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

Summary

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

Abstract

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.

Introduction

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, de....

Protocol

Vanderbilt University'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

1. Prepare 1640 RPMI: 10% FBS, 0.10 mM HEPES, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol and 1% penicillin/streptomycin.
2. Euthanize 10–12 week-old C57bl/6 male mice by CO₂ inhalation. Spray the chest and sides of the mouse with 70% ethanol. On the left side, carefully open the skin and the peritoneal c....

Results

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 naïve recipient mice. Ten days later, mice are implanted with osmotic minipumps for low-dose angiotensin II (140 ng/kg/min).......

Discussion

In the current protocol, we have optimized procedures to isolate CD11c⁺ DCs from spleens of mice and adoptively transfer them into naïve 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........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
APC/Cy7 anti-mouse CD11c	Biolegend	117324
autoMACS Running Buffer	Miltenyi Biotec	130-091-221
CD11c MicroBeads Ultrapure	Miltenyi Biotec	130-108-338
Collagenase D	Roche	11088866001
DNase I	Roche	10104159001
DPBS without calcium and magnesium	Corning	21-031-CV
FcR Blocking Reagent	Miltenyi Biotec	130-092-575
FITC anti-mouse CD45	Biolegend	103108
GentleMACS C tube	Miltenyi Biotec	130-096-334
GentleMACS dissociator device	Miltenyi Biotec	130-093-235	Use protocol: Spleen 04.01
LIVE/DEAD fixable violet dead cell stain kit	Invitrogen	L34964
LS Columns	Miltenyi Biotec	130-042-401
QuadroMACs Seperator	Miltenyi Biotec	130-090-976
RPMI 1640 medium	Gibco	11835-030