Taylorellae are actively phagocytised by A. castellanii. Bacterial uptake assay by trypan blue quenching. Acanthamoeba castellanii cells were infected with fluorescein-labelled E. coli, T. equigenitalis, T. asinigenitalis or L. pneumophila at an MOI of 50, in the presence, when indicated, of either 10 μM of cytochalasin D—an actin polymerization inhibitor (+CytoD)—or 2 μM of Wortmanin—a PI3K inhibitor (+Wort). After 30 min of incubation, the medium was replaced by trypan blue solution to quench the fluorescence of non-internalised bacteria. The fluorescence of internalised bacteria was measured using an excitation level of 485 nm and an emission of 530 nm. Fluorescence data were corrected for differences in labelling efficiency between the tested strains. Each bar represents the mean of triplicate wells and error bars represent the standard deviations. Significant differences from the control, determined by an unpaired tailed Student t test, are indicated by *(p = 0.058) and **(p < 0.05).
Allombert et al. BMC Microbiology 2014 14:69 doi:10.1186/1471-2180-14-69