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Measurement of Export to the Cytosol in Dendritic Cells Using a Cytofluorimetry-Based Assay

  • Omar I. Vivar
  • Joao G. MagalhaesEmail author
  • Sebastian Amigorena
Part of the Methods in Molecular Biology book series (MIMB, volume 1423)

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.

Key words

β-lactamase Cross-presentation Dendritic cell Cytosolic export CCF4 

Notes

Acknowledgments

This work was financed by Institut Curie, Institut National de la Santé et de la Recherche Médicale (INSERM), the European Research Council (2008 Advanced Grant 233062 PhagoDC), Agence National de Recherche (ANR-11-LABX-0043). O.I.V. received funding from Association pour la Recherche sur le Cancer.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Omar I. Vivar
    • 1
  • Joao G. Magalhaes
    • 1
    Email author
  • Sebastian Amigorena
    • 1
  1. 1.INSERM U932 and Institut Curie, Centre de rechercheParisFrance

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