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)


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 



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.


  1. 1.
    Joffre OP, Segura E, Savina A, Amigorena S (2012) Cross-presentation by dendritic cells. Nat Rev Immunol 12(8):557–569CrossRefPubMedGoogle Scholar
  2. 2.
    Mantegazza AR, Magalhaes JG, Amigorena S, Marks MS (2013) Presentation of phagocytosed antigens by MHC class I and II. Traffic 14(2):135–152CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Kovacsovics-Bankowski M, Rock KL (1995) A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules. Science 267(5195):243–246CrossRefPubMedGoogle Scholar
  4. 4.
    Cebrian I, Visentin G, Blanchard N, Jouve M, Bobard A, Moita C, Enninga J, Moita LF, Amigorena S, Savina A (2011) Sec22b regulates phagosomal maturation and antigen crosspresentation by dendritic cells. Cell 147(6):1355–1368CrossRefPubMedGoogle Scholar
  5. 5.
    Segura E, Durand M, Amigorena S (2013) Similar antigen cross-presentation capacity and phagocytic functions in all freshly isolated human lymphoid organ-resident dendritic cells. J Exp Med 210(5):1035–1047CrossRefPubMedPubMedCentralGoogle Scholar

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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