#160;130-092-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">FITC anti-mouse CD45</td>
			<td style="width:160px;">Biolegend</td>
			<td align="right" style="width:140px;">103108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GentleMACS C tube</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GentleMACS dissociator device</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Use protocol: Spleen 04.01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD fixable violet dead cell stain kit</td>
			<td>Invitrogen</td>
			<td>L34964</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuadroMACs Seperator&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 medium&#160;</td>
			<td style="width:160px;">Gibco</td>
			<td style="width:140px;">11835-030</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and adoptive transfer of high salt treated antigen-presenting dendritic cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Figure 1 represents a schematic of the described steps above. Isolated murine spleens are sorted for CD11c+ DCs by magnetic cell sorting and plated in either normal salt media (NS; 150 mmol NaCl) or high salt media (HS; 190 mmol NaCl) for 48 h. CD11c+ DCs are then adoptively transferred by retro-orbital injection to na&#239;ve recipient mice. Ten days later, mice are implanted with osmotic minipumps for low-dose angiotensin II (140 ng/kg...

Watch this Scientific Journal Video about Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells at JoVE.com

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

This method can answer key questions in the fields of hypertension and cardiovascular disease including the study of CD11c positive dendritic cells and how they potentiate the role of hypertensive injury. The main advantage of this technique is the isolation of CD11c positive dendritic cells from solid organs and the study of their physiological role in various disease states. So this process can be applied to multiple immune cell types, including B lymphocytes, T lymphocytes, monocytes, and macrophages.Visualization of the technique is critical to understand dendritic cell isolation, specifically the adoptive transfer process into recipient mice. To begin, spray the chest and sides of the euthanized mouse with 70%ethanol. On the left side, carefully open the skin and the peritoneal cavity to expose the spleen.Using small forceps, cautiously move the intestines and liver to the left side of the mouse. Then use small scissors to gently excise the spleen. Place each excised spleen into a labeled C tube containing three milliliters of RPMI 1640 medium.First add collagenase D and DNase to prepared RPMI 1640 medium to prepare the spleen digestion solution. Place the C tube onto a semi-automated homogenizer and ensure that the tube is tightly placed on the rotating arm. Press the start button on the homogenizer to run the spleen 04.01 protocol for 60 seconds.After this, detach the tube from the homogenizer and incubate the samples at 37 degrees Celsius under continuous at 20 rpm for 15 minutes. Next, use a pipette to transfer the solution into a 50 milliliter conical tube after filtering it through a 40 micrometer strainer. Wash the strainer with 10 milliliters of dPBS.Centrifuge the cells at 300 times g and at four degrees Celsius for 10 minutes. Then aspirate the supernatant completely and wash the pellet by re-suspending it in 10 milliliter of dPBS. Centrifuge the suspension at 300 times g and at four degrees Celsius for 10 minutes.Re-suspend the cell pellet in five milliliters of dPBS. Use a hemocytometer and Trypan blue exclusion to count the number of cells. Next centrifuge at 300 times g and at four degrees Celsius for 10 minutes to pellet the cells.Aspirate the supernatant completely and re-suspend the pellet in 400 milliliters of Magnetically Activated Cell-sorting buffer. Then add 100 milliliters of CD11c microbeads per 100 million cells. Vortex the cell suspension and incubate for 10 minutes in a refrigerator at four degrees Celsius.After this, add 10 milliliters of MACs buffer to wash the cells and centrifuge at 300 times g and at four degrees Celsius for 10 minutes. Aspirate the supernatant completely and re-suspend the cells in 500 milliliters of MACs buffer. Place the LS column in the magnetic field of the mag separator and place a 15 milliliter conical tube under each column to collect the flow through.Next, rinse the columns with three milliliters of buffer. Place the cell suspension onto each column and collect the flow through which contains unlabeled cells. Wash the columns with three milliliters of MACs buffer and collect the unlabeled cells that pass through.Wash the LS columns