Name: isolation and adoptive transfer of high salt treated antigen-presenting dendritic cells
Uploaded: 2019-03-05T13:30:00
Description: Watch this Scientific Journal Video about Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells at JoVE.com

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. 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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. 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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. 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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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	<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 ...

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