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Method Article

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

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July 8th, 2025

In This Article

Abstract

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

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

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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