A Method for the High Salt Treatment of Dendritic Cells and their Adoptive Transfer into Mice

Abstract

Source: Van Beusecum, J. P., et al. Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells. J. Vis. Exp. (2019).

This video demonstrates a procedure for the adoptive transfer of high salt-treated antigen-presenting dendritic cells (DCs) into mice. Murine CD11c+ dendritic cells (DCs) are exposed to a medium containing high amounts of sodium, forming isolevuglandin (IsoLG)-protein adducts in the cells. These adducts serve as neoantigens and are presented on the surface of DCs. The high salt-treated cells are injected into mice through adoptive cell transfer.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Isolation of Spleens from Mice Prepare 1640 RPMI (Roswell Park Memorial institute): 10% fetal bovine serum (FBS), 0.10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM sodium pyruvate, 50 µM β-mercaptoethanol and 1% penicillin/streptomycin. Euthanize 10–12 week-old C57bl/6 ma…

Materials

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