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© Andreas Kühbacher, Edith Gouin, Jason Mercer, Mario Emmenlauer, Christophe Dehio, Pascale Cossart and Javier Pizarro-Cerda
Immunofluorescence and segmentation analysis of HeLa cells infected with Listeria monocytogenes. Cells were labelled with DAPI to mark DNA (blue), with phalloidin to mark actin (red) and with anti-Internalin C antibodies to identify infected cells (yellow). Nuclei and cytoplasms were segmented using the public image analysis software CellProfiler
Scientific Fields
Diseases
Organisms
Applications
Technique
Starting Date
10
Apr 2016
Status
Ongoing
Members
7
Structures
2
Publications
1

About

We developed a novel image-based microscopy assay which allows to discriminate Listeria monocytogenes cellular entry from vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We generated a L. monocytogenes strain expressing a β-lactamase covalently attached to the bacterial cell wall, which can cleave a Förster resonance energy transfer (FRET) probe CCF4 loaded in the cytoplasm of target cells: the probe will be only cleaved by bacteria which escape their phagosomes. To discriminate between non-invading bacteria and bacteria trapped in phagosomes we perform subsequently a differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay.  

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