Related Experiment Video
Updated: Mar 21, 2026
Author Spotlight: Exploring the Mechanisms of MicroRNA Loading into Extracellular Vesicles in Cancer Progression
Published on: October 6, 2023
Measurement of Export to the Cytosol in Dendritic Cells Using a Cytofluorimetry-Based Assay
Omar I Vivar1, Joao G Magalhaes2, Sebastian Amigorena1
1INSERM U932 and Institut Curie, Centre de recherche, 26 Rue d'Ulm, 75005, Paris, France.
Insights
Dendritic cells (DCs) present external antigens via cross-presentation. A new flow cytometry assay quantifies antigen export to the cytosol in DCs, crucial for immune responses.
Area of Science:
- Immunology
- Cell Biology
Background:
- Cross-presentation of exogenous antigens on MHC class I is a critical immune function of dendritic cells (DCs).
- This process relies on the translocation of antigens from endocytic vesicles to the cytosol.
Purpose of the Study:
- To develop and validate a novel cytofluorimetry-based assay for quantifying antigen export to the cytosol in DCs.
- To analyze the kinetics and efficiency of this crucial step in cross-presentation.
Main Methods:
- Dendritic cells (DCs) were loaded with a Förster Resonance Energy Transfer (FRET)-sensitive cytosolic substrate of beta-lactamase (CCF4).
- Antigen export to the cytosol was measured by flow cytometry, detecting the cleavage of the FRET dye and subsequent fluorescence shift.
Main Results:
- The assay provides a quantitative measurement of antigen export to the cytosol.
- This method allows for the analysis of the kinetics and efficiency of antigen translocation in DC populations.
Conclusions:
- A new, quantitative flow cytometry assay has been established to study antigen export in dendritic cells.
- This assay is valuable for investigating the mechanisms and efficiency of cross-presentation.
Abstract:
The presentation of exogenous antigens on MHC class I molecules, known as cross-presentation, is a key function of dendritic cells (DCs). Cross-presentation via the cytosolic pathway involves antigen export from endocytic compartments to the cytosol. We have recently developed a cytofluorimetry-based assay to examine the kinetics and the efficiency of antigen export to the cytosol in DC populations. In this assay, DCs are loaded with a FRET-sensitive cytosolic substrate of β-lactamase, CCF4. Following uptake of β-lactamase by the DCs, the enzyme undergoes export to the cytosol leading to cleavage of the FRET dye. This cleavage and switch of fluorescence are analyzed by flow cytometry, allowing a quantitative measurement of this event.
More Related Videos
08:31Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols
Published on: October 18, 2016
08:53Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells
Published on: May 16, 2017