Figure 8.

Detoxified listeriolysin O (dtLLO) preserves the binding property of LLO to the plasma membrane of human immune cells. Human peripheral blood mononuclear cells (PBMCs) were pulsed with dtLLO (red line) or wild‐type LLO (wtLLO) (blue line) for 10 or 20 min. Non‐pulsed cells were included (black line) as a negative control. The pulsed cells were fixed and stained as described previously in the Materials and methods section and analysed by flow cytometry (a,c). Bars (b,d) represent the mean fluorescence intensity for each variable in (a) and (c). To evaluate the role of cholesterol in the binding of the toxoid to the plasma membranes, PBMCs were treated (pink line) or not (red line) with methyl‐β‐cyclodextrin (mβCD) for 30 min at 37ºC, washed and then pulsed with dtLLO for 10 or 20 min, then the cells were fixed, stained and analysed by flow cytometry (e,g). Non‐pulsed cells were treated (blue line) or not (black line), with mβCD as controls. Bars (f,h) represent the mean fluorescence intensity for each variable in (e) and (g). One representative experiment of three independent experiments is shown. [Colour figure can be viewed at wileyonlinelibrary.com]