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.
CITATION STYLE
Vivar, O. I., Magalhaes, J. G., & Amigorena, S. (2016). Measurement of export to the cytosol in dendritic cells using a cytofluorimetry-based assay. Methods in Molecular Biology, 1423, 183–198. https://doi.org/10.1007/978-1-4939-3606-9_13
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