A subscription to JoVE is required to view this content. Sign in or start your free trial.
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.
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.
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....
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
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.......
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........
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.
....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 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved