Labeling and tracking of acidic organelles in macrophages cells infected with <i>C</i>. <i>diphtheriae</i>.

<p>J774E cells were incubated with LysoTracker Red DND-99 for 120 min before cells were infected with <i>C</i>. <i>glutamicum</i> ATCC 13032 pEPR1p45<i>gfp</i> and <i>C</i>. <i>diphtheriae</i> strains DSM43988 pEPR1p45<i>gfp</i>, DSM43989 pEPR1p45<i>gfp</i> and DSM44123 pEPR1p45<i>gfp</i> at an MOI of 10 for 30 min. Extracellular bacteria were killed by the addition of gentamicin and after 2, 4 and 20 h, cells were fixed. Nuclei were stained with DAPI, the cytoskeleton with Alexa Fluor 647 Phalloidin and micrographs were taken using the confocal laser-scanning microscope Leica SP5 II and analysed with the LAS software suite. Non-pathogenic <i>C</i>. <i>glutamicum</i> immediately co-localize with acidic compartments (2 h) whereas <i>C</i>. <i>diphtheriae</i> strains only show co-localization after longer incubation time (4 h to 20 h). Representative pictures are shown. Scale bars: 10 μm.