Serratia Invasion to Epithelial Cells Figure 4. The S. marcescens-containing vacuoles are predominantly non acidic and non degradative compartments. A) CHO-EGFP-LC3 cells were infected with wild-type S. marcescens. After 60 min p.i, cells were incubated with 3 mM of LysoTracker to label acidic compartments. Cells were fixed at 240 or 360 min p.i. and intracellular bacteria were detected with antibodies anti-Serratia coupled with a secondary antibody labeled with Alexa Fluor 647. The percentages of colocalization of bacteria with EGFP-LC3 and/or with Lysotracker were determined by fluorescence microscopy. At least 300 infected cells were counted for each condition. The average 6 S.D. is shown for two independent experiments, *and **p,0.001. B) Representative confocal laser captured images of CHO-EGFP-LC3 cells (green fluorescence, d) infected with S. marcescens (blue fluorescence, b) and incubated with Lysotracker (red fluorescence, c) at 360 min p.i. are shown. Inset image (f) shows higher magnification of the boxed area in the merged image (e), and highlights a non-acidic, autophagic SeCV. Arrows point at acidic vesicles. Bars: 10 mm. C) CHO-EGFP-LC3 cells were pre-incubated with 10 mg/ml of DQ-BSA for 4 hours to label degradative compartments. Subsequently, cells were infected with wild-type S. marcescens and fixed at 240 or 360 min p.i. Intracellular bacteria were detected with antibodies anti-Serratia coupled with a secondary antibody PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e24054