Research Article

Cholesterol Oxidase Binds TLR2 and Modulates Functional Responses of Human Macrophages

Figure 2

FITC-ChoD binding to THP-1-derived macrophages determined by confocal microscopy and flow cytometry. Macrophages were incubated FITC-ChoD (24 μg/mL, equivalent to 0.5 U/mL) (a) or FITC-BSA (24 μg/mL) as a reference protein (b) and then stained with CellTrackerRed (red fluorescence, cytoplasm) and Hoechst 33258 (blue fluorescence, nuclei). Cells were analyzed by confocal microscopy (40x objective). Extracellular localization of FITC-ChoD is marked by white arrow (a). Intracellular localization of FITC-BSA is marked by yellow arrow (b). (c, d) Binding of FITC-labeled ChoD to macrophage membranes before and after incubation with unlabeled ChoD. Cells incubated with or without ChoD (0.5 U/mL) for 30 min were subsequently incubated with FITC-ChoD (24 μg/mL, equivalent to 0.5 U/mL) for 1 h. Fluorescence was measured by flow cytometry and expressed as MFI. The fluorescence intensity of cells not treated with FITC-ChoD was defined as autofluorescence and was subtracted from the fluorescence values of corresponding FITC trials. Data are means ± SEM of MFI values from three independent experiments (c). A representative histogram shows the fluorescence of (1) cells incubated with FITC-ChoD, (2) cells preincubated with unlabeled ChoD and then with FITC-ChoD, and (3) autofluorescence of untreated cells (d).