Method Article

Flow Cytometry-Based Analysis of Dendritic Cell Activation Using Immune Complexes

July 8th, 2025

In This Article

Abstract

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Source: Santana-Magal, N., et al. Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes. J. Vis. Exp. (2018)

This video demonstrates a protocol for assessing the activation of dendritic cells using immunoglobulin G (IgG)-coated tumor cells. Dendritic cells internalize the IgG-coated tumor cells, process the tumor antigens, and present them on the surface through MHC-II molecules, along with the co-expression of the costimulatory molecule CD86. These dendritic cells then undergo MHC-II and CD86-targeted immunostaining and flow cytometry analysis to identify their activation state.

Protocol

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All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Isolation of Tumor-Associated Monocyte-Derived Dendritic Cells

  1. In a laminar flow hood, remove tumors from CO2 euthanized mice and place tumors in RPMI medium without fetal bovine serum (FBS).  
    NOTE: Shaving the mice and spraying them with 70% ethanol before removing the tumors is highly recommended to minimize potential contaminations from the fur. Be sure that the tumor does not exceed 25 - 40 mm2 since larger tumors have fewer de....

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Ficoll-Paque PREMIUMGE-Healthcare17-5442-02
OptiPrepStemCell Technologies7820
CD45 MicroBeadsMiltenyi130-052-301
EasySep Monocyte Isolation KitStemCell Technologies19861
Collagenase IVSigmaC9697-50MGTest each lot for endotoxin
DNase ISigmaDN25-10MG
HBSSThermoFisher14025092
FBSThermoFisher16140071Test each lot for endotoxin
PE-CD11cBiolegend117307
APC-CD11bBiolegend101211
Brilliant Violet 650 MHCIIBiolegend107641
AF48- CD86Biolegend105017
APC/Cy7-Ly-C6Biolegend108423
PE/Cy7-CD15Biolegend135523

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Tags

Flow CytometryDendritic Cell ActivationImmune ComplexesMHC II ExpressionCD86 CostimulationCell DissociationAntibody StainingDead Cell StainingTumor Antigen ProcessingCell Surface Presentation

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