two additional times using three milliliters of MACs buffer per wash. After this, remove the LS columns from the magnetic separator. Add five milliliters of MACs buffer to each column and immediately plunge each column into a clear 15 milliliter conical tube to flush out the magnetically labeled cells.Use a hemocytometer and Trypan blue exclusion to determine the CD11c positive DC number as outlined in the text protocol. Then dilute the cell sample at a one to one dilution in 0.4%Trypan blue solution. First, prepare the cell pellets in DC culture media as outlined in the text protocol.Pipette 900 microliters of the DC suspension into 24-well flat bottom falcon plate. Add 100 microliters of the high salt DC culture media to each well, for a final sodium concentration of 190 millimolar. Label the plate and place it in a humidified carbon dioxide incubator at 37 degrees Celsius for 48 hours.After 48 hours remove the plate from the incubator. Pipette the cell culture media in a single well up and down to re-suspend the CD11c positive DCs. Transfer all this suspension in a corresponding labeled 1.6 milliliter tube.Repeat this for each individual well that was plated. Centrifuge all of the tubes at 300 times g and at four degrees Celsius for 10 minutes. Aspirate the supernatant and re-suspend the DC pellet with 100 microliters of sterile dPBS.Next, draw the DC solution from the one of the tubes into a one milliliter syringe, equipped with a 27 gauge half-inch needle. Place the naive male 10 week old C57-black-6 mouse under 2%isoflurane to achieve a stable surgical plane. Check the level of anesthesia with a lack of pedal reflex.Once a stable surgical plane is achieved, slowly and carefully introduce the needle into the retro-orbital space at an angle of approximately 30 degrees. Slowly and smoothly inject the CD11c positive DC suspension. Once the injection is complete, carefully remove the needle for the retro-orbital space, making sure not to damage the eye.Then remove the mice from the nose cone and monitor them for approximately 30 minutes during recovery from anesthesia. A typical blood pressure analysis is shown here following the adoptive transfer of DCs and osmotic mini pumps that have been implanted for low dose Angiotensin II infusion. It should be noted that this low dose of Angiotensin II is a suppressor dose that does not increase blood pressure in a normal mouse.Mice that receive normal salt-treated CD11c positive DCs maintain a normal blood pressure during the infusion, while the mice that receive high-salt treated CD11c positive DCs exhibit an increase in systolic blood pressure. Flow cytometry analysis is then performed utilizing the gating strategy shown here to determine the purity of the isolated CD11c positive cells. An increased enrichment of the CD11c positive cells is seen compared to the total splenocytes.Different CD11c Microbead concentrations are utilized to troubleshoot the manufacturer's protocol. Magnetic separate of splenocytes using 100 microliters of Microbeads yields about 65%of CD11c positive cells, while using 200 microliters yields 55%and using 300 microliters yields 50%An FcR blocker is included along with 100 microliters of Microbeads. Following the magnetic separation, the blockade of FcR yields approximately 65%CD11c positive cells.Some splenocytes are then incubated with 100 microliters of Microbeads and magnetically separated through an LS column. The separated cells are incubated with an additional 100 microliters of CD11c and separated again through an LS column. This double isolation process yielded 92%CD11c positive cells.When attempting this procedure, it is important to confirm the purity of the isolated cells by flow cytometry. Following magnetic isolation of dendritic cells they can be cultured to determine their activation status by molecular techniques including proteomics and single-cell sequencing. Since its development, it is now possible to study the function and specific role of dendritic cells in hypertension.Researchers are now exploring the specific antigens in hypertension.

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Immunology and Infection

5.5K vistas.  Vanderbilt University Medical Center. Este método puede responder a preguntas clave en los campos de la hipertensión y las enfermedades cardiovasculares, incluido el estudio de las células dendríticas positivas CD11c y cómo potencian el papel de la lesión hipertensa. La principal ventaja de esta técnica es el aislamiento de células dendríticas positivas CD11c de órganos sólidos y el estudio de su papel fisiológico en varios estados de la enfermedad. Así que este proceso se puede aplicar a múltiples tipos de células...