Generation and Use of Reporter Cell Lines to Analyze Integrity of Salmonella-Containing Vacuoles by Automated Live-Cell Imaging and Quantification.

The stable expression of fluorescent reporters in cell lines is crucial for the analysis of host-pathogen interactions. Here, an optimized protocol for generating a HeLa cell line that indicates membrane damage, including lentiviral particle production, transduction, functional tests, and fluorescence-activated sorting (FACS) of HeLa cells, is described. In addition, an image-based segmentation protocol is described for quantifying membrane damage to Salmonella-containing vacuoles (SCV) and the presence of cytosolic Salmonella enterica. This enables the analysis of virulence factors that affect the integrity of SCV and thus contribute to the intracellular lifestyle of the pathogen.